Datasets:

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acmc

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beamit-annotated_full_texts_dataset

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[ "Acid-Base Imbalance", "Acidosis", "Acidosis, Respiratory", "Adult", "Age Distribution", "Alkalosis", "Alkalosis, Respiratory", "Blood Gas Analysis", "Critical Illness", "Cross-Sectional Studies", "Female", "Humans", "Male", "Middle Aged", "Nepal", "Prevalence", "Sex Distribution", "Tertiary Care Centers", "Young Adult" ]

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[ "INTRODUCTION", "METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS", "Conflict of Interest" ]

[ "Smooth physiological and well balanced functioning of a body depends on a very tight balance between the concentrations of acids and bases in the blood. Acid-base balance is important in the healthy maintenance of the cellular functions of the body. When there is imbalance between acid and base components in the body, it leads to Acid-Base Disorder (ABD). ABD is generally well correlated with high rates of morbidity and mortality.1,2\nThe appropriate diagnosis of ABD in critically ill patients requires measurement of plasma electrolytes and arterial blood gases by Arterial Blood Analysis (ABG), which evaluates metabolic and respiratory functions (pCO2, pH and pO2). An early diagnosis established by ABG can help in guiding the treatment of such patients and provide the details related with seriousness of the case.3 The main aim of this study was to find the prevalence of acid-base disorders and biochemical findings of such disorders in patients in a tertairy care hospital.", "This is a descriptive cross-sectional study which was carried out between a periods of 1st September 2018 to 31st August 2019 at Nobel Medical College Teaching Hospital (NMCTH) after getting the approval from the Institutional Review Committee. All the patients, who presented to emergency department, Intensive Care Units (ICU), Neonatal ICU and in different wards undergoing ABG analysis in the department of Biochemistry, clinical laboratory services, were enrolled for the study. Whole sampling was done. All the participants had signed the informed consent for the study.\nArterial blood samples were collected from the patients presented in the different departments of NMCTH according to proper medical guidelines with all care on details such as site selection, collection procedures, sampling devices, sample handling etc. Sterile techniques were followed to prevent the site from being contaminated. Only those sample devices containing the proper amount of calcium-titrated heparin or lithium heparin as the anticoagulant were used to collect whole blood samples.\nArterial blood gas analysis was carried out by EDAN il5 blood gas and chemistry automated analyzer, which holds test cartridge in a portable and automated system that measures pH and blood gas, metabolites and electrolytes. It utilizes potentiometry and amperometry to determine the concentration of blood gas and blood chemistries. The test cartridge contains the fill port, the fluidic chamber, electrical contacts and an array of sensors. Different types of test cartridge contain different sensors.\nData were entered and calculations were done in Microsoft Excel, point estimate at 95% Confidence Interval was calculated along with frequency and proportion for binary data.", "A total of 1144 patients (admitted in Emergency department, ICU, NICU and in different wards) were evaluated for ABG analysis by sending their blood sample to the clinical laboratory services, NMCTH. Out of 1144 patients, the prevalence of acid base disorders was 718 (62.76%) at 95% Confidence Interval (59.96-65.56%). Simple and mixed acid base disorders were observed in 332 (46.24%) and 386 (53.76%) patients respectively (Figure 1). the ABD patients, we found that 403 (56.1%) were male and 315 (43.8%) were female.\nAmongst the patients with simple ABD, metabolic acidosis was identified in 103 (14.3%), metabolic alkalosis in 44 (6.1%), respiratory acidosis in 51 (7.1%) and respiratory alkalosis in 134 (18.7%) (Figure 2). In the mixed ABD, it was noted that the maximum were suffering with metabolic acidosis and respiratory alkalosis 204 (28.4%). The other mixed ABD in our study were metabolic alkalosis and respiratory acidosis 112 (15.6%), metabolic acidosis and respiratory acidosis 44 (6.1%) and metabolic alkalosis and respiratory alkalosis 26 (3.6%) (Figure 3). The findings of biochemical parameters of ABD patients are mentioned (Table 1).\nWe have also evaluated our study by age group wise. Respiratory alkalosis was observed in 51 (7.1%) patients with age more than 60 years and in 43 (5.98%) patients with age 41-60 years. Metabolic acidosis, respiratory acidosis and metabolic alkalosis were also most commonly seen in more than 60 years in 38 (5.2%), 22 (3.0%), 19 (2.6%) patients respectively and in 41-60 years in 35 (4.9%), 18 (2.5%), 16 (2.2%) patients respectively. The data for number of patients suffering with simple ABD in other age group (Table 2). Mixed ABD was also mostly seen in higher age groups and the most common mixed ABD in our study population was metabolic acidosis and respiratory alkalosis, which was observed in 82 (11.4%) patients of more than 60 years age and in 68 (9.4%) patients of 41-60 age groups. Another major mixed ABD observed in our study was metabolic alkalosis and respiratory acidosis, which was seen in 41 (5.7%) and in 32 (4.4%) in >60 years age and 41-60 age group of patients respectively.", "Acid base disorder is very common in critically ill patients and also strongly associated with mortality. Therefore, assessment of acid base status of such patients is an integral component of their treatment. Our findings have shown that the incidence of ABD in critically ill patients from the Emergency and other intensive care units of NMCTH is 63%. It can be compared with previous available reports, which elicits that the incidence rate of ABD in such patients in Korea,4 China,5 Italy,6 and Turkey7 is 66.4%, 94.2-97.3%, 56% and 71% respectively.\nIt was noted in our study that ABD was more common in male than female, which is similar to the findings reported earlier.4,7 In our study, it was seen that 29% of patients were suffering from simple ABD, whereas 34% of cases were of mixed ABD. In simple ABD cases, respiratory alkalosis was the most common but in mixed ABD, the maximum cases were of metabolic acidosis and respiratory alkalosis. Similar finding was observed in few studies, which reported as respiratory alkalosis as the most common type of simple ABD8,9 but Song ZF, et al.5 reported metabolic acidosis and respiratory alkalosis as not commonly observed in mixed ABD. In contrast to our results, a study carried out in China5 reported metabolic acidosis as the commonest simple ABD in sick people, whereas other studies conducted in USA10 and Scotland11 found metabolic alkalosis as the commonest type of simple ABD. Hodgkin JE, et al.10 reported metabolic acidosis and respiratory alkalosis (mixed ABD) as a less commonly observed ABD, which is different with our finding.\nIn our study, it was observed that ABD was more frequently seen in elderly patients with advanced age (>60 years). The reason for such a finding might be because of more incidence and severity of the diseases in advanced age group patients and they are also more likely to develop obstructive lung disease or kidney problem, which contributes the severity of ABD significantly. In addition to that, according to Nabata T, et al.12 various drugs and medication also affects acid-base status in advanced age patients.\nThe different biochemical parameters were estimated and were the basis of categorizing the cases as different types of ABD. The hallmark of metabolic acidosis was the decrease in blood pH and bicarbonate level with hypokalemia whereas for respiratory acidosis, it was decrease in blood pH with increase in pCO2 level and no change in sodium, potassium and chloride level. Similarly, the hall mark of metabolic alkalosis was increase in blood PH and bicarbonate level with hypokalemia, hyponatremia and hypochloremia, whereas increase in blood pH with decrease in pCO2 level with hypokalemia, hyponatremia and low ionized calcium were the hallmark for respiratory alkalosis. Similarly among the mixed ABD cases, normal blood pH, decrease pCO2, decrease bicarbonate with normal sodium, potassium and chloride level were the findings of metabolic acidosis and respiratory alkalosis. The findings of metabolic alkalosis and respiratory alkalosis were increase in blood pH, normal pCO2, increased bicarbonate, normal chloride, normal sodium and low potassium level. Nearly normal or slightly increased blood pH, increased pCO2, increased bicarbonate, normal or slightly decreased sodium, potassium level and normal chloride level were observed in metabolic alkalosis and respiratory acidosis. In case of metabolic acidosis and respiratory acidosis, decreased blood PH, normal or slightly increased pCO2, slightly decreased bicarbonate, normal sodium, potassium and chloride level were found.", "Acid base disorders are found to be the most common disorder among critically ill patients presented in the Emergency and other intensive care units. Therefore, the evaluation of arterial blood gas analysis becomes very important in understanding pathophysiology, making a diagnosis, planning effective treatment and monitoring progress. Mixed ABD was the most frequently observed case. Respiratory alkalosis was the most common among simple ABD case whereas metabolic acidosis and respiratory alkalosis was common in mixed ABD. Elderly people were more suffering with all types of ABD. Male suffered more from ABD than female.", "\nNone.\n" ]

[ "intro", "methods", "results", "discussion", "conclusions", "COI-statement" ]

[ "\nacidosis\n", "\nalkalosis\n", "\ncritical illness\n", "\npH\n" ]

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[ "abd", "patients", "alkalosis", "respiratory", "metabolic", "acidosis", "blood", "respiratory alkalosis", "mixed", "metabolic acidosis" ]

[ "case metabolic acidosis", "acidosis metabolic", "suffering metabolic acidosis", "reported metabolic acidosis", "acid base disorders" ]

[ "Antineoplastic Agents", "Cell Death", "Cell Line, Tumor", "Cell Survival", "Doxorubicin", "Drug Carriers", "Drug Delivery Systems", "Drug Liberation", "Dynamic Light Scattering", "Endocytosis", "Humans", "Hydrogels", "Nanogels", "Peptides" ]

[ "Materials and Methods", "Peptide Synthesis", "Hydrogels and Nanogels Formulation", "Doxorubicin Filled Hydrogels and Nanogels", "Doxil Filled Hydrogels", "Circular Dichroism (CD)", "Confocal Analysis", "Dynamic Light Scattering (DLS) Measurements", "Dox Release from Hydrogels and Nanogels", "Cell Line", "Cell Viability Evaluation by MTT", "Immunofluorescence Experiments", "Statistical Analyses", "Results and Discussion", "Formulation and Characterization of Dox Filled HGs", "Formulation and Characterization of Dox Filled NGs", "Drug Release from HGs and NGs", "Cytotoxicity Assays", "Determination of the Cellular Uptake by Immunofluorescence", "Conclusion" ]

[ "Protected Nα-Fmoc-amino acid derivatives, coupling reagents, and Rink amide MBHA (4-methylbenzhydrylamine) resin were purchased from Calbiochem-Novabiochem (Laufelfingen, Switzerland). The monodisperse Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc- AdOO-OH, PEG2) was purchased from Neosystem (Strasbourg, France). Lyophilized Fmoc-FF powder was purchased from Bachem (Bubendorf, Switzerland). Doxorubicin chlorohydrate (Dox.HCl) was purchased from Sigma Aldrich (Milan, Italy). Pegylated liposomal Dox (commercial name of Doxil) vials were kindly gifted by the Italian Cancer Institute in Naples (Italy) “Fondazione G. Pascale”. TWEEN®60, SPAN®60, and all other chemicals and solvents were purchased from Sigma-Aldrich, Fluka (Bucks, Switzerland) or LabScan (Stillorgan, Dublin, Ireland) and were used as received unless otherwise stated. All solutions were obtained by weight using doubly distilled water as a solvent. UV-Vis spectra were recorded on a Thermo Fisher Scientific Inc (Wilmington, Delaware USA) Nanodrop 2000c, equipped with a 1.0 cm quartz cuvette (Hellma).\nPeptide Synthesis (FY)3 and its PEGylated analogue PEG8-(FY)3 peptide derivative were synthesized by peptide solid phase synthesis (SPPS) procedures with a Fmoc/tBu chemistry approach as previously described39,42,43 and purified by RP-HPLC chromatography.\n(FY)3 and its PEGylated analogue PEG8-(FY)3 peptide derivative were synthesized by peptide solid phase synthesis (SPPS) procedures with a Fmoc/tBu chemistry approach as previously described39,42,43 and purified by RP-HPLC chromatography.\nHydrogels and Nanogels Formulation Pure Fmoc-FF hydrogel and mixed Fmoc-FF/PEG8-(FY)3 and Fmoc-FF/(FY)3 hydrogels (1/1 or 2/1, v/v) were prepared as previously described using the “solvent-switch method”44 to a final concentration of 0.5 wt%.38 Briefly, each peptide component (Fmoc-FF, (FY)3 and PEG8-(FY)3) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mg/mL. Pure Fmoc-FF hydrogel (400 µL) was obtained by diluting 20 µL of the Fmoc-FF stock solution with 380 μL of double distilled water under stirring (5 seconds). Preparation of all the mixed hydrogels was achieved analogously, by combining the peptides stock solutions at the desired volume/volume ratios. Pure and mixed nanogel formulations were prepared as previously described according to the top-down method.41 Briefly, the gel disk obtained into a silicone mold was homogenized at 35,000 min−1 for 5 min into 4 mL of an aqueous solution containing TWEEN 60/SPAN 60 at a w/w ratio of 52/48 (3.10–5 mol) for pure Fmoc-FF NGs and only TWEEN 60 (3.10–5 mol) for mixed Fmoc-FF/(FY)3 NGs. The resulting suspensions were then tip sonicated for 5 min at 9 W.\nPure Fmoc-FF hydrogel and mixed Fmoc-FF/PEG8-(FY)3 and Fmoc-FF/(FY)3 hydrogels (1/1 or 2/1, v/v) were prepared as previously described using the “solvent-switch method”44 to a final concentration of 0.5 wt%.38 Briefly, each peptide component (Fmoc-FF, (FY)3 and PEG8-(FY)3) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mg/mL. Pure Fmoc-FF hydrogel (400 µL) was obtained by diluting 20 µL of the Fmoc-FF stock solution with 380 μL of double distilled water under stirring (5 seconds). Preparation of all the mixed hydrogels was achieved analogously, by combining the peptides stock solutions at the desired volume/volume ratios. Pure and mixed nanogel formulations were prepared as previously described according to the top-down method.41 Briefly, the gel disk obtained into a silicone mold was homogenized at 35,000 min−1 for 5 min into 4 mL of an aqueous solution containing TWEEN 60/SPAN 60 at a w/w ratio of 52/48 (3.10–5 mol) for pure Fmoc-FF NGs and only TWEEN 60 (3.10–5 mol) for mixed Fmoc-FF/(FY)3 NGs. The resulting suspensions were then tip sonicated for 5 min at 9 W.\nDoxorubicin Filled Hydrogels and Nanogels Dox filled hydrogels were prepared as described above by adding 380 µL of an aqueous Dox solution at a concentration of 4.0.10–3 mol L−1 to the peptide stock solutions. Dox filled nanogels were prepared according to the top-down methodology as previously described.41 Shorty, a disk of pure or mixed hydrogel loaded with Dox was prepared adding the stock solution of peptide (100 mg/mL) to 900 μL of an aqueous solution of Dox (0.018 mol L−1), which allows to reach a drug weight/lipid weight ratio of 0.250. Then, the hydrogel disk was homogenized, and tip sonicated into 4 mL of TWEEN 60/SPAN 60 mixture or TWEEN 60, according to the empty formulation. Purification of Dox filled nanogels from free Dox was achieved by gel filtration on a pre-packed column Sephadex G-50. The drug loading content (DLC), defined as gDox encapsulated/g(surfactant+peptide), was quantified by subtraction of the free Dox from the total amount of loaded Dox. The Dox concentration was determined by UV-Vis spectroscopy using calibration curves obtained by measuring absorbance at λ = 480 nm.\nDox filled hydrogels were prepared as described above by adding 380 µL of an aqueous Dox solution at a concentration of 4.0.10–3 mol L−1 to the peptide stock solutions. Dox filled nanogels were prepared according to the top-down methodology as previously described.41 Shorty, a disk of pure or mixed hydrogel loaded with Dox was prepared adding the stock solution of peptide (100 mg/mL) to 900 μL of an aqueous solution of Dox (0.018 mol L−1), which allows to reach a drug weight/lipid weight ratio of 0.250. Then, the hydrogel disk was homogenized, and tip sonicated into 4 mL of TWEEN 60/SPAN 60 mixture or TWEEN 60, according to the empty formulation. Purification of Dox filled nanogels from free Dox was achieved by gel filtration on a pre-packed column Sephadex G-50. The drug loading content (DLC), defined as gDox encapsulated/g(surfactant+peptide), was quantified by subtraction of the free Dox from the total amount of loaded Dox. The Dox concentration was determined by UV-Vis spectroscopy using calibration curves obtained by measuring absorbance at λ = 480 nm.\nDoxil Filled Hydrogels Doxil filled hydrogels were prepared analogously to Dox filled ones, by adding 380 µL of the commercial Doxil solution opportunely diluted in order to achieve the same Dox concentration (4.0.10–3 mol L−1) that was in the HGs.\nDoxil filled hydrogels were prepared analogously to Dox filled ones, by adding 380 µL of the commercial Doxil solution opportunely diluted in order to achieve the same Dox concentration (4.0.10–3 mol L−1) that was in the HGs.\nCircular Dichroism (CD) Far-UV CD spectra of Dox filled hydrogels and nanogels were collected on a Jasco J-810 spectropolarimeter equipped with a NesLab RTE111 thermal controller unit at 25°C. 100 µL of a hydrated DMSO stock solution (immediately after its generation) were placed on a 0.1 mm quartz cell. The measurements were recorded as function of the time (every 8 minutes) in the range of wavelength between 300 and 200 nm. Other experimental settings were: scan speed = 50 nm min−1, sensitivity = 10 mdeg, time constant = 16 s, bandwidth = 1 nm. Each spectrum was obtained by averaging three scans. All the spectra are reported in optical density (O.D.).\nFar-UV CD spectra of Dox filled hydrogels and nanogels were collected on a Jasco J-810 spectropolarimeter equipped with a NesLab RTE111 thermal controller unit at 25°C. 100 µL of a hydrated DMSO stock solution (immediately after its generation) were placed on a 0.1 mm quartz cell. The measurements were recorded as function of the time (every 8 minutes) in the range of wavelength between 300 and 200 nm. Other experimental settings were: scan speed = 50 nm min−1, sensitivity = 10 mdeg, time constant = 16 s, bandwidth = 1 nm. Each spectrum was obtained by averaging three scans. All the spectra are reported in optical density (O.D.).\nConfocal Analysis For confocal microscopy, Dox filled hydrogels were drop-casted and spread on a glass slide, air-dried at room temperature, and examined by confocal microscopy. Confocal images were obtained with a Leica TCS-SMD-SP5 confocal microscope (λexc = 488 nm and λem = 505–600 nm). 0.8-µm thick optical slices were acquired with a 63× or 40×/1.4 NA objective.\nFor confocal microscopy, Dox filled hydrogels were drop-casted and spread on a glass slide, air-dried at room temperature, and examined by confocal microscopy. Confocal images were obtained with a Leica TCS-SMD-SP5 confocal microscope (λexc = 488 nm and λem = 505–600 nm). 0.8-µm thick optical slices were acquired with a 63× or 40×/1.4 NA objective.\nDynamic Light Scattering (DLS) Measurements Mean diameters and diffusion coefficients (D) of empty and Dox filled NGs were estimated by DLS using a Zetasizer Nano ZS (Malvern Instruments, Westborough, MA). Instrumental settings for the measurements are a backscatter detector at 173° in automatic modality, room temperature and disposable sizing cuvette as cell. DLS measurements in triplicate were carried out on aqueous samples after centrifugation at room temperature at 13,000 rpm for 5 minutes.\nMean diameters and diffusion coefficients (D) of empty and Dox filled NGs were estimated by DLS using a Zetasizer Nano ZS (Malvern Instruments, Westborough, MA). Instrumental settings for the measurements are a backscatter detector at 173° in automatic modality, room temperature and disposable sizing cuvette as cell. DLS measurements in triplicate were carried out on aqueous samples after centrifugation at room temperature at 13,000 rpm for 5 minutes.\nDox Release from Hydrogels and Nanogels Dox and Doxil release from hydrogels were evaluated in a conic tube (1.5 mL) using 400 µL of drug filled hydrogels (0.5% wt) adding, on the top of them, 800 µL of 0.100 mol L−1 phosphate buffer. At each time-point, 400 µL of this solution was removed and replaced with an equal fresh aliquot. Released Dox was quantified by UV-Vis spectrum of the supernatant at 480 nm. All the release experiments were performed in triplicates. The extent of Dox release was reported as percentage of the ratio between the amount of released drug and the total quantity of drug initially loaded into hydrogels. Dox release from nanogels was achieved using the well-known dialysis method.45 Briefly, Dox loaded nanogel suspension (1.0 mL) was placed into a dialysis bag (MW cut-off = 3500 Da). This bag was immersed under stirring for 72 h, at 37°C into 20 mL of phosphate buffer. Then, 2 mL of the dialyzed solution was replaced with an equal amount of fresh solution at different time points. Fluorescence measurements of each fraction of the dialyzed solution were recorded at room temperature with a spectrofluorophotometer Jasco (Model FP-750, Japan) in a quartz cell with 1.0 cm path length. The other settings were as follows: excitation and emission bandwidths = 5 nm, recording speed = 125 nm/min, and excitation wavelength = 480 nm, emission range from 490 to 700 nm. The amount of doxorubicin contained in each fraction was estimated using an analytical titration curve previously recorded for the free Dox in the same spectral range. Analogously to HGs, Dox release profile from NGs was reported as percentage of released drug/total drug loaded into NGs.\nDox and Doxil release from hydrogels were evaluated in a conic tube (1.5 mL) using 400 µL of drug filled hydrogels (0.5% wt) adding, on the top of them, 800 µL of 0.100 mol L−1 phosphate buffer. At each time-point, 400 µL of this solution was removed and replaced with an equal fresh aliquot. Released Dox was quantified by UV-Vis spectrum of the supernatant at 480 nm. All the release experiments were performed in triplicates. The extent of Dox release was reported as percentage of the ratio between the amount of released drug and the total quantity of drug initially loaded into hydrogels. Dox release from nanogels was achieved using the well-known dialysis method.45 Briefly, Dox loaded nanogel suspension (1.0 mL) was placed into a dialysis bag (MW cut-off = 3500 Da). This bag was immersed under stirring for 72 h, at 37°C into 20 mL of phosphate buffer. Then, 2 mL of the dialyzed solution was replaced with an equal amount of fresh solution at different time points. Fluorescence measurements of each fraction of the dialyzed solution were recorded at room temperature with a spectrofluorophotometer Jasco (Model FP-750, Japan) in a quartz cell with 1.0 cm path length. The other settings were as follows: excitation and emission bandwidths = 5 nm, recording speed = 125 nm/min, and excitation wavelength = 480 nm, emission range from 490 to 700 nm. The amount of doxorubicin contained in each fraction was estimated using an analytical titration curve previously recorded for the free Dox in the same spectral range. Analogously to HGs, Dox release profile from NGs was reported as percentage of released drug/total drug loaded into NGs.\nCell Line Human breast cancer cell line MDA-MB-231 was obtained from IRCCS-SDN Biobank (10.5334/ojb.26) and growth in Dulbecco’s Modified Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMAX. Cells were incubated at 37°C and 5% CO2 and seeded in T-25 culture flasks.\nHuman breast cancer cell line MDA-MB-231 was obtained from IRCCS-SDN Biobank (10.5334/ojb.26) and growth in Dulbecco’s Modified Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMAX. Cells were incubated at 37°C and 5% CO2 and seeded in T-25 culture flasks.\nCell Viability Evaluation by MTT For MTT assays (Sigma Aldrich, Germany), MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well. After 24 h free Dox at 1µmol L−1, nanogels and hydrogels (these latter preformed into a hollow plastic chamber sealed at one end with a porous membrane) were added to the wells. Cells were treated with the hydrogels for 24 h and with nanogel solutions for 72 h. At the end of the treatment, cell viability was assessed by the MTT assay. For the IC50 determination of Dox, MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well and treated with different concentrations (0.25, 0.5, 1, 2 and 4 µmol L−1) of Dox for 24 h. At the end of the treatment, cell viability was assessed by the MTT assay. In brief, MTT, dissolved in DMEM in the absence of phenol red (Sigma-Aldrich), was added to the cells at a final concentration of 0.5 mg/mL. After 4 h incubation at 37°C, the culture medium was removed, and the resulting formazan salts were dissolved by adding isopropanol containing 0.1 mol L−1 HCl and 10% Triton-X100. Absorbance values of blue formazan were determined at 490 nm using an automatic plate reader (EL 800, Biotek). Cell survival was expressed as percentage of viable cells in the presence of hydrogels or nanogels, compared to control cells grown in their absence. MTT assay was repeated twice with similar results.\nFor MTT assays (Sigma Aldrich, Germany), MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well. After 24 h free Dox at 1µmol L−1, nanogels and hydrogels (these latter preformed into a hollow plastic chamber sealed at one end with a porous membrane) were added to the wells. Cells were treated with the hydrogels for 24 h and with nanogel solutions for 72 h. At the end of the treatment, cell viability was assessed by the MTT assay. For the IC50 determination of Dox, MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well and treated with different concentrations (0.25, 0.5, 1, 2 and 4 µmol L−1) of Dox for 24 h. At the end of the treatment, cell viability was assessed by the MTT assay. In brief, MTT, dissolved in DMEM in the absence of phenol red (Sigma-Aldrich), was added to the cells at a final concentration of 0.5 mg/mL. After 4 h incubation at 37°C, the culture medium was removed, and the resulting formazan salts were dissolved by adding isopropanol containing 0.1 mol L−1 HCl and 10% Triton-X100. Absorbance values of blue formazan were determined at 490 nm using an automatic plate reader (EL 800, Biotek). Cell survival was expressed as percentage of viable cells in the presence of hydrogels or nanogels, compared to control cells grown in their absence. MTT assay was repeated twice with similar results.\nImmunofluorescence Experiments MDA-MB-231 cells were treated for 24 h with 1µmol L−1 free Dox or 1.13 mmol L−1 Dox loaded Fmoc-FF/(FY)3 hydrogels and Fmoc-FF/(FY)3 nanogels. Cells were fixed at -20 °C for 15 minutes with a -80 °C pre-cooled solution of methanol/acetone (1/1, v/v). Subsequently, cells were subjected to blocking with solution of 3% (w/v) BSA in PBS pH 7.4 at room temperature (RT). Anti B-actin (A2228, Sigma Aldrich) monoclonal antibody was diluted 1:200 in a solution of PBS + 1% (w/v) BSA and then all slides were incubated for 4 h at + 4°C. After three washing steps in PBS for 5 minutes each, FITC-conjugated anti-mouse secondary antibody (ab7064, Abcam, UK) diluted 1:400 in a solution of PBS + 1% (w/v) BSA was incubated for 1 h at 4°C in the dark. After additional three washing steps in PBS, a solution of 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fischer Scientific D1306) diluted 35,000-fold in PBS was used and it was left to act for 10 minutes at RT in the dark to color the nuclei. Images were obtained using an automated upright microscope system (Leica DM5500 B) coupled with Leica Cytovision software.\nStatistical Analyses All statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.\nAll statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.\nMDA-MB-231 cells were treated for 24 h with 1µmol L−1 free Dox or 1.13 mmol L−1 Dox loaded Fmoc-FF/(FY)3 hydrogels and Fmoc-FF/(FY)3 nanogels. Cells were fixed at -20 °C for 15 minutes with a -80 °C pre-cooled solution of methanol/acetone (1/1, v/v). Subsequently, cells were subjected to blocking with solution of 3% (w/v) BSA in PBS pH 7.4 at room temperature (RT). Anti B-actin (A2228, Sigma Aldrich) monoclonal antibody was diluted 1:200 in a solution of PBS + 1% (w/v) BSA and then all slides were incubated for 4 h at + 4°C. After three washing steps in PBS for 5 minutes each, FITC-conjugated anti-mouse secondary antibody (ab7064, Abcam, UK) diluted 1:400 in a solution of PBS + 1% (w/v) BSA was incubated for 1 h at 4°C in the dark. After additional three washing steps in PBS, a solution of 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fischer Scientific D1306) diluted 35,000-fold in PBS was used and it was left to act for 10 minutes at RT in the dark to color the nuclei. Images were obtained using an automated upright microscope system (Leica DM5500 B) coupled with Leica Cytovision software.\nStatistical Analyses All statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.\nAll statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.", "(FY)3 and its PEGylated analogue PEG8-(FY)3 peptide derivative were synthesized by peptide solid phase synthesis (SPPS) procedures with a Fmoc/tBu chemistry approach as previously described39,42,43 and purified by RP-HPLC chromatography.", "Pure Fmoc-FF hydrogel and mixed Fmoc-FF/PEG8-(FY)3 and Fmoc-FF/(FY)3 hydrogels (1/1 or 2/1, v/v) were prepared as previously described using the “solvent-switch method”44 to a final concentration of 0.5 wt%.38 Briefly, each peptide component (Fmoc-FF, (FY)3 and PEG8-(FY)3) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mg/mL. Pure Fmoc-FF hydrogel (400 µL) was obtained by diluting 20 µL of the Fmoc-FF stock solution with 380 μL of double distilled water under stirring (5 seconds). Preparation of all the mixed hydrogels was achieved analogously, by combining the peptides stock solutions at the desired volume/volume ratios. Pure and mixed nanogel formulations were prepared as previously described according to the top-down method.41 Briefly, the gel disk obtained into a silicone mold was homogenized at 35,000 min−1 for 5 min into 4 mL of an aqueous solution containing TWEEN 60/SPAN 60 at a w/w ratio of 52/48 (3.10–5 mol) for pure Fmoc-FF NGs and only TWEEN 60 (3.10–5 mol) for mixed Fmoc-FF/(FY)3 NGs. The resulting suspensions were then tip sonicated for 5 min at 9 W.", "Dox filled hydrogels were prepared as described above by adding 380 µL of an aqueous Dox solution at a concentration of 4.0.10–3 mol L−1 to the peptide stock solutions. Dox filled nanogels were prepared according to the top-down methodology as previously described.41 Shorty, a disk of pure or mixed hydrogel loaded with Dox was prepared adding the stock solution of peptide (100 mg/mL) to 900 μL of an aqueous solution of Dox (0.018 mol L−1), which allows to reach a drug weight/lipid weight ratio of 0.250. Then, the hydrogel disk was homogenized, and tip sonicated into 4 mL of TWEEN 60/SPAN 60 mixture or TWEEN 60, according to the empty formulation. Purification of Dox filled nanogels from free Dox was achieved by gel filtration on a pre-packed column Sephadex G-50. The drug loading content (DLC), defined as gDox encapsulated/g(surfactant+peptide), was quantified by subtraction of the free Dox from the total amount of loaded Dox. The Dox concentration was determined by UV-Vis spectroscopy using calibration curves obtained by measuring absorbance at λ = 480 nm.", "Doxil filled hydrogels were prepared analogously to Dox filled ones, by adding 380 µL of the commercial Doxil solution opportunely diluted in order to achieve the same Dox concentration (4.0.10–3 mol L−1) that was in the HGs.", "Far-UV CD spectra of Dox filled hydrogels and nanogels were collected on a Jasco J-810 spectropolarimeter equipped with a NesLab RTE111 thermal controller unit at 25°C. 100 µL of a hydrated DMSO stock solution (immediately after its generation) were placed on a 0.1 mm quartz cell. The measurements were recorded as function of the time (every 8 minutes) in the range of wavelength between 300 and 200 nm. Other experimental settings were: scan speed = 50 nm min−1, sensitivity = 10 mdeg, time constant = 16 s, bandwidth = 1 nm. Each spectrum was obtained by averaging three scans. All the spectra are reported in optical density (O.D.).", "For confocal microscopy, Dox filled hydrogels were drop-casted and spread on a glass slide, air-dried at room temperature, and examined by confocal microscopy. Confocal images were obtained with a Leica TCS-SMD-SP5 confocal microscope (λexc = 488 nm and λem = 505–600 nm). 0.8-µm thick optical slices were acquired with a 63× or 40×/1.4 NA objective.", "Mean diameters and diffusion coefficients (D) of empty and Dox filled NGs were estimated by DLS using a Zetasizer Nano ZS (Malvern Instruments, Westborough, MA). Instrumental settings for the measurements are a backscatter detector at 173° in automatic modality, room temperature and disposable sizing cuvette as cell. DLS measurements in triplicate were carried out on aqueous samples after centrifugation at room temperature at 13,000 rpm for 5 minutes.", "Dox and Doxil release from hydrogels were evaluated in a conic tube (1.5 mL) using 400 µL of drug filled hydrogels (0.5% wt) adding, on the top of them, 800 µL of 0.100 mol L−1 phosphate buffer. At each time-point, 400 µL of this solution was removed and replaced with an equal fresh aliquot. Released Dox was quantified by UV-Vis spectrum of the supernatant at 480 nm. All the release experiments were performed in triplicates. The extent of Dox release was reported as percentage of the ratio between the amount of released drug and the total quantity of drug initially loaded into hydrogels. Dox release from nanogels was achieved using the well-known dialysis method.45 Briefly, Dox loaded nanogel suspension (1.0 mL) was placed into a dialysis bag (MW cut-off = 3500 Da). This bag was immersed under stirring for 72 h, at 37°C into 20 mL of phosphate buffer. Then, 2 mL of the dialyzed solution was replaced with an equal amount of fresh solution at different time points. Fluorescence measurements of each fraction of the dialyzed solution were recorded at room temperature with a spectrofluorophotometer Jasco (Model FP-750, Japan) in a quartz cell with 1.0 cm path length. The other settings were as follows: excitation and emission bandwidths = 5 nm, recording speed = 125 nm/min, and excitation wavelength = 480 nm, emission range from 490 to 700 nm. The amount of doxorubicin contained in each fraction was estimated using an analytical titration curve previously recorded for the free Dox in the same spectral range. Analogously to HGs, Dox release profile from NGs was reported as percentage of released drug/total drug loaded into NGs.", "Human breast cancer cell line MDA-MB-231 was obtained from IRCCS-SDN Biobank (10.5334/ojb.26) and growth in Dulbecco’s Modified Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMAX. Cells were incubated at 37°C and 5% CO2 and seeded in T-25 culture flasks.", "For MTT assays (Sigma Aldrich, Germany), MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well. After 24 h free Dox at 1µmol L−1, nanogels and hydrogels (these latter preformed into a hollow plastic chamber sealed at one end with a porous membrane) were added to the wells. Cells were treated with the hydrogels for 24 h and with nanogel solutions for 72 h. At the end of the treatment, cell viability was assessed by the MTT assay. For the IC50 determination of Dox, MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well and treated with different concentrations (0.25, 0.5, 1, 2 and 4 µmol L−1) of Dox for 24 h. At the end of the treatment, cell viability was assessed by the MTT assay. In brief, MTT, dissolved in DMEM in the absence of phenol red (Sigma-Aldrich), was added to the cells at a final concentration of 0.5 mg/mL. After 4 h incubation at 37°C, the culture medium was removed, and the resulting formazan salts were dissolved by adding isopropanol containing 0.1 mol L−1 HCl and 10% Triton-X100. Absorbance values of blue formazan were determined at 490 nm using an automatic plate reader (EL 800, Biotek). Cell survival was expressed as percentage of viable cells in the presence of hydrogels or nanogels, compared to control cells grown in their absence. MTT assay was repeated twice with similar results.", "MDA-MB-231 cells were treated for 24 h with 1µmol L−1 free Dox or 1.13 mmol L−1 Dox loaded Fmoc-FF/(FY)3 hydrogels and Fmoc-FF/(FY)3 nanogels. Cells were fixed at -20 °C for 15 minutes with a -80 °C pre-cooled solution of methanol/acetone (1/1, v/v). Subsequently, cells were subjected to blocking with solution of 3% (w/v) BSA in PBS pH 7.4 at room temperature (RT). Anti B-actin (A2228, Sigma Aldrich) monoclonal antibody was diluted 1:200 in a solution of PBS + 1% (w/v) BSA and then all slides were incubated for 4 h at + 4°C. After three washing steps in PBS for 5 minutes each, FITC-conjugated anti-mouse secondary antibody (ab7064, Abcam, UK) diluted 1:400 in a solution of PBS + 1% (w/v) BSA was incubated for 1 h at 4°C in the dark. After additional three washing steps in PBS, a solution of 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fischer Scientific D1306) diluted 35,000-fold in PBS was used and it was left to act for 10 minutes at RT in the dark to color the nuclei. Images were obtained using an automated upright microscope system (Leica DM5500 B) coupled with Leica Cytovision software.\nStatistical Analyses All statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.\nAll statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.", "All statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.", "Classical administrations of active pharmaceutical ingredients (APIs) are affected by issues related to systemic toxicity, drug chemical instability and repeated dosing requirement. As previously mentioned, HGs offer convenient drug delivery approaches, allowing them to overcome these drawbacks thanks to their tunable features, modular degradability and easy formulation. Macroscopically, hydrogel-based materials belong to two different categories based on their size: hydrogels (HGs) and nanogels (NGs). The specific size-feature determines the route of administration: transepithelial drug delivery or in situ gelation process of implants for HGs and systemic, oral and pulmonary administration of APIs for nanogels.46 The knowledge about the encapsulation procedures, the efficiency of drugs loading, and the cytotoxicity profiles can steer the development of efficient vehicles. For instance, swollen HGs matrices can be loaded in different ways, acting at macroscopic level, on the fibrillary network, the mesh size or at molecular scales. Moreover, the drug release can be differently modulated according to covalent or non-covalent approaches, in which drug molecules are chemically bound on the HG matrix by stable or cleavable linkers, or they can establish electrostatic interactions with HGs building blocks. These different drug loading strategies could affect the experimental conditions of preparation (eg, solvents, pH and salt content) and in turn the biological profile of the resulting vehicle. In this perspective, comparative studies on analogies and differences between HGs and NGs may assume a great importance.\nFormulation and Characterization of Dox Filled HGs HGs filled with the anticancer drug Dox were prepared according to the “solvent-switch” method.41 This method consists of the addition of an aqueous solution of Dox directly into the stock peptide solution (100 mg/mL in DMSO). After few seconds of vortex, the resulting opaque metastable mixture is vertically incubated at room temperature until the formation of a limpid, translucent self-supportive hydrogel. Dox filled hydrogels Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 at two different ratios (1/1 or 2/1, v/v) were successfully formulated. Instead, in the same experimental conditions, Dox filled Fmoc-FF hydrogel appears not completely homogenous (see Figure S1). On the other hand, we observed a progressive improvement of the hydrogel homogeneity by increasing the amount (up to 0.018 mol L−1) of Dox to encapsulate (Figure S1). This behavior could be attributed to a shield effect of the charges occurring between the drug and the peptidic matrix. Although Dox filled Fmoc-FF hydrogel can be prepared, the Dox concentration is too high compared with mixed HGs and, for this reason, we did not further focus our attention on this sample. For mixed HGs, no syneresis was detected after 10 days. This specific no water-loss is an important advantage offered by HGs, thus allowing a precise modulation of water-soluble drug loaded in the system. According to this experimental evidence, we can assume that all the Dox was efficiently and quantitatively entrapped in the hydrogel matrix.\nThe resulting Dox filled hydrogels were further characterized in their xerogel form by confocal microscopy. Confocal images of one sample (Fmoc-FF/(FY)3, 1/1) are reported in Figure 2A and B. These images clearly show the characteristic intricate network of entangled fibers,47 and their red color suggests the tight interaction of Dox with the network constituents. The amount of drug we were able to encapsulate in mixed HGs was 2.32 mg/mL. This quantity corresponds to a drug loading content (DLC) of 0.440 and to an encapsulation ratio (ER%, defined as the weight percentage of drug encapsulated in the HG on the total drug added during preparation) of 100%, respectively. This high encapsulation degree can be explained as consequence of the electrostatic interactions between the positive charge on the drug and the negative one present on the C-terminus of the Fmoc-FF peptide. Analogously to free Dox, also Doxil was efficiently encapsulated into mixed hydrogels at the same drug concentration. This is surprising due to the negative Z potential value (see Figure S2) we measured for Doxil (ζ ~ -11 mV) into the experimental conditions ([Dox] =1.13 mmol L−1) used to achieve the encapsulation into the hydrogel.Figure 2Characterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nCharacterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nA macroscopic evaluation of the gelation kinetics for mixed drug-filled hydrogels can be done by simply following the opaque-to-transparent optic transition. As previously observed for the corresponding empty systems, a different time (between 20 and 40 minutes) is required for the gelation process of each mixed hydrogel.38 In particular, we observed a more rapid formation for mixed HGs containing PEG8-(FY)3 (24 and 30 minutes for 2/1 and 1/1, respectively) with respect to those containing (FY)3 (35 and 40 minutes for 2/1 and 1/1, respectively). Analogously to empty matrices, the gelation kinetics of Dox filled HGs is faster by increasing the amount of the Fmoc-FF. This result may be ascribable to the capability of Fmoc-FF to gelificate in a very quick time (~2 min).35 On the contrary, we observe a faster gelation for HGs containing PEG in respect to HGs lacking polymer. This result is probably due to the additional interactions occurring between the hydrophilic PEG structure and the hydrophilic daunosamine moiety of Dox. As an alternative, gelation kinetics of hydrogels can be more accurately determined by following other structural or mechanical transitions occurring over time, related to alteration of UV-Vis absorbance, storage modulus (G’) or the dichroic signal.48 In this perspective, we recorded the CD spectra of Dox filled hydrogels at several time points in the spectral region between 300 and 200 nm (see Figure 2C–F). From the inspection of all the CD spectra, it clearly appears an evolution of the dichroic signal towards a stable state, in which we can assume that the HG has reached its final arrangement. The co-existence of several conformational states in the solution is also confirmed by isosbestic points between 220 and 240 nm and around 267 nm for PEG8-(FY)3 containing HGs. By plotting the optical density (OD) in the relative minima for each sample, we were able to extrapolate the gelation times (see Figure S3). These times, evaluated from the CD measurements, were found in good agreement with the gelation times observed following the transition from the opaque to limpid form.\nHGs filled with the anticancer drug Dox were prepared according to the “solvent-switch” method.41 This method consists of the addition of an aqueous solution of Dox directly into the stock peptide solution (100 mg/mL in DMSO). After few seconds of vortex, the resulting opaque metastable mixture is vertically incubated at room temperature until the formation of a limpid, translucent self-supportive hydrogel. Dox filled hydrogels Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 at two different ratios (1/1 or 2/1, v/v) were successfully formulated. Instead, in the same experimental conditions, Dox filled Fmoc-FF hydrogel appears not completely homogenous (see Figure S1). On the other hand, we observed a progressive improvement of the hydrogel homogeneity by increasing the amount (up to 0.018 mol L−1) of Dox to encapsulate (Figure S1). This behavior could be attributed to a shield effect of the charges occurring between the drug and the peptidic matrix. Although Dox filled Fmoc-FF hydrogel can be prepared, the Dox concentration is too high compared with mixed HGs and, for this reason, we did not further focus our attention on this sample. For mixed HGs, no syneresis was detected after 10 days. This specific no water-loss is an important advantage offered by HGs, thus allowing a precise modulation of water-soluble drug loaded in the system. According to this experimental evidence, we can assume that all the Dox was efficiently and quantitatively entrapped in the hydrogel matrix.\nThe resulting Dox filled hydrogels were further characterized in their xerogel form by confocal microscopy. Confocal images of one sample (Fmoc-FF/(FY)3, 1/1) are reported in Figure 2A and B. These images clearly show the characteristic intricate network of entangled fibers,47 and their red color suggests the tight interaction of Dox with the network constituents. The amount of drug we were able to encapsulate in mixed HGs was 2.32 mg/mL. This quantity corresponds to a drug loading content (DLC) of 0.440 and to an encapsulation ratio (ER%, defined as the weight percentage of drug encapsulated in the HG on the total drug added during preparation) of 100%, respectively. This high encapsulation degree can be explained as consequence of the electrostatic interactions between the positive charge on the drug and the negative one present on the C-terminus of the Fmoc-FF peptide. Analogously to free Dox, also Doxil was efficiently encapsulated into mixed hydrogels at the same drug concentration. This is surprising due to the negative Z potential value (see Figure S2) we measured for Doxil (ζ ~ -11 mV) into the experimental conditions ([Dox] =1.13 mmol L−1) used to achieve the encapsulation into the hydrogel.Figure 2Characterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nCharacterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nA macroscopic evaluation of the gelation kinetics for mixed drug-filled hydrogels can be done by simply following the opaque-to-transparent optic transition. As previously observed for the corresponding empty systems, a different time (between 20 and 40 minutes) is required for the gelation process of each mixed hydrogel.38 In particular, we observed a more rapid formation for mixed HGs containing PEG8-(FY)3 (24 and 30 minutes for 2/1 and 1/1, respectively) with respect to those containing (FY)3 (35 and 40 minutes for 2/1 and 1/1, respectively). Analogously to empty matrices, the gelation kinetics of Dox filled HGs is faster by increasing the amount of the Fmoc-FF. This result may be ascribable to the capability of Fmoc-FF to gelificate in a very quick time (~2 min).35 On the contrary, we observe a faster gelation for HGs containing PEG in respect to HGs lacking polymer. This result is probably due to the additional interactions occurring between the hydrophilic PEG structure and the hydrophilic daunosamine moiety of Dox. As an alternative, gelation kinetics of hydrogels can be more accurately determined by following other structural or mechanical transitions occurring over time, related to alteration of UV-Vis absorbance, storage modulus (G’) or the dichroic signal.48 In this perspective, we recorded the CD spectra of Dox filled hydrogels at several time points in the spectral region between 300 and 200 nm (see Figure 2C–F). From the inspection of all the CD spectra, it clearly appears an evolution of the dichroic signal towards a stable state, in which we can assume that the HG has reached its final arrangement. The co-existence of several conformational states in the solution is also confirmed by isosbestic points between 220 and 240 nm and around 267 nm for PEG8-(FY)3 containing HGs. By plotting the optical density (OD) in the relative minima for each sample, we were able to extrapolate the gelation times (see Figure S3). These times, evaluated from the CD measurements, were found in good agreement with the gelation times observed following the transition from the opaque to limpid form.\nFormulation and Characterization of Dox Filled NGs The preparation of pure Fmoc-FF and mixed nanogels loaded with Dox was achieved according to the top-down methodology.41 In this approach, macroscopic discs of hydrogels (1.0%wt) loaded with Dox (0.018 mol L−1) were prepared into silicone molds according to the above-described protocol. Successively, these discs were homogenized into an aqueous solution of TWEEN®60 (polyethylene glycol sorbitan monostearate) and SPAN®60 (Sorbitan stearate) as stabilizing agents. The combination of this couple of surfactants permits to achieve a hydrophilic/lipophilic balance (HLB) value ranged between 4.7 and 14.9. Due to the similar behavior exhibited by the four mixed HG formulations in terms of DLC and stability, we decided to prepare only one of the four mixed formulations, ie, Fmoc-FF/(FY)3 (1/1, v/v). The best Dox filled NG formulations, in terms of size and stability, were obtained using TWEEN®60/SPAN®60 at 58/42 ratio (HLB= 10) or at 100/0 ratio (HLB= 14.9) for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v), respectively. Indeed, contrarily to the pure Fmoc-FF formulation, the mixed one was found unstable when prepared with a value of HLB = 10. The resulting suspensions, further submicronized using tip sonicator, were purified from free drug using size exclusion chromatography. The red coloration of purified formulations macroscopically suggested the incorporation of the anthracycline in the NGs vehicle. Due to its spectroscopic features (λabs = 480 nm; λem= 560, 590 nm), the amount of Dox encapsulated was analytically monitored and evaluated by UV-Vis spectroscopy, by checking the absorbance in the maximum at λabs= 480 nm. DLC and ER% values for pure Fmoc-FF nanogel were 0.137% and 63%, respectively. This DLC value is similar to that of the commercially available liposomal formulations Myocet (DLC=0.127) and Doxil (DLC=0.250). Therefore, the insertion of (FY)3 peptide in the preparation causes a slight decrease of both the DLC (0.093) and the ER% (45%) with respect to the pure formulation. The lower encapsulation degree observed for mixed NGs versus pure ones can be explained considering the different amount of the net negative charge present in the two nanogels. Indeed, the peptide (FY)3 has an amidated C-terminus, significantly less acidic compared to the carboxylic one, and a basic non protected N-terminal amino group that can support a positive charge after protonation. This latter phenomenon contributes to the reduction of the total negative charge in the inner sphere of NGs, which determines lower attractive forces between the drug and the peptide system. The size of empty and filled Fmoc-FF/(FY)3 NGs, measured by Dynamic Light Scattering (DLS), was 168 and 214 nm, respectively (see Figure 3). This increase in size (~22%) is comparable to the increase of dimensions previously observed for empty (174 nm) and filled (241 nm) Fmoc-FF NGs.Figure 3DLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.\nDLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.\nThe preparation of pure Fmoc-FF and mixed nanogels loaded with Dox was achieved according to the top-down methodology.41 In this approach, macroscopic discs of hydrogels (1.0%wt) loaded with Dox (0.018 mol L−1) were prepared into silicone molds according to the above-described protocol. Successively, these discs were homogenized into an aqueous solution of TWEEN®60 (polyethylene glycol sorbitan monostearate) and SPAN®60 (Sorbitan stearate) as stabilizing agents. The combination of this couple of surfactants permits to achieve a hydrophilic/lipophilic balance (HLB) value ranged between 4.7 and 14.9. Due to the similar behavior exhibited by the four mixed HG formulations in terms of DLC and stability, we decided to prepare only one of the four mixed formulations, ie, Fmoc-FF/(FY)3 (1/1, v/v). The best Dox filled NG formulations, in terms of size and stability, were obtained using TWEEN®60/SPAN®60 at 58/42 ratio (HLB= 10) or at 100/0 ratio (HLB= 14.9) for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v), respectively. Indeed, contrarily to the pure Fmoc-FF formulation, the mixed one was found unstable when prepared with a value of HLB = 10. The resulting suspensions, further submicronized using tip sonicator, were purified from free drug using size exclusion chromatography. The red coloration of purified formulations macroscopically suggested the incorporation of the anthracycline in the NGs vehicle. Due to its spectroscopic features (λabs = 480 nm; λem= 560, 590 nm), the amount of Dox encapsulated was analytically monitored and evaluated by UV-Vis spectroscopy, by checking the absorbance in the maximum at λabs= 480 nm. DLC and ER% values for pure Fmoc-FF nanogel were 0.137% and 63%, respectively. This DLC value is similar to that of the commercially available liposomal formulations Myocet (DLC=0.127) and Doxil (DLC=0.250). Therefore, the insertion of (FY)3 peptide in the preparation causes a slight decrease of both the DLC (0.093) and the ER% (45%) with respect to the pure formulation. The lower encapsulation degree observed for mixed NGs versus pure ones can be explained considering the different amount of the net negative charge present in the two nanogels. Indeed, the peptide (FY)3 has an amidated C-terminus, significantly less acidic compared to the carboxylic one, and a basic non protected N-terminal amino group that can support a positive charge after protonation. This latter phenomenon contributes to the reduction of the total negative charge in the inner sphere of NGs, which determines lower attractive forces between the drug and the peptide system. The size of empty and filled Fmoc-FF/(FY)3 NGs, measured by Dynamic Light Scattering (DLS), was 168 and 214 nm, respectively (see Figure 3). This increase in size (~22%) is comparable to the increase of dimensions previously observed for empty (174 nm) and filled (241 nm) Fmoc-FF NGs.Figure 3DLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.\nDLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.\nDrug Release from HGs and NGs Drug release from both hydrogels and nanogels was evaluated in 0.100 mol L−1 phosphate buffer solution up to 72 hours and the corresponding release profiles are reported in Figure 4A and B, respectively. Two different procedures were used for determination of Dox release from hydrogels and nanogels. In the first case, Dox or Doxil filled hydrogels, prepared into a conic tube, were directly put in contact with a double volume of physiological solution, cyclically replaced with a fresh one. Instead, in the second case, the release of Dox from nanogels was studied using a dialysis membrane immersed in phosphate buffer at 37°C. We assumed that the crossing of the free Dox through the dialysis membrane occurred quickly, thus, the overall release of the free drug from the peptide-based nanostructures to the dialysis bag medium could be considered to be rate determining for the process. The API release from the systems was considered undergone to a diffusion process. The Dox amount released was estimated by UV-Vis (at λabs= 480 nm) or by fluorescence spectroscopies (at λem= 590 nm) for HGs and NGs, respectively. The extent of drug was reported as a percentage of the ratio between the amount of released drug and the total drug initially loaded. From the inspection of Figure 4A, we can observe a similar release profile for all the Dox filled hydrogels during the first 24 hours. However, after 72 hours, each mixed gel exhibits a different release, with the lowest one for Fmoc-FF/(FY)3 (2/1) (16%). A low drug release (21%) was also observed for the other mixed HGs Fmoc-FF/(FY)3 (1/1). The same trend was also observed for the PEG8-(FY)3 containing hydrogels with a release of 19% and 28% for 2/1 and 1/1 ratios, respectively. These results agree with our expectations that PEGylation of the peptide could affect the rigidity and permeability of the hydrogels matrix and, in turn, the drug release. As expected, the amount of drug released from the hydrogel encapsulating Doxil (~8.5%) was lower than the drug released from the corresponding hydrogel (21%). In Figure 4B is reported the Dox release (%), which after 72 h, is around 20% and 40% for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels, respectively. Analogously to HGs, also for NGs the drug release is more appreciable during the first 8–12 h. The higher release observed for mixed NGs with respect to pure ones can be interpreted as a direct consequence of the lower electrostatic interactions occurring between the NG and the drug.Figure 4Drug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.\nDrug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.\nDrug release from both hydrogels and nanogels was evaluated in 0.100 mol L−1 phosphate buffer solution up to 72 hours and the corresponding release profiles are reported in Figure 4A and B, respectively. Two different procedures were used for determination of Dox release from hydrogels and nanogels. In the first case, Dox or Doxil filled hydrogels, prepared into a conic tube, were directly put in contact with a double volume of physiological solution, cyclically replaced with a fresh one. Instead, in the second case, the release of Dox from nanogels was studied using a dialysis membrane immersed in phosphate buffer at 37°C. We assumed that the crossing of the free Dox through the dialysis membrane occurred quickly, thus, the overall release of the free drug from the peptide-based nanostructures to the dialysis bag medium could be considered to be rate determining for the process. The API release from the systems was considered undergone to a diffusion process. The Dox amount released was estimated by UV-Vis (at λabs= 480 nm) or by fluorescence spectroscopies (at λem= 590 nm) for HGs and NGs, respectively. The extent of drug was reported as a percentage of the ratio between the amount of released drug and the total drug initially loaded. From the inspection of Figure 4A, we can observe a similar release profile for all the Dox filled hydrogels during the first 24 hours. However, after 72 hours, each mixed gel exhibits a different release, with the lowest one for Fmoc-FF/(FY)3 (2/1) (16%). A low drug release (21%) was also observed for the other mixed HGs Fmoc-FF/(FY)3 (1/1). The same trend was also observed for the PEG8-(FY)3 containing hydrogels with a release of 19% and 28% for 2/1 and 1/1 ratios, respectively. These results agree with our expectations that PEGylation of the peptide could affect the rigidity and permeability of the hydrogels matrix and, in turn, the drug release. As expected, the amount of drug released from the hydrogel encapsulating Doxil (~8.5%) was lower than the drug released from the corresponding hydrogel (21%). In Figure 4B is reported the Dox release (%), which after 72 h, is around 20% and 40% for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels, respectively. Analogously to HGs, also for NGs the drug release is more appreciable during the first 8–12 h. The higher release observed for mixed NGs with respect to pure ones can be interpreted as a direct consequence of the lower electrostatic interactions occurring between the NG and the drug.Figure 4Drug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.\nDrug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.\nCytotoxicity Assays Cytotoxicity of mixed Fmoc-FF/(FY)3 (1/1, v/v) hydrogel encapsulating Dox or its liposomal formulation Doxil, at the same Dox concentration, was evaluated after 24 hours of incubation on MDA-MB-231 breast cancer cell line, using MTT assay. The cytotoxicity of the free drug, as well as of the empty hydrogels, was studied in the same conditions. HGs preparation was directly achieved into the hollow plastic chamber sealed at one end with a porous membrane. This experimental setting for hydrogels allows to mimic its conditions of utilization, in which this biocompatible support loaded with the chemotherapeutic agent is grafted at the level of the tumor lesion, where a controlled and constant drug release is achieved. During our experiments, hydrogels remain in contact with the cells for all the duration of the treatment. As shown in Figure 5A, the cell viability of empty hydrogels was found to be more than 95%. This percentage of cell survival is similar to that previously observed by us for Fmoc-FF and Fmoc-FF/(FY)3 hydrogels co-incubated with the Chinese Hamster Ovarian (CHO) cell line.38 Dox loaded hydrogels significantly reduced viability of MDA-MB-231 cells after only 24 h of incubation (49%) as well as free Dox at a concentration of 1µmol L−1 (55%). This Dox concentration corresponds to its IC50 on MDA-MB-231 cells (see Supplementary Figure S4). Due to its lower drug release, hydrogels encapsulating Doxil showed a slightly lower cytotoxicity, with a cell viability of 57%.Figure 5MDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).Abbreviation: n.s., not significant.\nMDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).\nOn the other hand, Figure 5B reports the cell viability of the same cell line treated with pure Fmoc-FF and mixed Fmoc-FF(FY)3 nanogels, empty or loaded with the drug, in comparison to free Dox and Doxil. Initially, we evaluated the cytotoxicity of empty nanogels as function of the total peptide concentration at three different time points (24, 48 and 72 h). We observed that there is a significant decrease in the cell viability during the first 48 h of incubation with nanogels. After this time of incubation, cell viability improves during the next 24 h (see Figure S5). This effect may be due to a temporary inhibition of the cell cycle induced by nanogels. Based on these results, cytotoxicity of NGs encapsulating Dox was checked after 72 h of incubation. Analogously to HGs, Dox loaded nanogels are able to significantly reduce breast cancer cells viability, with a cell viability of 7% and 25% for pure and mixed hydrogels, respectively. The higher cytotoxicity of pure HG with respect to the mixed one is probably attributable to the intrinsic toxicity of the empty gel.\nCytotoxicity of mixed Fmoc-FF/(FY)3 (1/1, v/v) hydrogel encapsulating Dox or its liposomal formulation Doxil, at the same Dox concentration, was evaluated after 24 hours of incubation on MDA-MB-231 breast cancer cell line, using MTT assay. The cytotoxicity of the free drug, as well as of the empty hydrogels, was studied in the same conditions. HGs preparation was directly achieved into the hollow plastic chamber sealed at one end with a porous membrane. This experimental setting for hydrogels allows to mimic its conditions of utilization, in which this biocompatible support loaded with the chemotherapeutic agent is grafted at the level of the tumor lesion, where a controlled and constant drug release is achieved. During our experiments, hydrogels remain in contact with the cells for all the duration of the treatment. As shown in Figure 5A, the cell viability of empty hydrogels was found to be more than 95%. This percentage of cell survival is similar to that previously observed by us for Fmoc-FF and Fmoc-FF/(FY)3 hydrogels co-incubated with the Chinese Hamster Ovarian (CHO) cell line.38 Dox loaded hydrogels significantly reduced viability of MDA-MB-231 cells after only 24 h of incubation (49%) as well as free Dox at a concentration of 1µmol L−1 (55%). This Dox concentration corresponds to its IC50 on MDA-MB-231 cells (see Supplementary Figure S4). Due to its lower drug release, hydrogels encapsulating Doxil showed a slightly lower cytotoxicity, with a cell viability of 57%.Figure 5MDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).Abbreviation: n.s., not significant.\nMDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).\nOn the other hand, Figure 5B reports the cell viability of the same cell line treated with pure Fmoc-FF and mixed Fmoc-FF(FY)3 nanogels, empty or loaded with the drug, in comparison to free Dox and Doxil. Initially, we evaluated the cytotoxicity of empty nanogels as function of the total peptide concentration at three different time points (24, 48 and 72 h). We observed that there is a significant decrease in the cell viability during the first 48 h of incubation with nanogels. After this time of incubation, cell viability improves during the next 24 h (see Figure S5). This effect may be due to a temporary inhibition of the cell cycle induced by nanogels. Based on these results, cytotoxicity of NGs encapsulating Dox was checked after 72 h of incubation. Analogously to HGs, Dox loaded nanogels are able to significantly reduce breast cancer cells viability, with a cell viability of 7% and 25% for pure and mixed hydrogels, respectively. The higher cytotoxicity of pure HG with respect to the mixed one is probably attributable to the intrinsic toxicity of the empty gel.\nDetermination of the Cellular Uptake by Immunofluorescence The intracellular internalization of Dox loaded HGs and NGs was assessed using immunofluorescence analysis on MDA-MB-231 cells treated for 24 h. Internalization of free Dox and Doxil are also reported for comparison. As indicated by the overlapping of red fluorescence associated to Dox with blue signal associated to DAPI (nucleus), the free drug can internalize into the nucleus after 24 h of incubation at 37°C (Figure 6A). Differently, Dox loaded Fmoc-FF/(FY)3 mixed hydrogel induces the internalization of Dox at peri-nuclear level since Dox signal is not perfectly overlapped with DAPI, but it is also partially detectable in the cytoplasm together with the green Actin signal (Figure 6B). The same behavior was previously observed for other liposomal Dox formulations.49,50 At the same time, doxorubicin conveyed through the nanogels remains in the cytoplasm of treated cells (Figure 6C), whereas by using Doxil the drug diffuses equally between the cytoplasm and the nucleus (Figure 6D). The different intracellular distribution of the drug in Figure 6 suggests an internalization mechanism alternative to the diffusion for the drug delivered by supramolecular systems like HGs, NGs and liposomes. One of the most accredited hypotheses is that nanogels are able to bind themselves primarily to the cellular membrane and then to enter the tumor cell via the endocytosis pathway.51,52Figure 6Immunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.\nImmunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.\nThe intracellular internalization of Dox loaded HGs and NGs was assessed using immunofluorescence analysis on MDA-MB-231 cells treated for 24 h. Internalization of free Dox and Doxil are also reported for comparison. As indicated by the overlapping of red fluorescence associated to Dox with blue signal associated to DAPI (nucleus), the free drug can internalize into the nucleus after 24 h of incubation at 37°C (Figure 6A). Differently, Dox loaded Fmoc-FF/(FY)3 mixed hydrogel induces the internalization of Dox at peri-nuclear level since Dox signal is not perfectly overlapped with DAPI, but it is also partially detectable in the cytoplasm together with the green Actin signal (Figure 6B). The same behavior was previously observed for other liposomal Dox formulations.49,50 At the same time, doxorubicin conveyed through the nanogels remains in the cytoplasm of treated cells (Figure 6C), whereas by using Doxil the drug diffuses equally between the cytoplasm and the nucleus (Figure 6D). The different intracellular distribution of the drug in Figure 6 suggests an internalization mechanism alternative to the diffusion for the drug delivered by supramolecular systems like HGs, NGs and liposomes. One of the most accredited hypotheses is that nanogels are able to bind themselves primarily to the cellular membrane and then to enter the tumor cell via the endocytosis pathway.51,52Figure 6Immunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.\nImmunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.", "HGs filled with the anticancer drug Dox were prepared according to the “solvent-switch” method.41 This method consists of the addition of an aqueous solution of Dox directly into the stock peptide solution (100 mg/mL in DMSO). After few seconds of vortex, the resulting opaque metastable mixture is vertically incubated at room temperature until the formation of a limpid, translucent self-supportive hydrogel. Dox filled hydrogels Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 at two different ratios (1/1 or 2/1, v/v) were successfully formulated. Instead, in the same experimental conditions, Dox filled Fmoc-FF hydrogel appears not completely homogenous (see Figure S1). On the other hand, we observed a progressive improvement of the hydrogel homogeneity by increasing the amount (up to 0.018 mol L−1) of Dox to encapsulate (Figure S1). This behavior could be attributed to a shield effect of the charges occurring between the drug and the peptidic matrix. Although Dox filled Fmoc-FF hydrogel can be prepared, the Dox concentration is too high compared with mixed HGs and, for this reason, we did not further focus our attention on this sample. For mixed HGs, no syneresis was detected after 10 days. This specific no water-loss is an important advantage offered by HGs, thus allowing a precise modulation of water-soluble drug loaded in the system. According to this experimental evidence, we can assume that all the Dox was efficiently and quantitatively entrapped in the hydrogel matrix.\nThe resulting Dox filled hydrogels were further characterized in their xerogel form by confocal microscopy. Confocal images of one sample (Fmoc-FF/(FY)3, 1/1) are reported in Figure 2A and B. These images clearly show the characteristic intricate network of entangled fibers,47 and their red color suggests the tight interaction of Dox with the network constituents. The amount of drug we were able to encapsulate in mixed HGs was 2.32 mg/mL. This quantity corresponds to a drug loading content (DLC) of 0.440 and to an encapsulation ratio (ER%, defined as the weight percentage of drug encapsulated in the HG on the total drug added during preparation) of 100%, respectively. This high encapsulation degree can be explained as consequence of the electrostatic interactions between the positive charge on the drug and the negative one present on the C-terminus of the Fmoc-FF peptide. Analogously to free Dox, also Doxil was efficiently encapsulated into mixed hydrogels at the same drug concentration. This is surprising due to the negative Z potential value (see Figure S2) we measured for Doxil (ζ ~ -11 mV) into the experimental conditions ([Dox] =1.13 mmol L−1) used to achieve the encapsulation into the hydrogel.Figure 2Characterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nCharacterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nA macroscopic evaluation of the gelation kinetics for mixed drug-filled hydrogels can be done by simply following the opaque-to-transparent optic transition. As previously observed for the corresponding empty systems, a different time (between 20 and 40 minutes) is required for the gelation process of each mixed hydrogel.38 In particular, we observed a more rapid formation for mixed HGs containing PEG8-(FY)3 (24 and 30 minutes for 2/1 and 1/1, respectively) with respect to those containing (FY)3 (35 and 40 minutes for 2/1 and 1/1, respectively). Analogously to empty matrices, the gelation kinetics of Dox filled HGs is faster by increasing the amount of the Fmoc-FF. This result may be ascribable to the capability of Fmoc-FF to gelificate in a very quick time (~2 min).35 On the contrary, we observe a faster gelation for HGs containing PEG in respect to HGs lacking polymer. This result is probably due to the additional interactions occurring between the hydrophilic PEG structure and the hydrophilic daunosamine moiety of Dox. As an alternative, gelation kinetics of hydrogels can be more accurately determined by following other structural or mechanical transitions occurring over time, related to alteration of UV-Vis absorbance, storage modulus (G’) or the dichroic signal.48 In this perspective, we recorded the CD spectra of Dox filled hydrogels at several time points in the spectral region between 300 and 200 nm (see Figure 2C–F). From the inspection of all the CD spectra, it clearly appears an evolution of the dichroic signal towards a stable state, in which we can assume that the HG has reached its final arrangement. The co-existence of several conformational states in the solution is also confirmed by isosbestic points between 220 and 240 nm and around 267 nm for PEG8-(FY)3 containing HGs. By plotting the optical density (OD) in the relative minima for each sample, we were able to extrapolate the gelation times (see Figure S3). These times, evaluated from the CD measurements, were found in good agreement with the gelation times observed following the transition from the opaque to limpid form.", "The preparation of pure Fmoc-FF and mixed nanogels loaded with Dox was achieved according to the top-down methodology.41 In this approach, macroscopic discs of hydrogels (1.0%wt) loaded with Dox (0.018 mol L−1) were prepared into silicone molds according to the above-described protocol. Successively, these discs were homogenized into an aqueous solution of TWEEN®60 (polyethylene glycol sorbitan monostearate) and SPAN®60 (Sorbitan stearate) as stabilizing agents. The combination of this couple of surfactants permits to achieve a hydrophilic/lipophilic balance (HLB) value ranged between 4.7 and 14.9. Due to the similar behavior exhibited by the four mixed HG formulations in terms of DLC and stability, we decided to prepare only one of the four mixed formulations, ie, Fmoc-FF/(FY)3 (1/1, v/v). The best Dox filled NG formulations, in terms of size and stability, were obtained using TWEEN®60/SPAN®60 at 58/42 ratio (HLB= 10) or at 100/0 ratio (HLB= 14.9) for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v), respectively. Indeed, contrarily to the pure Fmoc-FF formulation, the mixed one was found unstable when prepared with a value of HLB = 10. The resulting suspensions, further submicronized using tip sonicator, were purified from free drug using size exclusion chromatography. The red coloration of purified formulations macroscopically suggested the incorporation of the anthracycline in the NGs vehicle. Due to its spectroscopic features (λabs = 480 nm; λem= 560, 590 nm), the amount of Dox encapsulated was analytically monitored and evaluated by UV-Vis spectroscopy, by checking the absorbance in the maximum at λabs= 480 nm. DLC and ER% values for pure Fmoc-FF nanogel were 0.137% and 63%, respectively. This DLC value is similar to that of the commercially available liposomal formulations Myocet (DLC=0.127) and Doxil (DLC=0.250). Therefore, the insertion of (FY)3 peptide in the preparation causes a slight decrease of both the DLC (0.093) and the ER% (45%) with respect to the pure formulation. The lower encapsulation degree observed for mixed NGs versus pure ones can be explained considering the different amount of the net negative charge present in the two nanogels. Indeed, the peptide (FY)3 has an amidated C-terminus, significantly less acidic compared to the carboxylic one, and a basic non protected N-terminal amino group that can support a positive charge after protonation. This latter phenomenon contributes to the reduction of the total negative charge in the inner sphere of NGs, which determines lower attractive forces between the drug and the peptide system. The size of empty and filled Fmoc-FF/(FY)3 NGs, measured by Dynamic Light Scattering (DLS), was 168 and 214 nm, respectively (see Figure 3). This increase in size (~22%) is comparable to the increase of dimensions previously observed for empty (174 nm) and filled (241 nm) Fmoc-FF NGs.Figure 3DLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.\nDLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.", "Drug release from both hydrogels and nanogels was evaluated in 0.100 mol L−1 phosphate buffer solution up to 72 hours and the corresponding release profiles are reported in Figure 4A and B, respectively. Two different procedures were used for determination of Dox release from hydrogels and nanogels. In the first case, Dox or Doxil filled hydrogels, prepared into a conic tube, were directly put in contact with a double volume of physiological solution, cyclically replaced with a fresh one. Instead, in the second case, the release of Dox from nanogels was studied using a dialysis membrane immersed in phosphate buffer at 37°C. We assumed that the crossing of the free Dox through the dialysis membrane occurred quickly, thus, the overall release of the free drug from the peptide-based nanostructures to the dialysis bag medium could be considered to be rate determining for the process. The API release from the systems was considered undergone to a diffusion process. The Dox amount released was estimated by UV-Vis (at λabs= 480 nm) or by fluorescence spectroscopies (at λem= 590 nm) for HGs and NGs, respectively. The extent of drug was reported as a percentage of the ratio between the amount of released drug and the total drug initially loaded. From the inspection of Figure 4A, we can observe a similar release profile for all the Dox filled hydrogels during the first 24 hours. However, after 72 hours, each mixed gel exhibits a different release, with the lowest one for Fmoc-FF/(FY)3 (2/1) (16%). A low drug release (21%) was also observed for the other mixed HGs Fmoc-FF/(FY)3 (1/1). The same trend was also observed for the PEG8-(FY)3 containing hydrogels with a release of 19% and 28% for 2/1 and 1/1 ratios, respectively. These results agree with our expectations that PEGylation of the peptide could affect the rigidity and permeability of the hydrogels matrix and, in turn, the drug release. As expected, the amount of drug released from the hydrogel encapsulating Doxil (~8.5%) was lower than the drug released from the corresponding hydrogel (21%). In Figure 4B is reported the Dox release (%), which after 72 h, is around 20% and 40% for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels, respectively. Analogously to HGs, also for NGs the drug release is more appreciable during the first 8–12 h. The higher release observed for mixed NGs with respect to pure ones can be interpreted as a direct consequence of the lower electrostatic interactions occurring between the NG and the drug.Figure 4Drug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.\nDrug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.", "Cytotoxicity of mixed Fmoc-FF/(FY)3 (1/1, v/v) hydrogel encapsulating Dox or its liposomal formulation Doxil, at the same Dox concentration, was evaluated after 24 hours of incubation on MDA-MB-231 breast cancer cell line, using MTT assay. The cytotoxicity of the free drug, as well as of the empty hydrogels, was studied in the same conditions. HGs preparation was directly achieved into the hollow plastic chamber sealed at one end with a porous membrane. This experimental setting for hydrogels allows to mimic its conditions of utilization, in which this biocompatible support loaded with the chemotherapeutic agent is grafted at the level of the tumor lesion, where a controlled and constant drug release is achieved. During our experiments, hydrogels remain in contact with the cells for all the duration of the treatment. As shown in Figure 5A, the cell viability of empty hydrogels was found to be more than 95%. This percentage of cell survival is similar to that previously observed by us for Fmoc-FF and Fmoc-FF/(FY)3 hydrogels co-incubated with the Chinese Hamster Ovarian (CHO) cell line.38 Dox loaded hydrogels significantly reduced viability of MDA-MB-231 cells after only 24 h of incubation (49%) as well as free Dox at a concentration of 1µmol L−1 (55%). This Dox concentration corresponds to its IC50 on MDA-MB-231 cells (see Supplementary Figure S4). Due to its lower drug release, hydrogels encapsulating Doxil showed a slightly lower cytotoxicity, with a cell viability of 57%.Figure 5MDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).Abbreviation: n.s., not significant.\nMDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).\nOn the other hand, Figure 5B reports the cell viability of the same cell line treated with pure Fmoc-FF and mixed Fmoc-FF(FY)3 nanogels, empty or loaded with the drug, in comparison to free Dox and Doxil. Initially, we evaluated the cytotoxicity of empty nanogels as function of the total peptide concentration at three different time points (24, 48 and 72 h). We observed that there is a significant decrease in the cell viability during the first 48 h of incubation with nanogels. After this time of incubation, cell viability improves during the next 24 h (see Figure S5). This effect may be due to a temporary inhibition of the cell cycle induced by nanogels. Based on these results, cytotoxicity of NGs encapsulating Dox was checked after 72 h of incubation. Analogously to HGs, Dox loaded nanogels are able to significantly reduce breast cancer cells viability, with a cell viability of 7% and 25% for pure and mixed hydrogels, respectively. The higher cytotoxicity of pure HG with respect to the mixed one is probably attributable to the intrinsic toxicity of the empty gel.", "The intracellular internalization of Dox loaded HGs and NGs was assessed using immunofluorescence analysis on MDA-MB-231 cells treated for 24 h. Internalization of free Dox and Doxil are also reported for comparison. As indicated by the overlapping of red fluorescence associated to Dox with blue signal associated to DAPI (nucleus), the free drug can internalize into the nucleus after 24 h of incubation at 37°C (Figure 6A). Differently, Dox loaded Fmoc-FF/(FY)3 mixed hydrogel induces the internalization of Dox at peri-nuclear level since Dox signal is not perfectly overlapped with DAPI, but it is also partially detectable in the cytoplasm together with the green Actin signal (Figure 6B). The same behavior was previously observed for other liposomal Dox formulations.49,50 At the same time, doxorubicin conveyed through the nanogels remains in the cytoplasm of treated cells (Figure 6C), whereas by using Doxil the drug diffuses equally between the cytoplasm and the nucleus (Figure 6D). The different intracellular distribution of the drug in Figure 6 suggests an internalization mechanism alternative to the diffusion for the drug delivered by supramolecular systems like HGs, NGs and liposomes. One of the most accredited hypotheses is that nanogels are able to bind themselves primarily to the cellular membrane and then to enter the tumor cell via the endocytosis pathway.51,52Figure 6Immunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.\nImmunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.", "The administration of drugs is often affected by several issues related to systemic toxicity, chemical instability and repeated dosing requirement. Hydrogels and nanogels can represent alternative drug delivery vehicles to conventional supramolecular structures, such as vesicles, liposomes and nanostructures already developed and in clinical use. Indeed, macroscopic tridimensional HG networks could allow transepithelial drug delivery or in situ gelation process for the formation of implants, whereas nanosized nanogels could be used for systemic, oral and pulmonary administration of drugs. Due to their high biocompatibility, good biodegradability and tunability, short or ultra-short peptides represent potential attractive alternatives for preparation of HGs and NGs with respect to natural and synthetic polymers. The peptide-based HGs and NGs here formulated are obtained by using the well-known hydrogelator Fmoc-FF alone or in combination with (FY)3 peptide or its PEGylated analogue PEG8-(FY)3 at two different ratios. NGs were prepared starting from the corresponding HGs using a top-down approach in which the macroscopic hydrogel is submicronized and stabilized with commercially available biocompatible surfactants. Due to the common structure of their inner peptidic network, both NGs and HGs allow to efficiently encapsulate Dox. The gelation kinetics (from 24 to 40 minutes) and the drug release (16–28%, after 72 h) from hydrogels are clearly influenced by the hydrogel peptide composition. Analogously, the DLC values (0.137 and 0.093 for pure and mixed NGs, respectively) and the release percentages (20–40%, after 72 h) in NGs are affected by their composition in terms of net charges. Cytotoxicity assays carried out on MDA-MB-231 breast cancer cell line pointed out a high cell viability (>95%) for empty HGs and NGs, and a reduced cell viability (49–57%) for Dox loaded HGs and NGs. Moreover, immunofluorescence assays show a different cellular localization for the Dox delivered by HGs and NGs with respect to the free Dox. Indeed, contrarily to the free Dox, localized in the nucleus, HGs and NGs allow internalization of the drug at the peri-nuclear level and in the cytoplasm, respectively. This result suggests a different internalization mechanism and a different intracellular distribution and Dox release between free Dox and Dox loaded in hydrogels/nanogels. All the in vitro data we collected and analyzed for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 nanogels loaded with Dox suggest their potential use in vivo, by systemic administration, to deliver the cytotoxic drug on tumor tissues and cells. Thanks to its characteristics the new Dox loaded peptide supramolecular systems here described could be considered as promising valid alternatives to the already available liposomal Dox formulations." ]

[ null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]

[ "Introduction", "Materials and Methods", "Peptide Synthesis", "Hydrogels and Nanogels Formulation", "Doxorubicin Filled Hydrogels and Nanogels", "Doxil Filled Hydrogels", "Circular Dichroism (CD)", "Confocal Analysis", "Dynamic Light Scattering (DLS) Measurements", "Dox Release from Hydrogels and Nanogels", "Cell Line", "Cell Viability Evaluation by MTT", "Immunofluorescence Experiments", "Statistical Analyses", "Results and Discussion", "Formulation and Characterization of Dox Filled HGs", "Formulation and Characterization of Dox Filled NGs", "Drug Release from HGs and NGs", "Cytotoxicity Assays", "Determination of the Cellular Uptake by Immunofluorescence", "Conclusion" ]

[ "Nowadays, cancer remains one of the leading causes of mortality worldwide, affecting more than 10 million new patients every year. Among cancerous diseases, breast cancer is the most common one for women in the United States with more than 200,000 newly diagnosed cases for year.1 Current treatment options include surgical resection, radiation, and chemotherapy. Chemotherapeutic regime establishes the treatment of patients with drugs (doxorubicin, docetaxel, paclitaxel, and tamoxifen) alone or in combination.2 Doxorubicin (Dox), also known as Adriamycin, is a natural antitumor antibiotic of the anthracycline class, which works as DNA intercalating agent and as an inhibitor of topoisomerase II.3 Despite its therapeutic potentiality, clinical use of Dox is hampered by its dose-limiting toxicity, represented by myelosuppression and cardiotoxic side effects, which cause increased cardiovascular risks.4 In addition, Dox-mediated cardiotoxicity was found to be cumulative and dose-dependent, with heart suffering from the very first dose and then with accumulative damage for each following anthracycline cycle.5,6 In order to overcome these drawbacks, Dox encapsulating nanoformulations has been proposed as an alternative strategy for its administration. Currently, two doxorubicin liposomal formulations, Caelyx®/Doxil® and Myocet®, and their bioequivalent formulations, are available in the clinic.7,8 The liposomal spatial confinement of Dox allows altering biodistribution of the drug, minimizing its toxicity, increasing the half-life and the therapeutic index and improving the pharmaceutical profile, thus leading to increased patient compliance.9,10\nIn the last years, other typologies of nanosized structures, including polymer-based aggregates,11 nanofibers,12 nanodisks,13 gold nanoparticles,14 graphene and graphene oxide15,16 and hydrogels (HGs), were evaluated and studied as novel Dox-delivery tools. HGs are self-supporting materials, structured as supramolecular hydrophilic networks associated with the construction of space-spanning structures characterized by a non-Newtonian behavior. The hydrophilic nature of HGs constituents allows entrapping a high volume of biological fluids and water during the swelling process.17 3D-connectivity in physical cross-linked HGs is related to aggregation/interaction of molecules, cooperating through non-covalent interactions or via chemical irreversible bonds. Due to their unique structure, HGs have been exploited as versatile tools for many different biomedical applications (such as 3D-extracellular matrices for wound healing systems,18 cell support for tissue engineering and regeneration,19,20 protein mimetics,21 ophthalmic compatible materials,22 and drug delivery systems23). Submicronization of HGs by top-down methodologies gives the possibility to generate smaller hydrogel particles with a size in the nano-range.24 These hydrogel nanoparticles, named nanogels (NGs), combine the same hydrated inner network of hydrogels with the size of injectable nanoparticles, such as micelles and liposomes.25 Due to their size, nanogel formulations could achieve a fast renal clearance, a feasible penetration through tissue barriers and a prolonged circulation time in the blood stream.\nHydrogels and nanogels can be prepared using synthetic26–28 or natural polymers29–32 or peptide sequences.33,34 With respect to polymers, peptides exhibit several advantages such as high biocompatibility, biodegradability and tunability. Fmoc-FF (Nα-fluorenylmethyloxycarbonyl diphenylalanine) hydrogelator (Figure 1) represents one of the most studied peptide sequences for hydrogels formulation, thanks to its capability to gelificate under physiological conditions, required for biomedical applications.35–37 Recently, we have described the preparation of pure Fmoc-FF and mixed Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 (at 2/1 or 1/1, v/v) hydrogels.38 PEG8-(FY)3 and (FY)3 are the PEGylated and the non-PEGylated versions of the (FY)3 hexapeptide, this latter containing as peptide framework the alternation of three tyrosine (Y) and three phenylalanine (F) residues.39 The rheological characterization of the mixed gels pointed out that the presence of PEG and its relative amount into the mixed gel causes a slowing down of the gelation kinetic and a decrease of the gel rigidity. This different behavior was attributed to the high flexibility and conformational freedom of the PEG chain in the mixed hydrogel. Independently of their composition, all the gels showed an in vitro cell viability higher than 95% after 24 h of incubation, thus suggesting their potential applications in the biomedical field.Figure 1Schematic representation of components and methodologies for the formulation of HGs and NGs. Chemical formulas for peptide-based components are reported in the legend.\nSchematic representation of components and methodologies for the formulation of HGs and NGs. Chemical formulas for peptide-based components are reported in the legend.\nHere we describe the Dox loading capability of hydrogels and nanogels (both pure and mixed) and their drug release properties over time. We also report the in vitro cytotoxicity of both empty and Dox filled HGs and NGs on MDA-MB-231 breast cancer cell line representative of Triple Negative Breast Cancer (TNBC), the most aggressive breast cancer subtype.40 These results are compared with empty and Dox filled nanogel formulations,41 obtained sub-micronizing hydrogels by a top-down method. We also checked the capability of peptide-based hydrogels to encapsulate supramolecular nanodrugs and we studied their effective cytotoxicity on cells. For instance, as nanodrug we chose the liposomal doxorubicin formulation Doxil. This strategy could allow to obtain a composite drug delivery platform for a multi-stage delivery of Dox.", "Protected Nα-Fmoc-amino acid derivatives, coupling reagents, and Rink amide MBHA (4-methylbenzhydrylamine) resin were purchased from Calbiochem-Novabiochem (Laufelfingen, Switzerland). The monodisperse Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc- AdOO-OH, PEG2) was purchased from Neosystem (Strasbourg, France). Lyophilized Fmoc-FF powder was purchased from Bachem (Bubendorf, Switzerland). Doxorubicin chlorohydrate (Dox.HCl) was purchased from Sigma Aldrich (Milan, Italy). Pegylated liposomal Dox (commercial name of Doxil) vials were kindly gifted by the Italian Cancer Institute in Naples (Italy) “Fondazione G. Pascale”. TWEEN®60, SPAN®60, and all other chemicals and solvents were purchased from Sigma-Aldrich, Fluka (Bucks, Switzerland) or LabScan (Stillorgan, Dublin, Ireland) and were used as received unless otherwise stated. All solutions were obtained by weight using doubly distilled water as a solvent. UV-Vis spectra were recorded on a Thermo Fisher Scientific Inc (Wilmington, Delaware USA) Nanodrop 2000c, equipped with a 1.0 cm quartz cuvette (Hellma).\nPeptide Synthesis (FY)3 and its PEGylated analogue PEG8-(FY)3 peptide derivative were synthesized by peptide solid phase synthesis (SPPS) procedures with a Fmoc/tBu chemistry approach as previously described39,42,43 and purified by RP-HPLC chromatography.\n(FY)3 and its PEGylated analogue PEG8-(FY)3 peptide derivative were synthesized by peptide solid phase synthesis (SPPS) procedures with a Fmoc/tBu chemistry approach as previously described39,42,43 and purified by RP-HPLC chromatography.\nHydrogels and Nanogels Formulation Pure Fmoc-FF hydrogel and mixed Fmoc-FF/PEG8-(FY)3 and Fmoc-FF/(FY)3 hydrogels (1/1 or 2/1, v/v) were prepared as previously described using the “solvent-switch method”44 to a final concentration of 0.5 wt%.38 Briefly, each peptide component (Fmoc-FF, (FY)3 and PEG8-(FY)3) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mg/mL. Pure Fmoc-FF hydrogel (400 µL) was obtained by diluting 20 µL of the Fmoc-FF stock solution with 380 μL of double distilled water under stirring (5 seconds). Preparation of all the mixed hydrogels was achieved analogously, by combining the peptides stock solutions at the desired volume/volume ratios. Pure and mixed nanogel formulations were prepared as previously described according to the top-down method.41 Briefly, the gel disk obtained into a silicone mold was homogenized at 35,000 min−1 for 5 min into 4 mL of an aqueous solution containing TWEEN 60/SPAN 60 at a w/w ratio of 52/48 (3.10–5 mol) for pure Fmoc-FF NGs and only TWEEN 60 (3.10–5 mol) for mixed Fmoc-FF/(FY)3 NGs. The resulting suspensions were then tip sonicated for 5 min at 9 W.\nPure Fmoc-FF hydrogel and mixed Fmoc-FF/PEG8-(FY)3 and Fmoc-FF/(FY)3 hydrogels (1/1 or 2/1, v/v) were prepared as previously described using the “solvent-switch method”44 to a final concentration of 0.5 wt%.38 Briefly, each peptide component (Fmoc-FF, (FY)3 and PEG8-(FY)3) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mg/mL. Pure Fmoc-FF hydrogel (400 µL) was obtained by diluting 20 µL of the Fmoc-FF stock solution with 380 μL of double distilled water under stirring (5 seconds). Preparation of all the mixed hydrogels was achieved analogously, by combining the peptides stock solutions at the desired volume/volume ratios. Pure and mixed nanogel formulations were prepared as previously described according to the top-down method.41 Briefly, the gel disk obtained into a silicone mold was homogenized at 35,000 min−1 for 5 min into 4 mL of an aqueous solution containing TWEEN 60/SPAN 60 at a w/w ratio of 52/48 (3.10–5 mol) for pure Fmoc-FF NGs and only TWEEN 60 (3.10–5 mol) for mixed Fmoc-FF/(FY)3 NGs. The resulting suspensions were then tip sonicated for 5 min at 9 W.\nDoxorubicin Filled Hydrogels and Nanogels Dox filled hydrogels were prepared as described above by adding 380 µL of an aqueous Dox solution at a concentration of 4.0.10–3 mol L−1 to the peptide stock solutions. Dox filled nanogels were prepared according to the top-down methodology as previously described.41 Shorty, a disk of pure or mixed hydrogel loaded with Dox was prepared adding the stock solution of peptide (100 mg/mL) to 900 μL of an aqueous solution of Dox (0.018 mol L−1), which allows to reach a drug weight/lipid weight ratio of 0.250. Then, the hydrogel disk was homogenized, and tip sonicated into 4 mL of TWEEN 60/SPAN 60 mixture or TWEEN 60, according to the empty formulation. Purification of Dox filled nanogels from free Dox was achieved by gel filtration on a pre-packed column Sephadex G-50. The drug loading content (DLC), defined as gDox encapsulated/g(surfactant+peptide), was quantified by subtraction of the free Dox from the total amount of loaded Dox. The Dox concentration was determined by UV-Vis spectroscopy using calibration curves obtained by measuring absorbance at λ = 480 nm.\nDox filled hydrogels were prepared as described above by adding 380 µL of an aqueous Dox solution at a concentration of 4.0.10–3 mol L−1 to the peptide stock solutions. Dox filled nanogels were prepared according to the top-down methodology as previously described.41 Shorty, a disk of pure or mixed hydrogel loaded with Dox was prepared adding the stock solution of peptide (100 mg/mL) to 900 μL of an aqueous solution of Dox (0.018 mol L−1), which allows to reach a drug weight/lipid weight ratio of 0.250. Then, the hydrogel disk was homogenized, and tip sonicated into 4 mL of TWEEN 60/SPAN 60 mixture or TWEEN 60, according to the empty formulation. Purification of Dox filled nanogels from free Dox was achieved by gel filtration on a pre-packed column Sephadex G-50. The drug loading content (DLC), defined as gDox encapsulated/g(surfactant+peptide), was quantified by subtraction of the free Dox from the total amount of loaded Dox. The Dox concentration was determined by UV-Vis spectroscopy using calibration curves obtained by measuring absorbance at λ = 480 nm.\nDoxil Filled Hydrogels Doxil filled hydrogels were prepared analogously to Dox filled ones, by adding 380 µL of the commercial Doxil solution opportunely diluted in order to achieve the same Dox concentration (4.0.10–3 mol L−1) that was in the HGs.\nDoxil filled hydrogels were prepared analogously to Dox filled ones, by adding 380 µL of the commercial Doxil solution opportunely diluted in order to achieve the same Dox concentration (4.0.10–3 mol L−1) that was in the HGs.\nCircular Dichroism (CD) Far-UV CD spectra of Dox filled hydrogels and nanogels were collected on a Jasco J-810 spectropolarimeter equipped with a NesLab RTE111 thermal controller unit at 25°C. 100 µL of a hydrated DMSO stock solution (immediately after its generation) were placed on a 0.1 mm quartz cell. The measurements were recorded as function of the time (every 8 minutes) in the range of wavelength between 300 and 200 nm. Other experimental settings were: scan speed = 50 nm min−1, sensitivity = 10 mdeg, time constant = 16 s, bandwidth = 1 nm. Each spectrum was obtained by averaging three scans. All the spectra are reported in optical density (O.D.).\nFar-UV CD spectra of Dox filled hydrogels and nanogels were collected on a Jasco J-810 spectropolarimeter equipped with a NesLab RTE111 thermal controller unit at 25°C. 100 µL of a hydrated DMSO stock solution (immediately after its generation) were placed on a 0.1 mm quartz cell. The measurements were recorded as function of the time (every 8 minutes) in the range of wavelength between 300 and 200 nm. Other experimental settings were: scan speed = 50 nm min−1, sensitivity = 10 mdeg, time constant = 16 s, bandwidth = 1 nm. Each spectrum was obtained by averaging three scans. All the spectra are reported in optical density (O.D.).\nConfocal Analysis For confocal microscopy, Dox filled hydrogels were drop-casted and spread on a glass slide, air-dried at room temperature, and examined by confocal microscopy. Confocal images were obtained with a Leica TCS-SMD-SP5 confocal microscope (λexc = 488 nm and λem = 505–600 nm). 0.8-µm thick optical slices were acquired with a 63× or 40×/1.4 NA objective.\nFor confocal microscopy, Dox filled hydrogels were drop-casted and spread on a glass slide, air-dried at room temperature, and examined by confocal microscopy. Confocal images were obtained with a Leica TCS-SMD-SP5 confocal microscope (λexc = 488 nm and λem = 505–600 nm). 0.8-µm thick optical slices were acquired with a 63× or 40×/1.4 NA objective.\nDynamic Light Scattering (DLS) Measurements Mean diameters and diffusion coefficients (D) of empty and Dox filled NGs were estimated by DLS using a Zetasizer Nano ZS (Malvern Instruments, Westborough, MA). Instrumental settings for the measurements are a backscatter detector at 173° in automatic modality, room temperature and disposable sizing cuvette as cell. DLS measurements in triplicate were carried out on aqueous samples after centrifugation at room temperature at 13,000 rpm for 5 minutes.\nMean diameters and diffusion coefficients (D) of empty and Dox filled NGs were estimated by DLS using a Zetasizer Nano ZS (Malvern Instruments, Westborough, MA). Instrumental settings for the measurements are a backscatter detector at 173° in automatic modality, room temperature and disposable sizing cuvette as cell. DLS measurements in triplicate were carried out on aqueous samples after centrifugation at room temperature at 13,000 rpm for 5 minutes.\nDox Release from Hydrogels and Nanogels Dox and Doxil release from hydrogels were evaluated in a conic tube (1.5 mL) using 400 µL of drug filled hydrogels (0.5% wt) adding, on the top of them, 800 µL of 0.100 mol L−1 phosphate buffer. At each time-point, 400 µL of this solution was removed and replaced with an equal fresh aliquot. Released Dox was quantified by UV-Vis spectrum of the supernatant at 480 nm. All the release experiments were performed in triplicates. The extent of Dox release was reported as percentage of the ratio between the amount of released drug and the total quantity of drug initially loaded into hydrogels. Dox release from nanogels was achieved using the well-known dialysis method.45 Briefly, Dox loaded nanogel suspension (1.0 mL) was placed into a dialysis bag (MW cut-off = 3500 Da). This bag was immersed under stirring for 72 h, at 37°C into 20 mL of phosphate buffer. Then, 2 mL of the dialyzed solution was replaced with an equal amount of fresh solution at different time points. Fluorescence measurements of each fraction of the dialyzed solution were recorded at room temperature with a spectrofluorophotometer Jasco (Model FP-750, Japan) in a quartz cell with 1.0 cm path length. The other settings were as follows: excitation and emission bandwidths = 5 nm, recording speed = 125 nm/min, and excitation wavelength = 480 nm, emission range from 490 to 700 nm. The amount of doxorubicin contained in each fraction was estimated using an analytical titration curve previously recorded for the free Dox in the same spectral range. Analogously to HGs, Dox release profile from NGs was reported as percentage of released drug/total drug loaded into NGs.\nDox and Doxil release from hydrogels were evaluated in a conic tube (1.5 mL) using 400 µL of drug filled hydrogels (0.5% wt) adding, on the top of them, 800 µL of 0.100 mol L−1 phosphate buffer. At each time-point, 400 µL of this solution was removed and replaced with an equal fresh aliquot. Released Dox was quantified by UV-Vis spectrum of the supernatant at 480 nm. All the release experiments were performed in triplicates. The extent of Dox release was reported as percentage of the ratio between the amount of released drug and the total quantity of drug initially loaded into hydrogels. Dox release from nanogels was achieved using the well-known dialysis method.45 Briefly, Dox loaded nanogel suspension (1.0 mL) was placed into a dialysis bag (MW cut-off = 3500 Da). This bag was immersed under stirring for 72 h, at 37°C into 20 mL of phosphate buffer. Then, 2 mL of the dialyzed solution was replaced with an equal amount of fresh solution at different time points. Fluorescence measurements of each fraction of the dialyzed solution were recorded at room temperature with a spectrofluorophotometer Jasco (Model FP-750, Japan) in a quartz cell with 1.0 cm path length. The other settings were as follows: excitation and emission bandwidths = 5 nm, recording speed = 125 nm/min, and excitation wavelength = 480 nm, emission range from 490 to 700 nm. The amount of doxorubicin contained in each fraction was estimated using an analytical titration curve previously recorded for the free Dox in the same spectral range. Analogously to HGs, Dox release profile from NGs was reported as percentage of released drug/total drug loaded into NGs.\nCell Line Human breast cancer cell line MDA-MB-231 was obtained from IRCCS-SDN Biobank (10.5334/ojb.26) and growth in Dulbecco’s Modified Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMAX. Cells were incubated at 37°C and 5% CO2 and seeded in T-25 culture flasks.\nHuman breast cancer cell line MDA-MB-231 was obtained from IRCCS-SDN Biobank (10.5334/ojb.26) and growth in Dulbecco’s Modified Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMAX. Cells were incubated at 37°C and 5% CO2 and seeded in T-25 culture flasks.\nCell Viability Evaluation by MTT For MTT assays (Sigma Aldrich, Germany), MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well. After 24 h free Dox at 1µmol L−1, nanogels and hydrogels (these latter preformed into a hollow plastic chamber sealed at one end with a porous membrane) were added to the wells. Cells were treated with the hydrogels for 24 h and with nanogel solutions for 72 h. At the end of the treatment, cell viability was assessed by the MTT assay. For the IC50 determination of Dox, MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well and treated with different concentrations (0.25, 0.5, 1, 2 and 4 µmol L−1) of Dox for 24 h. At the end of the treatment, cell viability was assessed by the MTT assay. In brief, MTT, dissolved in DMEM in the absence of phenol red (Sigma-Aldrich), was added to the cells at a final concentration of 0.5 mg/mL. After 4 h incubation at 37°C, the culture medium was removed, and the resulting formazan salts were dissolved by adding isopropanol containing 0.1 mol L−1 HCl and 10% Triton-X100. Absorbance values of blue formazan were determined at 490 nm using an automatic plate reader (EL 800, Biotek). Cell survival was expressed as percentage of viable cells in the presence of hydrogels or nanogels, compared to control cells grown in their absence. MTT assay was repeated twice with similar results.\nFor MTT assays (Sigma Aldrich, Germany), MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well. After 24 h free Dox at 1µmol L−1, nanogels and hydrogels (these latter preformed into a hollow plastic chamber sealed at one end with a porous membrane) were added to the wells. Cells were treated with the hydrogels for 24 h and with nanogel solutions for 72 h. At the end of the treatment, cell viability was assessed by the MTT assay. For the IC50 determination of Dox, MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well and treated with different concentrations (0.25, 0.5, 1, 2 and 4 µmol L−1) of Dox for 24 h. At the end of the treatment, cell viability was assessed by the MTT assay. In brief, MTT, dissolved in DMEM in the absence of phenol red (Sigma-Aldrich), was added to the cells at a final concentration of 0.5 mg/mL. After 4 h incubation at 37°C, the culture medium was removed, and the resulting formazan salts were dissolved by adding isopropanol containing 0.1 mol L−1 HCl and 10% Triton-X100. Absorbance values of blue formazan were determined at 490 nm using an automatic plate reader (EL 800, Biotek). Cell survival was expressed as percentage of viable cells in the presence of hydrogels or nanogels, compared to control cells grown in their absence. MTT assay was repeated twice with similar results.\nImmunofluorescence Experiments MDA-MB-231 cells were treated for 24 h with 1µmol L−1 free Dox or 1.13 mmol L−1 Dox loaded Fmoc-FF/(FY)3 hydrogels and Fmoc-FF/(FY)3 nanogels. Cells were fixed at -20 °C for 15 minutes with a -80 °C pre-cooled solution of methanol/acetone (1/1, v/v). Subsequently, cells were subjected to blocking with solution of 3% (w/v) BSA in PBS pH 7.4 at room temperature (RT). Anti B-actin (A2228, Sigma Aldrich) monoclonal antibody was diluted 1:200 in a solution of PBS + 1% (w/v) BSA and then all slides were incubated for 4 h at + 4°C. After three washing steps in PBS for 5 minutes each, FITC-conjugated anti-mouse secondary antibody (ab7064, Abcam, UK) diluted 1:400 in a solution of PBS + 1% (w/v) BSA was incubated for 1 h at 4°C in the dark. After additional three washing steps in PBS, a solution of 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fischer Scientific D1306) diluted 35,000-fold in PBS was used and it was left to act for 10 minutes at RT in the dark to color the nuclei. Images were obtained using an automated upright microscope system (Leica DM5500 B) coupled with Leica Cytovision software.\nStatistical Analyses All statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.\nAll statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.\nMDA-MB-231 cells were treated for 24 h with 1µmol L−1 free Dox or 1.13 mmol L−1 Dox loaded Fmoc-FF/(FY)3 hydrogels and Fmoc-FF/(FY)3 nanogels. Cells were fixed at -20 °C for 15 minutes with a -80 °C pre-cooled solution of methanol/acetone (1/1, v/v). Subsequently, cells were subjected to blocking with solution of 3% (w/v) BSA in PBS pH 7.4 at room temperature (RT). Anti B-actin (A2228, Sigma Aldrich) monoclonal antibody was diluted 1:200 in a solution of PBS + 1% (w/v) BSA and then all slides were incubated for 4 h at + 4°C. After three washing steps in PBS for 5 minutes each, FITC-conjugated anti-mouse secondary antibody (ab7064, Abcam, UK) diluted 1:400 in a solution of PBS + 1% (w/v) BSA was incubated for 1 h at 4°C in the dark. After additional three washing steps in PBS, a solution of 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fischer Scientific D1306) diluted 35,000-fold in PBS was used and it was left to act for 10 minutes at RT in the dark to color the nuclei. Images were obtained using an automated upright microscope system (Leica DM5500 B) coupled with Leica Cytovision software.\nStatistical Analyses All statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.\nAll statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.", "(FY)3 and its PEGylated analogue PEG8-(FY)3 peptide derivative were synthesized by peptide solid phase synthesis (SPPS) procedures with a Fmoc/tBu chemistry approach as previously described39,42,43 and purified by RP-HPLC chromatography.", "Pure Fmoc-FF hydrogel and mixed Fmoc-FF/PEG8-(FY)3 and Fmoc-FF/(FY)3 hydrogels (1/1 or 2/1, v/v) were prepared as previously described using the “solvent-switch method”44 to a final concentration of 0.5 wt%.38 Briefly, each peptide component (Fmoc-FF, (FY)3 and PEG8-(FY)3) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mg/mL. Pure Fmoc-FF hydrogel (400 µL) was obtained by diluting 20 µL of the Fmoc-FF stock solution with 380 μL of double distilled water under stirring (5 seconds). Preparation of all the mixed hydrogels was achieved analogously, by combining the peptides stock solutions at the desired volume/volume ratios. Pure and mixed nanogel formulations were prepared as previously described according to the top-down method.41 Briefly, the gel disk obtained into a silicone mold was homogenized at 35,000 min−1 for 5 min into 4 mL of an aqueous solution containing TWEEN 60/SPAN 60 at a w/w ratio of 52/48 (3.10–5 mol) for pure Fmoc-FF NGs and only TWEEN 60 (3.10–5 mol) for mixed Fmoc-FF/(FY)3 NGs. The resulting suspensions were then tip sonicated for 5 min at 9 W.", "Dox filled hydrogels were prepared as described above by adding 380 µL of an aqueous Dox solution at a concentration of 4.0.10–3 mol L−1 to the peptide stock solutions. Dox filled nanogels were prepared according to the top-down methodology as previously described.41 Shorty, a disk of pure or mixed hydrogel loaded with Dox was prepared adding the stock solution of peptide (100 mg/mL) to 900 μL of an aqueous solution of Dox (0.018 mol L−1), which allows to reach a drug weight/lipid weight ratio of 0.250. Then, the hydrogel disk was homogenized, and tip sonicated into 4 mL of TWEEN 60/SPAN 60 mixture or TWEEN 60, according to the empty formulation. Purification of Dox filled nanogels from free Dox was achieved by gel filtration on a pre-packed column Sephadex G-50. The drug loading content (DLC), defined as gDox encapsulated/g(surfactant+peptide), was quantified by subtraction of the free Dox from the total amount of loaded Dox. The Dox concentration was determined by UV-Vis spectroscopy using calibration curves obtained by measuring absorbance at λ = 480 nm.", "Doxil filled hydrogels were prepared analogously to Dox filled ones, by adding 380 µL of the commercial Doxil solution opportunely diluted in order to achieve the same Dox concentration (4.0.10–3 mol L−1) that was in the HGs.", "Far-UV CD spectra of Dox filled hydrogels and nanogels were collected on a Jasco J-810 spectropolarimeter equipped with a NesLab RTE111 thermal controller unit at 25°C. 100 µL of a hydrated DMSO stock solution (immediately after its generation) were placed on a 0.1 mm quartz cell. The measurements were recorded as function of the time (every 8 minutes) in the range of wavelength between 300 and 200 nm. Other experimental settings were: scan speed = 50 nm min−1, sensitivity = 10 mdeg, time constant = 16 s, bandwidth = 1 nm. Each spectrum was obtained by averaging three scans. All the spectra are reported in optical density (O.D.).", "For confocal microscopy, Dox filled hydrogels were drop-casted and spread on a glass slide, air-dried at room temperature, and examined by confocal microscopy. Confocal images were obtained with a Leica TCS-SMD-SP5 confocal microscope (λexc = 488 nm and λem = 505–600 nm). 0.8-µm thick optical slices were acquired with a 63× or 40×/1.4 NA objective.", "Mean diameters and diffusion coefficients (D) of empty and Dox filled NGs were estimated by DLS using a Zetasizer Nano ZS (Malvern Instruments, Westborough, MA). Instrumental settings for the measurements are a backscatter detector at 173° in automatic modality, room temperature and disposable sizing cuvette as cell. DLS measurements in triplicate were carried out on aqueous samples after centrifugation at room temperature at 13,000 rpm for 5 minutes.", "Dox and Doxil release from hydrogels were evaluated in a conic tube (1.5 mL) using 400 µL of drug filled hydrogels (0.5% wt) adding, on the top of them, 800 µL of 0.100 mol L−1 phosphate buffer. At each time-point, 400 µL of this solution was removed and replaced with an equal fresh aliquot. Released Dox was quantified by UV-Vis spectrum of the supernatant at 480 nm. All the release experiments were performed in triplicates. The extent of Dox release was reported as percentage of the ratio between the amount of released drug and the total quantity of drug initially loaded into hydrogels. Dox release from nanogels was achieved using the well-known dialysis method.45 Briefly, Dox loaded nanogel suspension (1.0 mL) was placed into a dialysis bag (MW cut-off = 3500 Da). This bag was immersed under stirring for 72 h, at 37°C into 20 mL of phosphate buffer. Then, 2 mL of the dialyzed solution was replaced with an equal amount of fresh solution at different time points. Fluorescence measurements of each fraction of the dialyzed solution were recorded at room temperature with a spectrofluorophotometer Jasco (Model FP-750, Japan) in a quartz cell with 1.0 cm path length. The other settings were as follows: excitation and emission bandwidths = 5 nm, recording speed = 125 nm/min, and excitation wavelength = 480 nm, emission range from 490 to 700 nm. The amount of doxorubicin contained in each fraction was estimated using an analytical titration curve previously recorded for the free Dox in the same spectral range. Analogously to HGs, Dox release profile from NGs was reported as percentage of released drug/total drug loaded into NGs.", "Human breast cancer cell line MDA-MB-231 was obtained from IRCCS-SDN Biobank (10.5334/ojb.26) and growth in Dulbecco’s Modified Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMAX. Cells were incubated at 37°C and 5% CO2 and seeded in T-25 culture flasks.", "For MTT assays (Sigma Aldrich, Germany), MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well. After 24 h free Dox at 1µmol L−1, nanogels and hydrogels (these latter preformed into a hollow plastic chamber sealed at one end with a porous membrane) were added to the wells. Cells were treated with the hydrogels for 24 h and with nanogel solutions for 72 h. At the end of the treatment, cell viability was assessed by the MTT assay. For the IC50 determination of Dox, MDA-MB-231 cells were seeded in 24-well plates at a density of 50 × 104 per well and treated with different concentrations (0.25, 0.5, 1, 2 and 4 µmol L−1) of Dox for 24 h. At the end of the treatment, cell viability was assessed by the MTT assay. In brief, MTT, dissolved in DMEM in the absence of phenol red (Sigma-Aldrich), was added to the cells at a final concentration of 0.5 mg/mL. After 4 h incubation at 37°C, the culture medium was removed, and the resulting formazan salts were dissolved by adding isopropanol containing 0.1 mol L−1 HCl and 10% Triton-X100. Absorbance values of blue formazan were determined at 490 nm using an automatic plate reader (EL 800, Biotek). Cell survival was expressed as percentage of viable cells in the presence of hydrogels or nanogels, compared to control cells grown in their absence. MTT assay was repeated twice with similar results.", "MDA-MB-231 cells were treated for 24 h with 1µmol L−1 free Dox or 1.13 mmol L−1 Dox loaded Fmoc-FF/(FY)3 hydrogels and Fmoc-FF/(FY)3 nanogels. Cells were fixed at -20 °C for 15 minutes with a -80 °C pre-cooled solution of methanol/acetone (1/1, v/v). Subsequently, cells were subjected to blocking with solution of 3% (w/v) BSA in PBS pH 7.4 at room temperature (RT). Anti B-actin (A2228, Sigma Aldrich) monoclonal antibody was diluted 1:200 in a solution of PBS + 1% (w/v) BSA and then all slides were incubated for 4 h at + 4°C. After three washing steps in PBS for 5 minutes each, FITC-conjugated anti-mouse secondary antibody (ab7064, Abcam, UK) diluted 1:400 in a solution of PBS + 1% (w/v) BSA was incubated for 1 h at 4°C in the dark. After additional three washing steps in PBS, a solution of 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fischer Scientific D1306) diluted 35,000-fold in PBS was used and it was left to act for 10 minutes at RT in the dark to color the nuclei. Images were obtained using an automated upright microscope system (Leica DM5500 B) coupled with Leica Cytovision software.\nStatistical Analyses All statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.\nAll statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.", "All statistical analyses were performed using the Graphpad Prism 6 (Graphpad Software, Graphpad Holdings, LLC, CA, USA). Numbers of biological and/or technical replicates, as well as a description of the statistical parameters, are stated in the figure legends. All experimental images are representative of at least two independent experiments. For statistical significance, a p–value less than 0.05 was considered, unless otherwise specified.", "Classical administrations of active pharmaceutical ingredients (APIs) are affected by issues related to systemic toxicity, drug chemical instability and repeated dosing requirement. As previously mentioned, HGs offer convenient drug delivery approaches, allowing them to overcome these drawbacks thanks to their tunable features, modular degradability and easy formulation. Macroscopically, hydrogel-based materials belong to two different categories based on their size: hydrogels (HGs) and nanogels (NGs). The specific size-feature determines the route of administration: transepithelial drug delivery or in situ gelation process of implants for HGs and systemic, oral and pulmonary administration of APIs for nanogels.46 The knowledge about the encapsulation procedures, the efficiency of drugs loading, and the cytotoxicity profiles can steer the development of efficient vehicles. For instance, swollen HGs matrices can be loaded in different ways, acting at macroscopic level, on the fibrillary network, the mesh size or at molecular scales. Moreover, the drug release can be differently modulated according to covalent or non-covalent approaches, in which drug molecules are chemically bound on the HG matrix by stable or cleavable linkers, or they can establish electrostatic interactions with HGs building blocks. These different drug loading strategies could affect the experimental conditions of preparation (eg, solvents, pH and salt content) and in turn the biological profile of the resulting vehicle. In this perspective, comparative studies on analogies and differences between HGs and NGs may assume a great importance.\nFormulation and Characterization of Dox Filled HGs HGs filled with the anticancer drug Dox were prepared according to the “solvent-switch” method.41 This method consists of the addition of an aqueous solution of Dox directly into the stock peptide solution (100 mg/mL in DMSO). After few seconds of vortex, the resulting opaque metastable mixture is vertically incubated at room temperature until the formation of a limpid, translucent self-supportive hydrogel. Dox filled hydrogels Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 at two different ratios (1/1 or 2/1, v/v) were successfully formulated. Instead, in the same experimental conditions, Dox filled Fmoc-FF hydrogel appears not completely homogenous (see Figure S1). On the other hand, we observed a progressive improvement of the hydrogel homogeneity by increasing the amount (up to 0.018 mol L−1) of Dox to encapsulate (Figure S1). This behavior could be attributed to a shield effect of the charges occurring between the drug and the peptidic matrix. Although Dox filled Fmoc-FF hydrogel can be prepared, the Dox concentration is too high compared with mixed HGs and, for this reason, we did not further focus our attention on this sample. For mixed HGs, no syneresis was detected after 10 days. This specific no water-loss is an important advantage offered by HGs, thus allowing a precise modulation of water-soluble drug loaded in the system. According to this experimental evidence, we can assume that all the Dox was efficiently and quantitatively entrapped in the hydrogel matrix.\nThe resulting Dox filled hydrogels were further characterized in their xerogel form by confocal microscopy. Confocal images of one sample (Fmoc-FF/(FY)3, 1/1) are reported in Figure 2A and B. These images clearly show the characteristic intricate network of entangled fibers,47 and their red color suggests the tight interaction of Dox with the network constituents. The amount of drug we were able to encapsulate in mixed HGs was 2.32 mg/mL. This quantity corresponds to a drug loading content (DLC) of 0.440 and to an encapsulation ratio (ER%, defined as the weight percentage of drug encapsulated in the HG on the total drug added during preparation) of 100%, respectively. This high encapsulation degree can be explained as consequence of the electrostatic interactions between the positive charge on the drug and the negative one present on the C-terminus of the Fmoc-FF peptide. Analogously to free Dox, also Doxil was efficiently encapsulated into mixed hydrogels at the same drug concentration. This is surprising due to the negative Z potential value (see Figure S2) we measured for Doxil (ζ ~ -11 mV) into the experimental conditions ([Dox] =1.13 mmol L−1) used to achieve the encapsulation into the hydrogel.Figure 2Characterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nCharacterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nA macroscopic evaluation of the gelation kinetics for mixed drug-filled hydrogels can be done by simply following the opaque-to-transparent optic transition. As previously observed for the corresponding empty systems, a different time (between 20 and 40 minutes) is required for the gelation process of each mixed hydrogel.38 In particular, we observed a more rapid formation for mixed HGs containing PEG8-(FY)3 (24 and 30 minutes for 2/1 and 1/1, respectively) with respect to those containing (FY)3 (35 and 40 minutes for 2/1 and 1/1, respectively). Analogously to empty matrices, the gelation kinetics of Dox filled HGs is faster by increasing the amount of the Fmoc-FF. This result may be ascribable to the capability of Fmoc-FF to gelificate in a very quick time (~2 min).35 On the contrary, we observe a faster gelation for HGs containing PEG in respect to HGs lacking polymer. This result is probably due to the additional interactions occurring between the hydrophilic PEG structure and the hydrophilic daunosamine moiety of Dox. As an alternative, gelation kinetics of hydrogels can be more accurately determined by following other structural or mechanical transitions occurring over time, related to alteration of UV-Vis absorbance, storage modulus (G’) or the dichroic signal.48 In this perspective, we recorded the CD spectra of Dox filled hydrogels at several time points in the spectral region between 300 and 200 nm (see Figure 2C–F). From the inspection of all the CD spectra, it clearly appears an evolution of the dichroic signal towards a stable state, in which we can assume that the HG has reached its final arrangement. The co-existence of several conformational states in the solution is also confirmed by isosbestic points between 220 and 240 nm and around 267 nm for PEG8-(FY)3 containing HGs. By plotting the optical density (OD) in the relative minima for each sample, we were able to extrapolate the gelation times (see Figure S3). These times, evaluated from the CD measurements, were found in good agreement with the gelation times observed following the transition from the opaque to limpid form.\nHGs filled with the anticancer drug Dox were prepared according to the “solvent-switch” method.41 This method consists of the addition of an aqueous solution of Dox directly into the stock peptide solution (100 mg/mL in DMSO). After few seconds of vortex, the resulting opaque metastable mixture is vertically incubated at room temperature until the formation of a limpid, translucent self-supportive hydrogel. Dox filled hydrogels Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 at two different ratios (1/1 or 2/1, v/v) were successfully formulated. Instead, in the same experimental conditions, Dox filled Fmoc-FF hydrogel appears not completely homogenous (see Figure S1). On the other hand, we observed a progressive improvement of the hydrogel homogeneity by increasing the amount (up to 0.018 mol L−1) of Dox to encapsulate (Figure S1). This behavior could be attributed to a shield effect of the charges occurring between the drug and the peptidic matrix. Although Dox filled Fmoc-FF hydrogel can be prepared, the Dox concentration is too high compared with mixed HGs and, for this reason, we did not further focus our attention on this sample. For mixed HGs, no syneresis was detected after 10 days. This specific no water-loss is an important advantage offered by HGs, thus allowing a precise modulation of water-soluble drug loaded in the system. According to this experimental evidence, we can assume that all the Dox was efficiently and quantitatively entrapped in the hydrogel matrix.\nThe resulting Dox filled hydrogels were further characterized in their xerogel form by confocal microscopy. Confocal images of one sample (Fmoc-FF/(FY)3, 1/1) are reported in Figure 2A and B. These images clearly show the characteristic intricate network of entangled fibers,47 and their red color suggests the tight interaction of Dox with the network constituents. The amount of drug we were able to encapsulate in mixed HGs was 2.32 mg/mL. This quantity corresponds to a drug loading content (DLC) of 0.440 and to an encapsulation ratio (ER%, defined as the weight percentage of drug encapsulated in the HG on the total drug added during preparation) of 100%, respectively. This high encapsulation degree can be explained as consequence of the electrostatic interactions between the positive charge on the drug and the negative one present on the C-terminus of the Fmoc-FF peptide. Analogously to free Dox, also Doxil was efficiently encapsulated into mixed hydrogels at the same drug concentration. This is surprising due to the negative Z potential value (see Figure S2) we measured for Doxil (ζ ~ -11 mV) into the experimental conditions ([Dox] =1.13 mmol L−1) used to achieve the encapsulation into the hydrogel.Figure 2Characterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nCharacterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nA macroscopic evaluation of the gelation kinetics for mixed drug-filled hydrogels can be done by simply following the opaque-to-transparent optic transition. As previously observed for the corresponding empty systems, a different time (between 20 and 40 minutes) is required for the gelation process of each mixed hydrogel.38 In particular, we observed a more rapid formation for mixed HGs containing PEG8-(FY)3 (24 and 30 minutes for 2/1 and 1/1, respectively) with respect to those containing (FY)3 (35 and 40 minutes for 2/1 and 1/1, respectively). Analogously to empty matrices, the gelation kinetics of Dox filled HGs is faster by increasing the amount of the Fmoc-FF. This result may be ascribable to the capability of Fmoc-FF to gelificate in a very quick time (~2 min).35 On the contrary, we observe a faster gelation for HGs containing PEG in respect to HGs lacking polymer. This result is probably due to the additional interactions occurring between the hydrophilic PEG structure and the hydrophilic daunosamine moiety of Dox. As an alternative, gelation kinetics of hydrogels can be more accurately determined by following other structural or mechanical transitions occurring over time, related to alteration of UV-Vis absorbance, storage modulus (G’) or the dichroic signal.48 In this perspective, we recorded the CD spectra of Dox filled hydrogels at several time points in the spectral region between 300 and 200 nm (see Figure 2C–F). From the inspection of all the CD spectra, it clearly appears an evolution of the dichroic signal towards a stable state, in which we can assume that the HG has reached its final arrangement. The co-existence of several conformational states in the solution is also confirmed by isosbestic points between 220 and 240 nm and around 267 nm for PEG8-(FY)3 containing HGs. By plotting the optical density (OD) in the relative minima for each sample, we were able to extrapolate the gelation times (see Figure S3). These times, evaluated from the CD measurements, were found in good agreement with the gelation times observed following the transition from the opaque to limpid form.\nFormulation and Characterization of Dox Filled NGs The preparation of pure Fmoc-FF and mixed nanogels loaded with Dox was achieved according to the top-down methodology.41 In this approach, macroscopic discs of hydrogels (1.0%wt) loaded with Dox (0.018 mol L−1) were prepared into silicone molds according to the above-described protocol. Successively, these discs were homogenized into an aqueous solution of TWEEN®60 (polyethylene glycol sorbitan monostearate) and SPAN®60 (Sorbitan stearate) as stabilizing agents. The combination of this couple of surfactants permits to achieve a hydrophilic/lipophilic balance (HLB) value ranged between 4.7 and 14.9. Due to the similar behavior exhibited by the four mixed HG formulations in terms of DLC and stability, we decided to prepare only one of the four mixed formulations, ie, Fmoc-FF/(FY)3 (1/1, v/v). The best Dox filled NG formulations, in terms of size and stability, were obtained using TWEEN®60/SPAN®60 at 58/42 ratio (HLB= 10) or at 100/0 ratio (HLB= 14.9) for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v), respectively. Indeed, contrarily to the pure Fmoc-FF formulation, the mixed one was found unstable when prepared with a value of HLB = 10. The resulting suspensions, further submicronized using tip sonicator, were purified from free drug using size exclusion chromatography. The red coloration of purified formulations macroscopically suggested the incorporation of the anthracycline in the NGs vehicle. Due to its spectroscopic features (λabs = 480 nm; λem= 560, 590 nm), the amount of Dox encapsulated was analytically monitored and evaluated by UV-Vis spectroscopy, by checking the absorbance in the maximum at λabs= 480 nm. DLC and ER% values for pure Fmoc-FF nanogel were 0.137% and 63%, respectively. This DLC value is similar to that of the commercially available liposomal formulations Myocet (DLC=0.127) and Doxil (DLC=0.250). Therefore, the insertion of (FY)3 peptide in the preparation causes a slight decrease of both the DLC (0.093) and the ER% (45%) with respect to the pure formulation. The lower encapsulation degree observed for mixed NGs versus pure ones can be explained considering the different amount of the net negative charge present in the two nanogels. Indeed, the peptide (FY)3 has an amidated C-terminus, significantly less acidic compared to the carboxylic one, and a basic non protected N-terminal amino group that can support a positive charge after protonation. This latter phenomenon contributes to the reduction of the total negative charge in the inner sphere of NGs, which determines lower attractive forces between the drug and the peptide system. The size of empty and filled Fmoc-FF/(FY)3 NGs, measured by Dynamic Light Scattering (DLS), was 168 and 214 nm, respectively (see Figure 3). This increase in size (~22%) is comparable to the increase of dimensions previously observed for empty (174 nm) and filled (241 nm) Fmoc-FF NGs.Figure 3DLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.\nDLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.\nThe preparation of pure Fmoc-FF and mixed nanogels loaded with Dox was achieved according to the top-down methodology.41 In this approach, macroscopic discs of hydrogels (1.0%wt) loaded with Dox (0.018 mol L−1) were prepared into silicone molds according to the above-described protocol. Successively, these discs were homogenized into an aqueous solution of TWEEN®60 (polyethylene glycol sorbitan monostearate) and SPAN®60 (Sorbitan stearate) as stabilizing agents. The combination of this couple of surfactants permits to achieve a hydrophilic/lipophilic balance (HLB) value ranged between 4.7 and 14.9. Due to the similar behavior exhibited by the four mixed HG formulations in terms of DLC and stability, we decided to prepare only one of the four mixed formulations, ie, Fmoc-FF/(FY)3 (1/1, v/v). The best Dox filled NG formulations, in terms of size and stability, were obtained using TWEEN®60/SPAN®60 at 58/42 ratio (HLB= 10) or at 100/0 ratio (HLB= 14.9) for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v), respectively. Indeed, contrarily to the pure Fmoc-FF formulation, the mixed one was found unstable when prepared with a value of HLB = 10. The resulting suspensions, further submicronized using tip sonicator, were purified from free drug using size exclusion chromatography. The red coloration of purified formulations macroscopically suggested the incorporation of the anthracycline in the NGs vehicle. Due to its spectroscopic features (λabs = 480 nm; λem= 560, 590 nm), the amount of Dox encapsulated was analytically monitored and evaluated by UV-Vis spectroscopy, by checking the absorbance in the maximum at λabs= 480 nm. DLC and ER% values for pure Fmoc-FF nanogel were 0.137% and 63%, respectively. This DLC value is similar to that of the commercially available liposomal formulations Myocet (DLC=0.127) and Doxil (DLC=0.250). Therefore, the insertion of (FY)3 peptide in the preparation causes a slight decrease of both the DLC (0.093) and the ER% (45%) with respect to the pure formulation. The lower encapsulation degree observed for mixed NGs versus pure ones can be explained considering the different amount of the net negative charge present in the two nanogels. Indeed, the peptide (FY)3 has an amidated C-terminus, significantly less acidic compared to the carboxylic one, and a basic non protected N-terminal amino group that can support a positive charge after protonation. This latter phenomenon contributes to the reduction of the total negative charge in the inner sphere of NGs, which determines lower attractive forces between the drug and the peptide system. The size of empty and filled Fmoc-FF/(FY)3 NGs, measured by Dynamic Light Scattering (DLS), was 168 and 214 nm, respectively (see Figure 3). This increase in size (~22%) is comparable to the increase of dimensions previously observed for empty (174 nm) and filled (241 nm) Fmoc-FF NGs.Figure 3DLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.\nDLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.\nDrug Release from HGs and NGs Drug release from both hydrogels and nanogels was evaluated in 0.100 mol L−1 phosphate buffer solution up to 72 hours and the corresponding release profiles are reported in Figure 4A and B, respectively. Two different procedures were used for determination of Dox release from hydrogels and nanogels. In the first case, Dox or Doxil filled hydrogels, prepared into a conic tube, were directly put in contact with a double volume of physiological solution, cyclically replaced with a fresh one. Instead, in the second case, the release of Dox from nanogels was studied using a dialysis membrane immersed in phosphate buffer at 37°C. We assumed that the crossing of the free Dox through the dialysis membrane occurred quickly, thus, the overall release of the free drug from the peptide-based nanostructures to the dialysis bag medium could be considered to be rate determining for the process. The API release from the systems was considered undergone to a diffusion process. The Dox amount released was estimated by UV-Vis (at λabs= 480 nm) or by fluorescence spectroscopies (at λem= 590 nm) for HGs and NGs, respectively. The extent of drug was reported as a percentage of the ratio between the amount of released drug and the total drug initially loaded. From the inspection of Figure 4A, we can observe a similar release profile for all the Dox filled hydrogels during the first 24 hours. However, after 72 hours, each mixed gel exhibits a different release, with the lowest one for Fmoc-FF/(FY)3 (2/1) (16%). A low drug release (21%) was also observed for the other mixed HGs Fmoc-FF/(FY)3 (1/1). The same trend was also observed for the PEG8-(FY)3 containing hydrogels with a release of 19% and 28% for 2/1 and 1/1 ratios, respectively. These results agree with our expectations that PEGylation of the peptide could affect the rigidity and permeability of the hydrogels matrix and, in turn, the drug release. As expected, the amount of drug released from the hydrogel encapsulating Doxil (~8.5%) was lower than the drug released from the corresponding hydrogel (21%). In Figure 4B is reported the Dox release (%), which after 72 h, is around 20% and 40% for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels, respectively. Analogously to HGs, also for NGs the drug release is more appreciable during the first 8–12 h. The higher release observed for mixed NGs with respect to pure ones can be interpreted as a direct consequence of the lower electrostatic interactions occurring between the NG and the drug.Figure 4Drug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.\nDrug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.\nDrug release from both hydrogels and nanogels was evaluated in 0.100 mol L−1 phosphate buffer solution up to 72 hours and the corresponding release profiles are reported in Figure 4A and B, respectively. Two different procedures were used for determination of Dox release from hydrogels and nanogels. In the first case, Dox or Doxil filled hydrogels, prepared into a conic tube, were directly put in contact with a double volume of physiological solution, cyclically replaced with a fresh one. Instead, in the second case, the release of Dox from nanogels was studied using a dialysis membrane immersed in phosphate buffer at 37°C. We assumed that the crossing of the free Dox through the dialysis membrane occurred quickly, thus, the overall release of the free drug from the peptide-based nanostructures to the dialysis bag medium could be considered to be rate determining for the process. The API release from the systems was considered undergone to a diffusion process. The Dox amount released was estimated by UV-Vis (at λabs= 480 nm) or by fluorescence spectroscopies (at λem= 590 nm) for HGs and NGs, respectively. The extent of drug was reported as a percentage of the ratio between the amount of released drug and the total drug initially loaded. From the inspection of Figure 4A, we can observe a similar release profile for all the Dox filled hydrogels during the first 24 hours. However, after 72 hours, each mixed gel exhibits a different release, with the lowest one for Fmoc-FF/(FY)3 (2/1) (16%). A low drug release (21%) was also observed for the other mixed HGs Fmoc-FF/(FY)3 (1/1). The same trend was also observed for the PEG8-(FY)3 containing hydrogels with a release of 19% and 28% for 2/1 and 1/1 ratios, respectively. These results agree with our expectations that PEGylation of the peptide could affect the rigidity and permeability of the hydrogels matrix and, in turn, the drug release. As expected, the amount of drug released from the hydrogel encapsulating Doxil (~8.5%) was lower than the drug released from the corresponding hydrogel (21%). In Figure 4B is reported the Dox release (%), which after 72 h, is around 20% and 40% for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels, respectively. Analogously to HGs, also for NGs the drug release is more appreciable during the first 8–12 h. The higher release observed for mixed NGs with respect to pure ones can be interpreted as a direct consequence of the lower electrostatic interactions occurring between the NG and the drug.Figure 4Drug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.\nDrug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.\nCytotoxicity Assays Cytotoxicity of mixed Fmoc-FF/(FY)3 (1/1, v/v) hydrogel encapsulating Dox or its liposomal formulation Doxil, at the same Dox concentration, was evaluated after 24 hours of incubation on MDA-MB-231 breast cancer cell line, using MTT assay. The cytotoxicity of the free drug, as well as of the empty hydrogels, was studied in the same conditions. HGs preparation was directly achieved into the hollow plastic chamber sealed at one end with a porous membrane. This experimental setting for hydrogels allows to mimic its conditions of utilization, in which this biocompatible support loaded with the chemotherapeutic agent is grafted at the level of the tumor lesion, where a controlled and constant drug release is achieved. During our experiments, hydrogels remain in contact with the cells for all the duration of the treatment. As shown in Figure 5A, the cell viability of empty hydrogels was found to be more than 95%. This percentage of cell survival is similar to that previously observed by us for Fmoc-FF and Fmoc-FF/(FY)3 hydrogels co-incubated with the Chinese Hamster Ovarian (CHO) cell line.38 Dox loaded hydrogels significantly reduced viability of MDA-MB-231 cells after only 24 h of incubation (49%) as well as free Dox at a concentration of 1µmol L−1 (55%). This Dox concentration corresponds to its IC50 on MDA-MB-231 cells (see Supplementary Figure S4). Due to its lower drug release, hydrogels encapsulating Doxil showed a slightly lower cytotoxicity, with a cell viability of 57%.Figure 5MDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).Abbreviation: n.s., not significant.\nMDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).\nOn the other hand, Figure 5B reports the cell viability of the same cell line treated with pure Fmoc-FF and mixed Fmoc-FF(FY)3 nanogels, empty or loaded with the drug, in comparison to free Dox and Doxil. Initially, we evaluated the cytotoxicity of empty nanogels as function of the total peptide concentration at three different time points (24, 48 and 72 h). We observed that there is a significant decrease in the cell viability during the first 48 h of incubation with nanogels. After this time of incubation, cell viability improves during the next 24 h (see Figure S5). This effect may be due to a temporary inhibition of the cell cycle induced by nanogels. Based on these results, cytotoxicity of NGs encapsulating Dox was checked after 72 h of incubation. Analogously to HGs, Dox loaded nanogels are able to significantly reduce breast cancer cells viability, with a cell viability of 7% and 25% for pure and mixed hydrogels, respectively. The higher cytotoxicity of pure HG with respect to the mixed one is probably attributable to the intrinsic toxicity of the empty gel.\nCytotoxicity of mixed Fmoc-FF/(FY)3 (1/1, v/v) hydrogel encapsulating Dox or its liposomal formulation Doxil, at the same Dox concentration, was evaluated after 24 hours of incubation on MDA-MB-231 breast cancer cell line, using MTT assay. The cytotoxicity of the free drug, as well as of the empty hydrogels, was studied in the same conditions. HGs preparation was directly achieved into the hollow plastic chamber sealed at one end with a porous membrane. This experimental setting for hydrogels allows to mimic its conditions of utilization, in which this biocompatible support loaded with the chemotherapeutic agent is grafted at the level of the tumor lesion, where a controlled and constant drug release is achieved. During our experiments, hydrogels remain in contact with the cells for all the duration of the treatment. As shown in Figure 5A, the cell viability of empty hydrogels was found to be more than 95%. This percentage of cell survival is similar to that previously observed by us for Fmoc-FF and Fmoc-FF/(FY)3 hydrogels co-incubated with the Chinese Hamster Ovarian (CHO) cell line.38 Dox loaded hydrogels significantly reduced viability of MDA-MB-231 cells after only 24 h of incubation (49%) as well as free Dox at a concentration of 1µmol L−1 (55%). This Dox concentration corresponds to its IC50 on MDA-MB-231 cells (see Supplementary Figure S4). Due to its lower drug release, hydrogels encapsulating Doxil showed a slightly lower cytotoxicity, with a cell viability of 57%.Figure 5MDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).Abbreviation: n.s., not significant.\nMDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).\nOn the other hand, Figure 5B reports the cell viability of the same cell line treated with pure Fmoc-FF and mixed Fmoc-FF(FY)3 nanogels, empty or loaded with the drug, in comparison to free Dox and Doxil. Initially, we evaluated the cytotoxicity of empty nanogels as function of the total peptide concentration at three different time points (24, 48 and 72 h). We observed that there is a significant decrease in the cell viability during the first 48 h of incubation with nanogels. After this time of incubation, cell viability improves during the next 24 h (see Figure S5). This effect may be due to a temporary inhibition of the cell cycle induced by nanogels. Based on these results, cytotoxicity of NGs encapsulating Dox was checked after 72 h of incubation. Analogously to HGs, Dox loaded nanogels are able to significantly reduce breast cancer cells viability, with a cell viability of 7% and 25% for pure and mixed hydrogels, respectively. The higher cytotoxicity of pure HG with respect to the mixed one is probably attributable to the intrinsic toxicity of the empty gel.\nDetermination of the Cellular Uptake by Immunofluorescence The intracellular internalization of Dox loaded HGs and NGs was assessed using immunofluorescence analysis on MDA-MB-231 cells treated for 24 h. Internalization of free Dox and Doxil are also reported for comparison. As indicated by the overlapping of red fluorescence associated to Dox with blue signal associated to DAPI (nucleus), the free drug can internalize into the nucleus after 24 h of incubation at 37°C (Figure 6A). Differently, Dox loaded Fmoc-FF/(FY)3 mixed hydrogel induces the internalization of Dox at peri-nuclear level since Dox signal is not perfectly overlapped with DAPI, but it is also partially detectable in the cytoplasm together with the green Actin signal (Figure 6B). The same behavior was previously observed for other liposomal Dox formulations.49,50 At the same time, doxorubicin conveyed through the nanogels remains in the cytoplasm of treated cells (Figure 6C), whereas by using Doxil the drug diffuses equally between the cytoplasm and the nucleus (Figure 6D). The different intracellular distribution of the drug in Figure 6 suggests an internalization mechanism alternative to the diffusion for the drug delivered by supramolecular systems like HGs, NGs and liposomes. One of the most accredited hypotheses is that nanogels are able to bind themselves primarily to the cellular membrane and then to enter the tumor cell via the endocytosis pathway.51,52Figure 6Immunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.\nImmunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.\nThe intracellular internalization of Dox loaded HGs and NGs was assessed using immunofluorescence analysis on MDA-MB-231 cells treated for 24 h. Internalization of free Dox and Doxil are also reported for comparison. As indicated by the overlapping of red fluorescence associated to Dox with blue signal associated to DAPI (nucleus), the free drug can internalize into the nucleus after 24 h of incubation at 37°C (Figure 6A). Differently, Dox loaded Fmoc-FF/(FY)3 mixed hydrogel induces the internalization of Dox at peri-nuclear level since Dox signal is not perfectly overlapped with DAPI, but it is also partially detectable in the cytoplasm together with the green Actin signal (Figure 6B). The same behavior was previously observed for other liposomal Dox formulations.49,50 At the same time, doxorubicin conveyed through the nanogels remains in the cytoplasm of treated cells (Figure 6C), whereas by using Doxil the drug diffuses equally between the cytoplasm and the nucleus (Figure 6D). The different intracellular distribution of the drug in Figure 6 suggests an internalization mechanism alternative to the diffusion for the drug delivered by supramolecular systems like HGs, NGs and liposomes. One of the most accredited hypotheses is that nanogels are able to bind themselves primarily to the cellular membrane and then to enter the tumor cell via the endocytosis pathway.51,52Figure 6Immunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.\nImmunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.", "HGs filled with the anticancer drug Dox were prepared according to the “solvent-switch” method.41 This method consists of the addition of an aqueous solution of Dox directly into the stock peptide solution (100 mg/mL in DMSO). After few seconds of vortex, the resulting opaque metastable mixture is vertically incubated at room temperature until the formation of a limpid, translucent self-supportive hydrogel. Dox filled hydrogels Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 at two different ratios (1/1 or 2/1, v/v) were successfully formulated. Instead, in the same experimental conditions, Dox filled Fmoc-FF hydrogel appears not completely homogenous (see Figure S1). On the other hand, we observed a progressive improvement of the hydrogel homogeneity by increasing the amount (up to 0.018 mol L−1) of Dox to encapsulate (Figure S1). This behavior could be attributed to a shield effect of the charges occurring between the drug and the peptidic matrix. Although Dox filled Fmoc-FF hydrogel can be prepared, the Dox concentration is too high compared with mixed HGs and, for this reason, we did not further focus our attention on this sample. For mixed HGs, no syneresis was detected after 10 days. This specific no water-loss is an important advantage offered by HGs, thus allowing a precise modulation of water-soluble drug loaded in the system. According to this experimental evidence, we can assume that all the Dox was efficiently and quantitatively entrapped in the hydrogel matrix.\nThe resulting Dox filled hydrogels were further characterized in their xerogel form by confocal microscopy. Confocal images of one sample (Fmoc-FF/(FY)3, 1/1) are reported in Figure 2A and B. These images clearly show the characteristic intricate network of entangled fibers,47 and their red color suggests the tight interaction of Dox with the network constituents. The amount of drug we were able to encapsulate in mixed HGs was 2.32 mg/mL. This quantity corresponds to a drug loading content (DLC) of 0.440 and to an encapsulation ratio (ER%, defined as the weight percentage of drug encapsulated in the HG on the total drug added during preparation) of 100%, respectively. This high encapsulation degree can be explained as consequence of the electrostatic interactions between the positive charge on the drug and the negative one present on the C-terminus of the Fmoc-FF peptide. Analogously to free Dox, also Doxil was efficiently encapsulated into mixed hydrogels at the same drug concentration. This is surprising due to the negative Z potential value (see Figure S2) we measured for Doxil (ζ ~ -11 mV) into the experimental conditions ([Dox] =1.13 mmol L−1) used to achieve the encapsulation into the hydrogel.Figure 2Characterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nCharacterization of mixed HGs. (A and B) Confocal microscopy image of Fmoc-FF/(FY)3 (1/1) xerogel loaded with Dox. Scale bar 200 (A) and 50 µm (B), respectively. CD spectrum of Fmoc-FF/(FY)3 (1/1) loaded with Dox in the visible region between 600 and 400 nm. CD spectra of DOX filled co-assembled hydrogels Fmoc-FF/(FY)3 (2/1) (C) Fmoc-FF/(FY)3 (1/1) (D) Fmoc-FF/PEG8-(FY)3 (2/1) (E) and Fmoc-FF/PEG8-(FY)3 (1/1) (F) as function of the time.\nA macroscopic evaluation of the gelation kinetics for mixed drug-filled hydrogels can be done by simply following the opaque-to-transparent optic transition. As previously observed for the corresponding empty systems, a different time (between 20 and 40 minutes) is required for the gelation process of each mixed hydrogel.38 In particular, we observed a more rapid formation for mixed HGs containing PEG8-(FY)3 (24 and 30 minutes for 2/1 and 1/1, respectively) with respect to those containing (FY)3 (35 and 40 minutes for 2/1 and 1/1, respectively). Analogously to empty matrices, the gelation kinetics of Dox filled HGs is faster by increasing the amount of the Fmoc-FF. This result may be ascribable to the capability of Fmoc-FF to gelificate in a very quick time (~2 min).35 On the contrary, we observe a faster gelation for HGs containing PEG in respect to HGs lacking polymer. This result is probably due to the additional interactions occurring between the hydrophilic PEG structure and the hydrophilic daunosamine moiety of Dox. As an alternative, gelation kinetics of hydrogels can be more accurately determined by following other structural or mechanical transitions occurring over time, related to alteration of UV-Vis absorbance, storage modulus (G’) or the dichroic signal.48 In this perspective, we recorded the CD spectra of Dox filled hydrogels at several time points in the spectral region between 300 and 200 nm (see Figure 2C–F). From the inspection of all the CD spectra, it clearly appears an evolution of the dichroic signal towards a stable state, in which we can assume that the HG has reached its final arrangement. The co-existence of several conformational states in the solution is also confirmed by isosbestic points between 220 and 240 nm and around 267 nm for PEG8-(FY)3 containing HGs. By plotting the optical density (OD) in the relative minima for each sample, we were able to extrapolate the gelation times (see Figure S3). These times, evaluated from the CD measurements, were found in good agreement with the gelation times observed following the transition from the opaque to limpid form.", "The preparation of pure Fmoc-FF and mixed nanogels loaded with Dox was achieved according to the top-down methodology.41 In this approach, macroscopic discs of hydrogels (1.0%wt) loaded with Dox (0.018 mol L−1) were prepared into silicone molds according to the above-described protocol. Successively, these discs were homogenized into an aqueous solution of TWEEN®60 (polyethylene glycol sorbitan monostearate) and SPAN®60 (Sorbitan stearate) as stabilizing agents. The combination of this couple of surfactants permits to achieve a hydrophilic/lipophilic balance (HLB) value ranged between 4.7 and 14.9. Due to the similar behavior exhibited by the four mixed HG formulations in terms of DLC and stability, we decided to prepare only one of the four mixed formulations, ie, Fmoc-FF/(FY)3 (1/1, v/v). The best Dox filled NG formulations, in terms of size and stability, were obtained using TWEEN®60/SPAN®60 at 58/42 ratio (HLB= 10) or at 100/0 ratio (HLB= 14.9) for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v), respectively. Indeed, contrarily to the pure Fmoc-FF formulation, the mixed one was found unstable when prepared with a value of HLB = 10. The resulting suspensions, further submicronized using tip sonicator, were purified from free drug using size exclusion chromatography. The red coloration of purified formulations macroscopically suggested the incorporation of the anthracycline in the NGs vehicle. Due to its spectroscopic features (λabs = 480 nm; λem= 560, 590 nm), the amount of Dox encapsulated was analytically monitored and evaluated by UV-Vis spectroscopy, by checking the absorbance in the maximum at λabs= 480 nm. DLC and ER% values for pure Fmoc-FF nanogel were 0.137% and 63%, respectively. This DLC value is similar to that of the commercially available liposomal formulations Myocet (DLC=0.127) and Doxil (DLC=0.250). Therefore, the insertion of (FY)3 peptide in the preparation causes a slight decrease of both the DLC (0.093) and the ER% (45%) with respect to the pure formulation. The lower encapsulation degree observed for mixed NGs versus pure ones can be explained considering the different amount of the net negative charge present in the two nanogels. Indeed, the peptide (FY)3 has an amidated C-terminus, significantly less acidic compared to the carboxylic one, and a basic non protected N-terminal amino group that can support a positive charge after protonation. This latter phenomenon contributes to the reduction of the total negative charge in the inner sphere of NGs, which determines lower attractive forces between the drug and the peptide system. The size of empty and filled Fmoc-FF/(FY)3 NGs, measured by Dynamic Light Scattering (DLS), was 168 and 214 nm, respectively (see Figure 3). This increase in size (~22%) is comparable to the increase of dimensions previously observed for empty (174 nm) and filled (241 nm) Fmoc-FF NGs.Figure 3DLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.\nDLS profiles for empty and Dox filled Fmoc-FF/(FY)3 (1/1, v/v) nanogels prepared according to the top-down method.", "Drug release from both hydrogels and nanogels was evaluated in 0.100 mol L−1 phosphate buffer solution up to 72 hours and the corresponding release profiles are reported in Figure 4A and B, respectively. Two different procedures were used for determination of Dox release from hydrogels and nanogels. In the first case, Dox or Doxil filled hydrogels, prepared into a conic tube, were directly put in contact with a double volume of physiological solution, cyclically replaced with a fresh one. Instead, in the second case, the release of Dox from nanogels was studied using a dialysis membrane immersed in phosphate buffer at 37°C. We assumed that the crossing of the free Dox through the dialysis membrane occurred quickly, thus, the overall release of the free drug from the peptide-based nanostructures to the dialysis bag medium could be considered to be rate determining for the process. The API release from the systems was considered undergone to a diffusion process. The Dox amount released was estimated by UV-Vis (at λabs= 480 nm) or by fluorescence spectroscopies (at λem= 590 nm) for HGs and NGs, respectively. The extent of drug was reported as a percentage of the ratio between the amount of released drug and the total drug initially loaded. From the inspection of Figure 4A, we can observe a similar release profile for all the Dox filled hydrogels during the first 24 hours. However, after 72 hours, each mixed gel exhibits a different release, with the lowest one for Fmoc-FF/(FY)3 (2/1) (16%). A low drug release (21%) was also observed for the other mixed HGs Fmoc-FF/(FY)3 (1/1). The same trend was also observed for the PEG8-(FY)3 containing hydrogels with a release of 19% and 28% for 2/1 and 1/1 ratios, respectively. These results agree with our expectations that PEGylation of the peptide could affect the rigidity and permeability of the hydrogels matrix and, in turn, the drug release. As expected, the amount of drug released from the hydrogel encapsulating Doxil (~8.5%) was lower than the drug released from the corresponding hydrogel (21%). In Figure 4B is reported the Dox release (%), which after 72 h, is around 20% and 40% for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels, respectively. Analogously to HGs, also for NGs the drug release is more appreciable during the first 8–12 h. The higher release observed for mixed NGs with respect to pure ones can be interpreted as a direct consequence of the lower electrostatic interactions occurring between the NG and the drug.Figure 4Drug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.\nDrug release profiles for: (A) multicomponent Fmoc-FF/(FY)3 and Fmoc-FF/PEG8-(FY)3 hydrogels up 72 hours; (B) pure Fmoc-FF and mixed Fmoc-FF/(FY)3 (1/1, v/v) nanogels.", "Cytotoxicity of mixed Fmoc-FF/(FY)3 (1/1, v/v) hydrogel encapsulating Dox or its liposomal formulation Doxil, at the same Dox concentration, was evaluated after 24 hours of incubation on MDA-MB-231 breast cancer cell line, using MTT assay. The cytotoxicity of the free drug, as well as of the empty hydrogels, was studied in the same conditions. HGs preparation was directly achieved into the hollow plastic chamber sealed at one end with a porous membrane. This experimental setting for hydrogels allows to mimic its conditions of utilization, in which this biocompatible support loaded with the chemotherapeutic agent is grafted at the level of the tumor lesion, where a controlled and constant drug release is achieved. During our experiments, hydrogels remain in contact with the cells for all the duration of the treatment. As shown in Figure 5A, the cell viability of empty hydrogels was found to be more than 95%. This percentage of cell survival is similar to that previously observed by us for Fmoc-FF and Fmoc-FF/(FY)3 hydrogels co-incubated with the Chinese Hamster Ovarian (CHO) cell line.38 Dox loaded hydrogels significantly reduced viability of MDA-MB-231 cells after only 24 h of incubation (49%) as well as free Dox at a concentration of 1µmol L−1 (55%). This Dox concentration corresponds to its IC50 on MDA-MB-231 cells (see Supplementary Figure S4). Due to its lower drug release, hydrogels encapsulating Doxil showed a slightly lower cytotoxicity, with a cell viability of 57%.Figure 5MDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).Abbreviation: n.s., not significant.\nMDA-MB-231 cell survival after doxorubicin treatments. (A) MTT assay was conducted on MDA-MB-231 cells treated for 24 h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF HG (gray bar), empty Fmoc-FF/(FY)3 (1/1, v/v) HG (light gray bar), mixed Fmoc-FF/(FY)3 (1/1, v/v) HG loaded with Dox (green bar) and loaded with Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels, compared to control cells grown in their absence. (B) MTT assay was conducted on MDA-MB-231 cells treated for 72h with 1µmol·L−1 of free Dox (red bar), empty Fmoc-FF NG (blue empty bar), empty Fmoc-FF/(FY)3 (1/1, v/v) NG (green empty bar), Dox loaded Fmoc-FF nanogels (blue fill bar), Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) NG (green fill bar) and Doxil (burgundy bar) in comparison with untreated cells (black bar). Cell survival was expressed as percentage of viable cells in the presence of hydrogels and nanogels compared to control cells grown in their absence (Error represents SD of four independent experiments. *p-value<0,05. Mann-Withey t-test).\nOn the other hand, Figure 5B reports the cell viability of the same cell line treated with pure Fmoc-FF and mixed Fmoc-FF(FY)3 nanogels, empty or loaded with the drug, in comparison to free Dox and Doxil. Initially, we evaluated the cytotoxicity of empty nanogels as function of the total peptide concentration at three different time points (24, 48 and 72 h). We observed that there is a significant decrease in the cell viability during the first 48 h of incubation with nanogels. After this time of incubation, cell viability improves during the next 24 h (see Figure S5). This effect may be due to a temporary inhibition of the cell cycle induced by nanogels. Based on these results, cytotoxicity of NGs encapsulating Dox was checked after 72 h of incubation. Analogously to HGs, Dox loaded nanogels are able to significantly reduce breast cancer cells viability, with a cell viability of 7% and 25% for pure and mixed hydrogels, respectively. The higher cytotoxicity of pure HG with respect to the mixed one is probably attributable to the intrinsic toxicity of the empty gel.", "The intracellular internalization of Dox loaded HGs and NGs was assessed using immunofluorescence analysis on MDA-MB-231 cells treated for 24 h. Internalization of free Dox and Doxil are also reported for comparison. As indicated by the overlapping of red fluorescence associated to Dox with blue signal associated to DAPI (nucleus), the free drug can internalize into the nucleus after 24 h of incubation at 37°C (Figure 6A). Differently, Dox loaded Fmoc-FF/(FY)3 mixed hydrogel induces the internalization of Dox at peri-nuclear level since Dox signal is not perfectly overlapped with DAPI, but it is also partially detectable in the cytoplasm together with the green Actin signal (Figure 6B). The same behavior was previously observed for other liposomal Dox formulations.49,50 At the same time, doxorubicin conveyed through the nanogels remains in the cytoplasm of treated cells (Figure 6C), whereas by using Doxil the drug diffuses equally between the cytoplasm and the nucleus (Figure 6D). The different intracellular distribution of the drug in Figure 6 suggests an internalization mechanism alternative to the diffusion for the drug delivered by supramolecular systems like HGs, NGs and liposomes. One of the most accredited hypotheses is that nanogels are able to bind themselves primarily to the cellular membrane and then to enter the tumor cell via the endocytosis pathway.51,52Figure 6Immunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.\nImmunofluorescence assay on cells. MDA-MB-231 cells treated with: (A) free Dox; (B) Dox loaded mixed Fmoc-FF/(FY)3 (1/1, v/v) HG; (C) Dox loaded Fmoc-FF/(FY)3 (1/1, v/v) NGs and (D) Doxil. Column I corresponds to Nuclei (DAPI, blue) and B-actin (FITC, green) staining. Column II corresponds to II β-actin (FITC, green) and doxorubicin (red) staining. Column III corresponds to Nuclei (DAPI, blue) and doxorubicin (red) staining. Column IV corresponds to Overlapping of FITC, PE and DAPI channels. Magnification 63×. Scale bars 20 µm.", "The administration of drugs is often affected by several issues related to systemic toxicity, chemical instability and repeated dosing requirement. Hydrogels and nanogels can represent alternative drug delivery vehicles to conventional supramolecular structures, such as vesicles, liposomes and nanostructures already developed and in clinical use. Indeed, macroscopic tridimensional HG networks could allow transepithelial drug delivery or in situ gelation process for the formation of implants, whereas nanosized nanogels could be used for systemic, oral and pulmonary administration of drugs. Due to their high biocompatibility, good biodegradability and tunability, short or ultra-short peptides represent potential attractive alternatives for preparation of HGs and NGs with respect to natural and synthetic polymers. The peptide-based HGs and NGs here formulated are obtained by using the well-known hydrogelator Fmoc-FF alone or in combination with (FY)3 peptide or its PEGylated analogue PEG8-(FY)3 at two different ratios. NGs were prepared starting from the corresponding HGs using a top-down approach in which the macroscopic hydrogel is submicronized and stabilized with commercially available biocompatible surfactants. Due to the common structure of their inner peptidic network, both NGs and HGs allow to efficiently encapsulate Dox. The gelation kinetics (from 24 to 40 minutes) and the drug release (16–28%, after 72 h) from hydrogels are clearly influenced by the hydrogel peptide composition. Analogously, the DLC values (0.137 and 0.093 for pure and mixed NGs, respectively) and the release percentages (20–40%, after 72 h) in NGs are affected by their composition in terms of net charges. Cytotoxicity assays carried out on MDA-MB-231 breast cancer cell line pointed out a high cell viability (>95%) for empty HGs and NGs, and a reduced cell viability (49–57%) for Dox loaded HGs and NGs. Moreover, immunofluorescence assays show a different cellular localization for the Dox delivered by HGs and NGs with respect to the free Dox. Indeed, contrarily to the free Dox, localized in the nucleus, HGs and NGs allow internalization of the drug at the peri-nuclear level and in the cytoplasm, respectively. This result suggests a different internalization mechanism and a different intracellular distribution and Dox release between free Dox and Dox loaded in hydrogels/nanogels. All the in vitro data we collected and analyzed for pure Fmoc-FF and mixed Fmoc-FF/(FY)3 nanogels loaded with Dox suggest their potential use in vivo, by systemic administration, to deliver the cytotoxic drug on tumor tissues and cells. Thanks to its characteristics the new Dox loaded peptide supramolecular systems here described could be considered as promising valid alternatives to the already available liposomal Dox formulations." ]

[ "intro", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]

[ "hydrogels", "nanogels", "drug delivery", "peptide materials", "doxorubicin", "in vitro assays" ]

[ 4098, 38, 231, 208, 41, 128, 71, 79, 324, 63, 293, 434, 78, 8157, 1148, 628, 588, 1044, 516, 489 ]

[ "dox", "fmoc", "ff", "fmoc ff", "fy", "hydrogels", "drug", "ff fy", "fmoc ff fy", "mixed" ]

[ "doxorubicin formulation doxil", "cell survival doxorubicin", "dox known adriamycin", "tamoxifen combination doxorubicin", "anticancer drug dox" ]

[ "Adolescent", "Adult", "Aged", "Child", "Female", "Ghana", "Hepatitis E", "Humans", "Male", "Middle Aged", "Young Adult" ]

[ "Background", "Serological markers of HEV infection", "Case fatality rate" ]

[ "Types of viral hepatitis (A, B, C, D and E) that cause acute and/or chronic infection and liver inflammation are considered to be of significant global public health importance [1]. Hepatitis E (HEV) infection has long been regarded as being endemic in resource-poor regions such as Africa and Southeast Asia, where frequent outbreaks occur due to poor sanitation and hygiene [2, 3]. In more advanced countries, sporadic cases of acute HEV have been traced to individuals with a past history of travel to endemic areas [4]. According to the World Health Organization (WHO), more than 20 million HEV infections occur annually across the world resulting in over 3 million symptomatic cases of hepatitis E [2]. Hepatitis E does not usually develop into a carrier state and most individuals recover fully from acute HEV infections [2, 5]. In a few instances, however, it may progress into fulminant hepatitis, with a case fatality rate of around 1–2% in the general population [6] and as high as 40% in pregnant women [7].\nThe 2010 Global Burden of Disease Study estimated that there were more than 56 000 HEV-related deaths annually [8]. Although, there is wide acceptance that HEV is of significant public health importance in Ghana, the burden of the disease has not been thoroughly documented [9]. In this paper, we review studies on HEV infection in Ghana to summarise the available evidence regarding incidence and prevalence of the disease. To our knowledge, this is the first systematic review that has attempted to summarise the prevalence and incidence of HEV infection in Ghana. This paper forms part of an ongoing work aimed at documenting the burden of common viral hepatitis in Ghana. Studies on the estimated burdens of other hepatitis types arising from this broad work have been published elsewhere [10, 11].", "Among the nine serological studies, the proportion of participants testing positive for any HEV antibody (total Ig) varied from 5.8% to 71.55%. The incidence of HEV infection as determined by the proportion of participants across the seven studies showing positive results for IgM was within the range of 0.7–45.9%. The highest incidence of HEV as determined by IgM seropositivity was reported for a cohort of blood donors (45.9%) [20] and pig handlers (38.1%) [18]. The only study conducted among HIV patients, however, reported a very low incidence rate, with the proportion of participants testing positive for IgM at 0.7% [22]. The seroprevalence of HEV infection (as determined by the proportion of individuals testing positive for IgG) across the seven studies was within the range of 0–45.3%. Seroprevalence of 0% was reported in a cohort of pig handlers [18] and 45.3% in a cohort of HIV-positive adults [22].", "Only one study reporting case fatality was identified. It reported a case fatality rate of 66.7% among pregnant women [25]. However, the study involved a small sample size of only three cases." ]

[ null, null, null ]

[ "Background", "Methods", "Results", "Serological markers of HEV infection", "Case fatality rate", "Discussion", "Conclusions" ]

[ "Types of viral hepatitis (A, B, C, D and E) that cause acute and/or chronic infection and liver inflammation are considered to be of significant global public health importance [1]. Hepatitis E (HEV) infection has long been regarded as being endemic in resource-poor regions such as Africa and Southeast Asia, where frequent outbreaks occur due to poor sanitation and hygiene [2, 3]. In more advanced countries, sporadic cases of acute HEV have been traced to individuals with a past history of travel to endemic areas [4]. According to the World Health Organization (WHO), more than 20 million HEV infections occur annually across the world resulting in over 3 million symptomatic cases of hepatitis E [2]. Hepatitis E does not usually develop into a carrier state and most individuals recover fully from acute HEV infections [2, 5]. In a few instances, however, it may progress into fulminant hepatitis, with a case fatality rate of around 1–2% in the general population [6] and as high as 40% in pregnant women [7].\nThe 2010 Global Burden of Disease Study estimated that there were more than 56 000 HEV-related deaths annually [8]. Although, there is wide acceptance that HEV is of significant public health importance in Ghana, the burden of the disease has not been thoroughly documented [9]. In this paper, we review studies on HEV infection in Ghana to summarise the available evidence regarding incidence and prevalence of the disease. To our knowledge, this is the first systematic review that has attempted to summarise the prevalence and incidence of HEV infection in Ghana. This paper forms part of an ongoing work aimed at documenting the burden of common viral hepatitis in Ghana. Studies on the estimated burdens of other hepatitis types arising from this broad work have been published elsewhere [10, 11].", "We conducted a systematic review of literature on HEV infection by following the recommendations outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [12]. Studies reporting HEV infection among Ghanaians were identified by searching for articles (published up to 4th August 2016) in the PubMed, ISI Web of Science, Google Scholar, African Journals Online and the WHO African Index Medicus databases.\nThe keywords used were ‘Hepatitis E’ OR ‘Hepatitis E virus’ OR ‘HEV’ OR ‘Hepatitis E antibody’* OR ‘non-A and non-B (nAnB)’ AND ‘epidemiology’ OR ‘prevalence’ OR ‘incidence’ AND ‘Ghana’. ‘Non-A’ and non-B (nAnB)’ were included as search terms because HEV has been identified as the causative agent in many epidemic and sporadic cases of enterically transmitted non-A and non-B viral hepatitis in a number of developing countries [13, 14]. We also searched the websites of the Ministry of Health (http://www.moh-ghana.org/) and the Ghana Health Service (http://www.ghanahealthservice.org/) to identify non-indexed studies and policy reports on the subject.\nIn this review, we included serological studies that reported on the detection of antibodies to HEV-immunoglobulin G (IgG) or immunoglobulin M (IgM) among Ghanaians. The detection of IgM was considered to represent sporadic acute hepatitis cases [5, 15] and was used to estimate the incidence of HEV infection. The detection of IgG was considered to represent remote infection and was used to estimate HEV seroprevalence among the population [15]. Studies assessing case fatality rate were also included.\nA 12-point scoring system, adapted from the Downs and Black checklist [16], was used to assess the quality of the individual studies. The scoring system included questions such as whether study’s objective was clearly defined, whether participant characteristics and whether study’s conclusion was sound and based on the results. Descriptive data such as the study population, year of publication, region, setting and the proportion of people who tested positive for IgG or IgM antibodies were summarised. Due to the heterogeneity of the studies, a formal meta-analysis was not conducted.", "Figure 1 summarises the steps used to retrieve the appropriate studies. A total of 208 articles were retrieved. After the titles and abstract were screened and duplicates were removed, 13 studies were selected for full text analysis. Out of these, 10 studies on HEV in Ghana were selected for the review [9, 17–25].Fig. 1PRISMA flow chart showing the process of selecting studies\n\nPRISMA flow chart showing the process of selecting studies\nThe selected studies (see Table 1) included nine 9 serological studies that reported on the presence of HEV antibodies and one study that focused on case fatality rate. Taken together, the studies involved a total sample population of 2 894 across six regions of Ghana. The regional distribution of studies was as follows: Ashanti (n = 3), Greater Accra (n = 6) and one study that involved five regions (Ashanti, Brong-Ahafo, Upper East, Upper West and Northern). The majority of the studies (80%, n = 8) were conducted solely among urban dwellers. Of the serological studies, one was conducted exclusively among children [17], two studies were conducted among pig handlers [18, 19], two among blood donors [20, 21], one involved adult HIV patients [22], one involved pregnant women [9], one involved persons with viral haemorrhagic fever symptoms [23] and one study involved patients with suspected hepatitis [24]. The ten studies were published between 1999 and 2016, however, the sampling took place between 1993 and 2013. In terms of the quality, 80% (n = 8) of the studies were deemed as being of high quality and 20% (n = 2) were considered to be of medium quality.Table 1Studies on hepatitis E in GhanaSerological Studies reporting markers of HEV infectionStudyNo.ReferencePublication yearSampling yearDesignRegionSample sizeSettingParticipantsAge of participantsAnti -HEV (%)QualitygradeTotal IgIgGIgM1Martinson et al. [17]19991993Cross sectionalAshanti803RuralChildren6-184.4n.sn.sHigh2Adjei et al. [9]20092008Cross sectionalGreater Accra157UrbanPregnant women13-4228.6610.1918.47High3Adjei et al. [18]20092008Cross sectionalGreater Accra105UrbanPig handlers12–6538.10.038.1High4Adjei et al. [19]20102008Cross sectionalGreater Accra353UrbanPig handlers15-7038.4819.2615.58High5Tettey [20]20112008Cross sectionalGreater Accra471UrbanBlood donors18-60 (m)19–42 (f)71.5525.645.9Medium6Meldal et al. [21]2012n.sCross sectionalAshanti239UrbanBlood donorsn.s10.54.65.9High7Feldt et al. [22]20132008-10Cross sectionalAshanti402UrbanAdult HIV patients40 (±9.6)46.0245.30.7High8Bonney et al. [23]20132009-13Hospital-based surveillance studyAshanti,Brong-Ahafo, Northern,Upper West, Upper East258MixedPatients with VHF symptoms23.5a\n9.75.83.9High9Ofosu-Appiah et al. [24]20162010-11Cross-sectionalGreater Accra103UrbanPatients with suspected hepatitis33.0 ± 14.55.8n.sn.sMediumStudies reporting Case Fatality RateReferencePublication yearSampling yearDesignRegionSample sizeSettingParticipantsAge of participantsCase fatality-rateQuality grade10Bonney et al. [25]20122010Case seriesGreater Accra3UrbanPregnant women31, 31, 1766.7%High\nVHF viral haemorrhagic fever; n.s not specified; m male; f female; IgG Immunoglobulin G; IgM immunoglobulin M; HEV RNA hepatitis E virus RNA\na median\n\nStudies on hepatitis E in Ghana\n\nVHF viral haemorrhagic fever; n.s not specified; m male; f female; IgG Immunoglobulin G; IgM immunoglobulin M; HEV RNA hepatitis E virus RNA\n\na median\n Serological markers of HEV infection Among the nine serological studies, the proportion of participants testing positive for any HEV antibody (total Ig) varied from 5.8% to 71.55%. The incidence of HEV infection as determined by the proportion of participants across the seven studies showing positive results for IgM was within the range of 0.7–45.9%. The highest incidence of HEV as determined by IgM seropositivity was reported for a cohort of blood donors (45.9%) [20] and pig handlers (38.1%) [18]. The only study conducted among HIV patients, however, reported a very low incidence rate, with the proportion of participants testing positive for IgM at 0.7% [22]. The seroprevalence of HEV infection (as determined by the proportion of individuals testing positive for IgG) across the seven studies was within the range of 0–45.3%. Seroprevalence of 0% was reported in a cohort of pig handlers [18] and 45.3% in a cohort of HIV-positive adults [22].\nAmong the nine serological studies, the proportion of participants testing positive for any HEV antibody (total Ig) varied from 5.8% to 71.55%. The incidence of HEV infection as determined by the proportion of participants across the seven studies showing positive results for IgM was within the range of 0.7–45.9%. The highest incidence of HEV as determined by IgM seropositivity was reported for a cohort of blood donors (45.9%) [20] and pig handlers (38.1%) [18]. The only study conducted among HIV patients, however, reported a very low incidence rate, with the proportion of participants testing positive for IgM at 0.7% [22]. The seroprevalence of HEV infection (as determined by the proportion of individuals testing positive for IgG) across the seven studies was within the range of 0–45.3%. Seroprevalence of 0% was reported in a cohort of pig handlers [18] and 45.3% in a cohort of HIV-positive adults [22].\n Case fatality rate Only one study reporting case fatality was identified. It reported a case fatality rate of 66.7% among pregnant women [25]. However, the study involved a small sample size of only three cases.\nOnly one study reporting case fatality was identified. It reported a case fatality rate of 66.7% among pregnant women [25]. However, the study involved a small sample size of only three cases.", "Among the nine serological studies, the proportion of participants testing positive for any HEV antibody (total Ig) varied from 5.8% to 71.55%. The incidence of HEV infection as determined by the proportion of participants across the seven studies showing positive results for IgM was within the range of 0.7–45.9%. The highest incidence of HEV as determined by IgM seropositivity was reported for a cohort of blood donors (45.9%) [20] and pig handlers (38.1%) [18]. The only study conducted among HIV patients, however, reported a very low incidence rate, with the proportion of participants testing positive for IgM at 0.7% [22]. The seroprevalence of HEV infection (as determined by the proportion of individuals testing positive for IgG) across the seven studies was within the range of 0–45.3%. Seroprevalence of 0% was reported in a cohort of pig handlers [18] and 45.3% in a cohort of HIV-positive adults [22].", "Only one study reporting case fatality was identified. It reported a case fatality rate of 66.7% among pregnant women [25]. However, the study involved a small sample size of only three cases.", "To our knowledge, this is the first systematic review that has attempted to thoroughly summarise evidence on the incidence and seroprevalence of HEV infection in Ghana. Our results indicate that HEV infection in Ghana may be quite high, with between 5.8% and 71.55% of Ghanaians showing serological markers of past or current infection in the studies under review. The discrepancies in the proportion of Ghanaians testing positive for HEV serological markers across the studies could be related to geographical (regional) differences in the levels of HEV infection across the country, as well as to the varied populations studied.\nAlmost 60% of the serological studies reported a higher proportion of participants testing positive for IgM than IgG. This by inference suggests that there is a higher HEV incidence compared to prevalence among Ghanaians, which may point to frequent outbreaks than a usual or common situation (usually IgG is higher than IgM). In HIV individuals, while the incidence (based on IgM detection) appears to be very low, the prevalence is very high. This may suggest that HEV infection in HIV-positive Ghanaians persists for longer periods. This longer persistence, which can sometimes lead to chronic states, is also increasingly being reported globally [26, 27]. This trend is explained by the limited immune system capacity of HIV patients to resolve acute infections and HEV may be regarded as just another form of opportunistic infection [27].\nOnly one study [9] was conducted among pregnant women, which reported that 28.7% showed serological markers of HEV infection. The highest proportion comprised women in the third trimester – an observation that is consistent with globally reported trends [28]. HEV infection in pregnant women may pose additional health risks due to the possible vertical transmission of the disease.\nThe two studies that focused on blood donors reported that 10.5% and 71.55% tested positive for serological markers of HEV infection. While the clinical relevance of transfusion-transmitted HEV remains unclear [29], studies have demonstrated the possibility of HEV transmission through blood transfusions in endemic areas, following retrospective evaluations of transfusion recipients [30]. As such, the high levels of HEV infection among blood donors in Ghana could pose serious concerns regarding the safety of blood supply [31]. Although, Ghana has a mandatory policy that requires the screening of all donated blood, this mainly covers HIV 1 and 2, hepatitis B, hepatitis C and syphilis [32]. Therefore, attention must also be paid to HEV, as the risk of spreading the virus through blood transfusion could be high, as this review suggests.\nThe studies conducted among pig handlers reported that over 38% showed serological markers of HEV infection. Adjei et al. [19] found that among pig handlers, the risk of HEV infection correlated with the level of contact with animals or their waste, and the absence of piped water on the farms. Although the studies on pig handlers in Ghana did not report on a particular genotype, studies elsewhere have suggested possible zoonotic transmission of HEV, especially of genotypes 3 and 4 [33, 34].\nThe study by Bonney et al. [25], although based on a small sample size (n = 3), reported an HEV case fatality rate of 66.7% among pregnant women in Ghana. This rate is high when compared with the case fatality rate reported in Sudan (31.1%) [35] and the Central African Republic (14.3%) [36], although perhaps it is better to do these comparisons when evidence of the HEV genotype becomes available.\nThe seemingly high levels of HEV infection among Ghanaians may be attributed to the low level of knowledge and awareness about the transmission pathways of common viral hepatitis in the country [37, 38]. Additionally, many communities in Ghana are densely populated and sanitation conditions remain precarious, with only around 13% of Ghanaians estimated to have access to improved sanitation [39]. All this may facilitate the outbreak of HEV, particularly of genotypes 1 and 2 [2, 3, 9]. There is evidence of high faecal-oral transmission of HEV in African countries [40], which suggests that public health measures may need to address issues such inadequate access to safe water and poor sanitation in an effort to control HEV outbreaks.\nCurrently, there is no specific treatment available for HEV infection. However, a recombinant vaccine to prevent the disease has been developed and recently approved in China, but is not yet available in any other country [41]. Early studies on this vaccine have demonstrated 95% efficacy in preventing HEV infection and clinical disease [42, 43]. Once this vaccine becomes widely available, it may be particularly useful for high-risk groups such as HIV patients, in whom HEV infection remains high.\nThis study had some limitations. The main one is the relatively small number of studies identified. This highlights that research and an understanding of the HEV infection burden in Ghana is less developed compared to research on and understanding of other viral infections. The majority of the studies (80%, n = 8) involved samples from only two regions of Ghana. Subsequently, no samples from four regions (Central, Volta, Eastern and Western) were included in the studies reviewed. Additionally, there are four major genotypes of hepatitis with different modes of transmission [44] and which may require different approaches for control. The studies reviewed, however, did not report on these genotypes. Furthermore, there are challenges in diagnosing HEV infections and while there are many diagnostic assays, not all have been rigorously tested and may at times present incongruous results [45]. For instance, currently the US Food and Drug Administration has not approved any serologic test for HEV diagnosis [46].", "Although this review is based on a limited number of studies, it does highlight a high level of HEV infection among Ghanaians. Preventive measures including educational interventions, screening of all donated blood and high-risk groups (e.g., HIV patients), as well as general improvement in sanitary and living conditions are needed to reduce the burden of the disease. Additionally, further research regarding the contribution of the various HEV genotypes is urgently needed to fully understand the burden of the disease in Ghana." ]

[ null, "materials|methods", "results", null, null, "discussion", "conclusion" ]

[ "Hepatitis E", "HEV", "Infectious diseases", "Viral hepatitis", "Systematic review", "Ghana" ]

[ 360, 186, 39 ]

[ "hev", "studies", "infection", "hev infection", "reported", "hepatitis", "positive", "ghana", "study", "igm" ]

[ "estimated burdens hepatitis", "incidence hev infection", "importance hepatitis hev", "prevalence incidence hev", "viral hepatitis country" ]

[ "Alveolar Bone Loss", "Crowns", "Dental Implants", "Dental Prosthesis Design", "Dental Prosthesis, Implant-Supported", "Dental Restoration Failure", "Follow-Up Studies", "Humans", "Periodontitis", "Retrospective Studies" ]

[ "INTRODUCTION", "Appendix of Materials and Methods", "AUTHOR CONTRIBUTIONS" ]

[ "The use of dental implants in supporting prosthetic rehabilitations for partially or totally edentulous patients showed a significant increase over the last decades. Several longitudinal studies\n1\n reported implant survival percentages ranging from 92.8% to 97.1% over a period up to 10 years, demonstrating that dental implants constitute a valid treatment option for the replacement of missing teeth. At this proposal, favorable results were recently provided for the use of short implants (6 mm‐length) as an effective therapy in the rehabilitation of upper maxillary and mandibular jaw atrophies.\n2\n, \n3\n Even if available 5‐year studies\n4\n support the use of these implants as a predictable method only if splinted with other implants, other current investigations propose the possibility of their use as single crown implants,\n5\n, \n6\n, \n7\n which may be suggested as a gold standard in terms of oral hygiene procedures.\n8\n\n\nMoreover, even if dental implants seem to demonstrate high percentages of survival and stable marginal bone levels at a long‐term follow‐up, insurgence of peri‐implant diseases still represents a critical issue.\n9\n Clinical signs of peri‐implant inflammation around soft tissues (bleeding on probing, erythema, swelling, and/or suppuration), without loss of supporting bone, typically regard the onset of peri‐mucositis.\n10\n, \n11\n Once this condition turns to a non‐reversible infection, in which periodontal bacteria are involved in massively reducing marginal bone levels, peri‐implantitis can be observed. Peri‐implantitis implies a non‐linear progressive bone destruction, with a faster progression rate compared to periodontitis,\n12\n, \n13\n due to specific microorganisms present at the implant site, host defense and absence of periodontal ligament.\n9\n, \n14\n It is thus essential to establish codified protocols to prevent or efficiently manage the development of peri‐implant diseases,\n15\n as peri‐implant inflammation is directly connected with the presence of plaque deposits.\n9\n, \n10\n, \n11\n\n\nAt this regard, history of periodontitis is considered a preponderant risk factor, among others, in determining the possible development of severe peri‐implant complications.\n16\n Several reviews and metanalysis\n17\n, \n18\n, \n19\n reveal that patients with a history of periodontitis, over a long term, show higher values of probing pocket depth, greater bone loss and higher prevalence of peri‐implantitis compared to periodontally healthy patients. The evidence concerning clinical and radiographic outcomes of short and ultra‐short implants placed in patients with treated periodontitis is still scarce and lacking long‐term homogeneous follow‐ups.\n20\n, \n21\n, \n22\n, \n23\n Furthermore, it is currently highly debated the use of these implants supporting single crowns specifically in this type of patients.\n22\n Sites with deep pocketing and gingival bleeding typical of periodontitis, once properly treated to achieve a successful resolution of the inflammation, remain however characterized by an unavoidable bone loss,\n24\n for which reduced‐length implants often represent the main option to re‐establish a functional fixed rehabilitation. Despite the advantages offered by this solution, strict maintenance protocols need to be established in periodontal patients rehabilitated with implants, to avoid an additional extensive marginal bone loss,\n25\n that could be definitively harmful in case of a short or ultra‐short implant.\nBased on the limits of the already published 3‐year investigations,\n8\n, \n22\n authors here hypothesized that longer‐term assessment is required for a proper clinical evaluation of effective conditions of locking‐taper implants rehabilitated with single crowns, especially if placed in patients with history of periodontitis. The aim of this 5‐year retrospective study, which adds complementary results to another investigation on the same sample of patients,\n23\n was to assess implant survival, marginal bone loss and implant success of 333 short and ultra‐short implants restored with single crowns in the maxillary and mandibular edentulous posterior regions, respectively, placed in patients with history of periodontitis (PP), and patients without history of periodontitis (NPP). As previously described,\n23\n a rigorous value of 1 mm was set as threshold for radiographically detectable marginal bone loss, measured during the time interval from loading to 5‐year follow‐up, and compatible with implant success and absence of signs of peri‐implantitis.", "The locking‐taper (Morse taper or Morse cone) dental implant system (Bicon Dental Implants, Bicon LLC) used in this study is characterized by a convergent crest module, platform switching, plateau root‐form design, and an Integra CP surface (hydroxyapatite treated and acid‐etched).\n8\n, \n22\n, \n23\n\n\nA complete clinical and radiographic evaluation (dental and periodontal status; panoramic and periapical radiograph, cone beam computed tomography) and basic periodontal treatment was performed before implant placement. A pre‐operative medication consisting of 2 g of Augmentin (875 mg amoxicillin plus 125 mg clavulanic acid), or 1 g of Klacid (Clarithromycin 500 mg) if allergic to penicillin, was given 1 h before surgery. All surgical procedures were performed under local anesthesia, using only Articain 4% with adrenaline 1:100 000 (Citocartin) or Articain 4% with adrenaline 1:100 000 (Citocartin) associated with oral sedation (Halcion 0.25 mg).\n8\n, \n22\n, \n23\n\n\nA full‐thickness flap was performed, and a high‐speed 2.0 mm‐diameter pilot drill (with a cutting edge at the apical portion and drilling at 1100 rpm) with external saline irrigation was used to perforate the cortical plate. Final pilot drilling length was determined by measuring residual bone height and adding at least 1.0 mm to the selected implant length to allow for a sub‐crestal implant placement. Latch reamers presenting a 0.5 mm progressive increase in diameter were used at 50 rpm, without external irrigation to widen the osteotomy until the final implant diameter was reached. The selected implant was manually inserted into the osteotomy, a healing plug was placed in the implant well, and autogenous bone collected during the slow speed preparation of the osteotomy was used to fill the gap between the implant and the bony walls. The incisions were closed by single polyglycolic acid sutures (Vycril, ACE Surgical Supply Co.). A post‐operative periapical radiograph was taken, post‐operative instructions were given as well as antibiotic and analgesic prescriptions.\n8\n, \n22\n, \n23\n\n\nAfter 4‐to‐6 months the implants were surgically uncovered, healing abutments were placed, and the mucosal flaps readapted around them. After three weeks of soft tissue healing, impressions were taken using a polyether material (3 M ESPE Impregum Impression Material). Definitive single‐crown porcelain or composite restorations were placed within 2 weeks. The choice for restorative materials (porcelain or composite) was based on patients' preference, which was guided by personal economic resources in most of the cases. The technique used for the composite restorations was the Integrated Abutment Crown (IAC), in which crowns are conventionally fabricated but also extra‐orally cemented to the abutment, excess cement is removed and then the one‐piece abutment and crown are inserted.\n28\n More precisely, the IAC is a cementless restoration for single‐tooth implants, where the crown is extra‐orally chemo‐mechanically bonded to the coronal part of a titanium alloy non‐shouldered or shouldered locking‐taper abutment, reduced using carbide burs to provide for smooth surface contours and subgingival margins: in this way, the implant abutment and the crown material constitute one unit. The crown is inserted into place by mean a gentle tapping using a 250‐g mallet, by mean a crown seating tip supplied by the manufacturer and a custom‐made acrylic tapping jig to ensure accurate proper seating. When composite material was preferred for the crown, a micro‐hybrid composite containing 73% by weight micro‐fine ceramic particles, embedded in an organic polymer matrix (Ceramage, Shofu Inc), was used. In case of choice of ceramic material, a bilayer crown was planned using a zirconia framework veneered with feldspar ceramic (Ceramica Natural ZiR, Tressis Italia srl).\n23\n\n\nRecall appointments were established to manage prosthetic complications as needed, and a maintenance program was designed to provide patients a professional oral hygiene session every 4 months.\n23\n\n\nPeri‐implant bone levels were measured through digitally scanned intraoral radiographs, performed with a paralleling technique, using Rinn centering devices (Rinn XCP Posterior Aiming Ring‐Yellow, Dentsply). This was done immediately after implant placement, at healing abutment placement, at prosthetic loading, and after 5 years of loading. Measurements were taken as previously described.\n8\n, \n22\n, \n23\n The implant‐abutment interface (IAI) was taken as a reference for measurements. CBL was measured on mesial and distal sides as the linear distance between the IAI and the highest point of the interproximal bone crest parallel to the lateral sides of the implant body: a positive value was given when the crest was located coronally to the IAI and a negative value when the crest was located apically to the IAI; for every implant, at each examination interval, an average mesial‐distal value was calculated. F‐BIC was defined as the first most coronal bone‐to‐implant relationship visible at the first line of contact, on both mesial and distal sides; if F‐BIC matches with IAI, the measurement was 0; if it is located apically, the measurement was a positive value.\n8\n, \n22\n, \n23\n As described in the literature, implants were divided into two groups on the basis of presenting a CIR less than or greater than two. The crown height was measured on the radiograph immediately after the prosthetic loading, from the most occlusal point to the IAI. Anatomical crown‐to‐implant ratio (in which the fulcrum is positioned at the interface between the implant shoulder and the crown‐abutment complex) was calculated by dividing the digital length of the crown by the digital length of the implant.\n8\n, \n22\n, \n23\n\n\nMeasurements were assessed with the aid of a software program (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA) which uses a measuring tool in conjunction with a magnification tool. To correct the distortion of the radiographic image, the apparent size of each implant (measured directly on the radiograph) was compared with the actual length to determine, with adequate accuracy and precision, the amount of any changes of the crestal bone around each implant. Setting up the implant length as a known initial reference, the measurements were made to the nearest 0.01 mm. One dentist who was not involved in the treatment of the patients completed all the measurements on periapical radiographs; the observation intervals of radiographs were masked to the examiner. Before the start of the study, this investigator was calibrated for adequate intra‐ and inter‐examiner levels of reproducibility in recording the radiographic parameters.\n8\n, \n22\n, \n23\n\n\nThe calibration for intra‐examiner reproducibility was done with double recording of 25 measurements (25 implants), with an interval of 24 hours between the first and second recording. Three basic parameters, directly connected to CBL, F‐BIC and CIR, were measured on three radiographs, utilized for this purpose: mesial CBL, mesial F‐BIC and crown height, all at prosthetic loading. An average value lower than 0.20 mm was considered reliable as threshold limit from a clinically point of view. According to Bland–Altman Method,\n32\n 3 plots were obtained, with respective average values of difference between each pair of measurements, together with confidence intervals (C.I.) of: 0.01 mm (95% C.I.[−0.06;0.09]) for mesial CBL at loading, 0.00 mm (95% C.I.[−0.04;0.04])for mesial F‐BIC at loading, and − 0.01 mm (95% C.I.[−0.12;0.11]) for crown height.\nFurthermore, the abovementioned exercise, according to the same method, was repeated by another dentist (always not involved in the treatments of patients) for inter‐examiner reproducibility. The following respective average values of difference between each pair of measurements, together with C.I., were obtained: 0.00 mm (95% C.I.[−0.05;0.06]) for mesial CBL at loading, 0.00 mm (95% C.I.[−0.05;0.06]) for mesial F‐BIC at loading, and 0.01 mm (95% C.I.[−0.04;0.06]) for crown height.\nFor data collection, a database including all patients evaluated in the study was created with Microsoft Excel. All data analysis was carried out using Stata v.13.0 for Macintosh (StataCorp). The normality assumptions for continuous data were assessed by using the Shapiro–Wilk test; mean and standard deviation were reported for normally distributed data, median and interquartile range (iqr) otherwise. For categorical data, absolute frequencies, percentages and 95% confidence intervals were reported. The association between categorical variables was tested with χ\n2 test; if any of the expected values was less than 5, a Fisher's exact test was performed. The comparison between the means of continuous variables in two different times was performed by using paired Student's “t” test or Wilcoxon matched‐pairs signed‐rank test. The comparison between the means of two different groups was performed using unpaired Student's “t”, or Wilcoxon rank‐sum test. The comparison of the means among more than two groups was done using one‐way analysis of variance (ANOVA), or Kruskal‐Wallis equality‐of‐populations rank test. A multivariate analysis (logistic regression) was carried out to find factors associated with implant success. Significance level was set at 0.05.\n8\n, \n22\n, \n23\n\n\nThe study presents compliance with the STROBE checklist guidelines.\n33\n\n", "Giorgio Lombardo: Concept and design, critical revision and approval of article. Annarita Signoriello: Data collection and analysis, planning of statistics, drafting article. Alessia Pardo, Xiomara Zilena Serpa Romero, Luisa Arévalo Tovar: Data collection and analysis. Luis Armando Vila Sierra, Pier Francesco Nocini: Critical revision and approval of article. Mauro Marincola: Concept and design, critical revision and approval of article" ]

[ null, null, null ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "Appendix of Materials and Methods", "RESULTS", "DISCUSSION", "CONCLUSIONS", "AUTHOR CONTRIBUTIONS", "CONFLICT OF INTEREST" ]

[ "The use of dental implants in supporting prosthetic rehabilitations for partially or totally edentulous patients showed a significant increase over the last decades. Several longitudinal studies\n1\n reported implant survival percentages ranging from 92.8% to 97.1% over a period up to 10 years, demonstrating that dental implants constitute a valid treatment option for the replacement of missing teeth. At this proposal, favorable results were recently provided for the use of short implants (6 mm‐length) as an effective therapy in the rehabilitation of upper maxillary and mandibular jaw atrophies.\n2\n, \n3\n Even if available 5‐year studies\n4\n support the use of these implants as a predictable method only if splinted with other implants, other current investigations propose the possibility of their use as single crown implants,\n5\n, \n6\n, \n7\n which may be suggested as a gold standard in terms of oral hygiene procedures.\n8\n\n\nMoreover, even if dental implants seem to demonstrate high percentages of survival and stable marginal bone levels at a long‐term follow‐up, insurgence of peri‐implant diseases still represents a critical issue.\n9\n Clinical signs of peri‐implant inflammation around soft tissues (bleeding on probing, erythema, swelling, and/or suppuration), without loss of supporting bone, typically regard the onset of peri‐mucositis.\n10\n, \n11\n Once this condition turns to a non‐reversible infection, in which periodontal bacteria are involved in massively reducing marginal bone levels, peri‐implantitis can be observed. Peri‐implantitis implies a non‐linear progressive bone destruction, with a faster progression rate compared to periodontitis,\n12\n, \n13\n due to specific microorganisms present at the implant site, host defense and absence of periodontal ligament.\n9\n, \n14\n It is thus essential to establish codified protocols to prevent or efficiently manage the development of peri‐implant diseases,\n15\n as peri‐implant inflammation is directly connected with the presence of plaque deposits.\n9\n, \n10\n, \n11\n\n\nAt this regard, history of periodontitis is considered a preponderant risk factor, among others, in determining the possible development of severe peri‐implant complications.\n16\n Several reviews and metanalysis\n17\n, \n18\n, \n19\n reveal that patients with a history of periodontitis, over a long term, show higher values of probing pocket depth, greater bone loss and higher prevalence of peri‐implantitis compared to periodontally healthy patients. The evidence concerning clinical and radiographic outcomes of short and ultra‐short implants placed in patients with treated periodontitis is still scarce and lacking long‐term homogeneous follow‐ups.\n20\n, \n21\n, \n22\n, \n23\n Furthermore, it is currently highly debated the use of these implants supporting single crowns specifically in this type of patients.\n22\n Sites with deep pocketing and gingival bleeding typical of periodontitis, once properly treated to achieve a successful resolution of the inflammation, remain however characterized by an unavoidable bone loss,\n24\n for which reduced‐length implants often represent the main option to re‐establish a functional fixed rehabilitation. Despite the advantages offered by this solution, strict maintenance protocols need to be established in periodontal patients rehabilitated with implants, to avoid an additional extensive marginal bone loss,\n25\n that could be definitively harmful in case of a short or ultra‐short implant.\nBased on the limits of the already published 3‐year investigations,\n8\n, \n22\n authors here hypothesized that longer‐term assessment is required for a proper clinical evaluation of effective conditions of locking‐taper implants rehabilitated with single crowns, especially if placed in patients with history of periodontitis. The aim of this 5‐year retrospective study, which adds complementary results to another investigation on the same sample of patients,\n23\n was to assess implant survival, marginal bone loss and implant success of 333 short and ultra‐short implants restored with single crowns in the maxillary and mandibular edentulous posterior regions, respectively, placed in patients with history of periodontitis (PP), and patients without history of periodontitis (NPP). As previously described,\n23\n a rigorous value of 1 mm was set as threshold for radiographically detectable marginal bone loss, measured during the time interval from loading to 5‐year follow‐up, and compatible with implant success and absence of signs of peri‐implantitis.", "A retrospective study with a 5‐year follow‐up was conducted in 2020 on patients who had been referred between February 2007 and June 2015, for edentulism (tooth loss caused by trauma, caries or periodontitis) in the posterior areas of maxilla and mandible at the Dental and Maxillo‐Facial Surgery Clinic at the University of Verona (Italy). The study was approved by the University Institutional Review Board (Prot. 34 939, CROWNMAXMAND, 30/05/18). The nature and aim of the study, together with the anonymity in the scientific use of data, were clearly presented in a written informed consent form, and signed by every patient. All procedures accorded with Helsinki Declaration and good clinical practice guidelines for research on human beings.\n23\n\n\nPatients enrolled for the study, which presented the same sample of the recently published 5‐year follow‐up retrospective study,\n23\n matched the following inclusion criteria\n8\n, \n22\n, \n23\n: aged between 18 and 90 years; having had single‐tooth replacement of at least one 8.0, 6.0, or 5.0 mm locking‐taper implant supporting a single crown; had reduced alveolar bone height and had no previous consent for bone augmentation procedures; had a history of treated periodontitis, or were never affected by any form of periodontitis; and who were compliant with a regular maintenance program (professional oral hygiene sessions every 4 months).\nPatients with a history of treated periodontitis were characterized by previously assessed forms of periodontitis, corresponding to stage I, II or III, and grade A or B, according to the latest updates on classification of periodontal and peri‐implant diseases.\n10\n, \n11\n, \n23\n, \n24\n These patients were subjects following a regular maintenance program on a reduced periodontium every 3 months to ensure gingival health at the time of implant placement. On the other hand, periodontally healthy patients were subjects never affected by any form of periodontitis.\n22\n, \n23\n\n\nExclusion criteria for the study were\n8\n, \n22\n, \n23\n: presence of active infection at an implant site; ASA status III, IV,V, and VI (according to the American Society of Anesthesiologists' classification), that is, severe systemic diseases or substantive functional limitations which contraindicated implant surgery (such as drug or alcohol abuse, uncontrolled diabetes mellitus, immunosuppression or immunodepression, severe autoimmune diseases, treatment or past treatment with intravenous amino‐bisphosphonates for metastatic bone diseases, radiotherapy to head or neck within 2 years prior to treatment, history of malignancy or chemotherapy within the previous year, treatment with oral amino‐bisphosphonates for more than 3 years, morbid obesity, active hepatitis, severe renal disease, severe cardiovascular conditions, recent history of myocardial infarction (MI) or transient ischemic attack (TIA)); untreated periodontitis; poor oral hygiene and motivation; current pregnancy or lactation; heavy smoking (more than 25 cigarettes per day); severe clenching or bruxism.\nAll surgical treatments were conducted by a single clinician, as previously described\n8\n, \n22\n, \n23\n [see Appendix]. Clinical and radiographic examinations\n8\n, \n22\n, \n23\n were performed during the follow‐up 5 years from loading time, one time per year at regular intervals. The post‐surgery evaluations and the follow‐up evaluations [see Appendix] were performed by two other operators both of whom were different from the clinician who performed the surgical phase.\nImplant lengths considered in the study were 8.0, 6.0, and 5.0 mm; implant diameters were 3, 3.5, 4.0, 4.5, 5.0, 6.0 and 6.5 mm. Covariates included were: sex, age, smoking history, history of periodontitis,\n10\n, \n11\n, \n24\n ASA status, number of oral hygiene sessions per year, use of interproximal oral hygiene devices, arch involved, tooth site, prosthetic material, crown‐to‐implant ratio (CIR).\n8\n, \n22\n, \n23\n\n\nA descriptive analysis was conducted between loading time and the 5‐year follow‐up time, according to covariates. This included assessment of crestal bone level (CBL, average bone level around implants at mesial and distal sides, expressed in mm), first bone‐to‐implant contact (F‐BIC, in mm)\n8\n, \n22\n, \n23\n, \n26\n with their variations ΔCBL (average bone loss) and ΔF‐BIC (average apical shift of the “first bone‐to‐implant contact point” position) [see Appendix].\nPeri‐implant soft tissues were assessed using a periodontal probe (Florida Probe; Florida Probes Company) and applying a force of mild intensity (0.25 N). For each implant site, four parameters were assessed. The Modified Bleeding Index (mBI), and the Modified Plaque Index (mPLI), as reported in the literature by Mombelli,\n27\n were used to record the appropriate values for the mesial, central, and distal on the buccal and lingual/palatal sides of each implant. Similarly, the peri‐implant probing depths (PPD) were performed on the same six sites. The amount of keratinized tissue (KT) was assessed by measuring the distance between the zenith of the buccal gingival margin and the mucogingival line.\n23\n\n\nBiological complications after loading were also assessed at the 5‐year recall appointment. According to the latest updates,\n10\n, \n11\n peri‐implant mucositis was defined as at least one soft‐tissue peri‐implant surface with positive BOP or pus on probing, PPD ≥4 mm, and no radiographically detectable bone loss. It should be noted that visual signs of inflammation can vary and that peri‐implant mucositis can exist around implants with variable levels of bone support.\n23\n\n\nWe diagnosed peri‐implantitis when an implant had simultaneously one surface with positive BOP or pus on probing, increasing PPD compared to previous examinations or PPD ≥5 mm in the absence of the previous examination data, and presence of radiographically detectable bone loss greater than 1 mm when compared with the loading measurements. As opposed to earlier 3‐year studies on locking‐taper implants,\n8\n, \n22\n the threshold for bone loss at a longer follow‐up of 5 years was set at 1 mm. This was done in recognition of the fact that in the present study implant length was highly reduced compared to other longer implant types, for which a threshold of 2 mm can be considered acceptable.\n10\n, \n11\n, \n13\n In case of 6.0 mm and 5.0 mm‐length implants, a marginal bone loss of 2 mm, representing slightly less than half of the entire implant length, appears to be underestimated after 5 years of follow‐up.\n23\n\n\nStudy outcomes were implant survival, marginal bone loss and implant success after 5 years of follow‐up, which were assessed according to covariates.\n23\n\n\nIn regard with implant survival, failure was considered as the need for implant removal either before loading (due to absence of osseointegration), or after loading (due to excessive bone loss). Implant survival was considered as the implant's state of being in function at the five‐year follow‐up evaluation, eg., symptom‐free, without mobility, radiolucency, or bone loss so severe as to warrant implant removal.\n22\n, \n23\n, \n28\n, \n29\n\n\nAmong survived implants, implant success was defined according to the following criteria\n30\n, \n31\n and to the defined bone loss threshold: absence of persistent pain, dysesthesia, or paraesthesia in the implant area; absence of peri‐implant infection with or without suppuration; absence of perceptible mobility of the implant; and finally, absence of persistent peri‐implant bone resorption greater than 1 mm during the time interval from loading to 5‐year follow‐up. Therefore, once the failed implants are excluded, implant success can be considered for survived implants without signs of peri‐implantitis\n23\n; on the other side, survived implants with signs of peri‐implantitis are not defined as successful.\nBy way of illustration, Figures 1 and 2 report some radiographic cases.\nSingle implant placed in 4.6 site (5 × 6 mm) in a patient without history of periodontal disease: (A) Pre‐operative radiograph before implant placement; (B) Radiograph obtained at time of placement; (C) Radiograph obtained at time of loading; (D) Radiograph obtained at 5‐year follow‐up\nSingle implants placed in 2.5 and 2.6 sites (4 × 8 mm and 4.5 × 6 mm) in a patient with history of periodontal disease: (A) Pre‐operative radiograph before implant placement; (B) Radiograph obtained at time of placement; (C) Radiograph obtained at time of loading; (D) Radiograph obtained at 5‐year follow‐up\nAppendix of Materials and Methods The locking‐taper (Morse taper or Morse cone) dental implant system (Bicon Dental Implants, Bicon LLC) used in this study is characterized by a convergent crest module, platform switching, plateau root‐form design, and an Integra CP surface (hydroxyapatite treated and acid‐etched).\n8\n, \n22\n, \n23\n\n\nA complete clinical and radiographic evaluation (dental and periodontal status; panoramic and periapical radiograph, cone beam computed tomography) and basic periodontal treatment was performed before implant placement. A pre‐operative medication consisting of 2 g of Augmentin (875 mg amoxicillin plus 125 mg clavulanic acid), or 1 g of Klacid (Clarithromycin 500 mg) if allergic to penicillin, was given 1 h before surgery. All surgical procedures were performed under local anesthesia, using only Articain 4% with adrenaline 1:100 000 (Citocartin) or Articain 4% with adrenaline 1:100 000 (Citocartin) associated with oral sedation (Halcion 0.25 mg).\n8\n, \n22\n, \n23\n\n\nA full‐thickness flap was performed, and a high‐speed 2.0 mm‐diameter pilot drill (with a cutting edge at the apical portion and drilling at 1100 rpm) with external saline irrigation was used to perforate the cortical plate. Final pilot drilling length was determined by measuring residual bone height and adding at least 1.0 mm to the selected implant length to allow for a sub‐crestal implant placement. Latch reamers presenting a 0.5 mm progressive increase in diameter were used at 50 rpm, without external irrigation to widen the osteotomy until the final implant diameter was reached. The selected implant was manually inserted into the osteotomy, a healing plug was placed in the implant well, and autogenous bone collected during the slow speed preparation of the osteotomy was used to fill the gap between the implant and the bony walls. The incisions were closed by single polyglycolic acid sutures (Vycril, ACE Surgical Supply Co.). A post‐operative periapical radiograph was taken, post‐operative instructions were given as well as antibiotic and analgesic prescriptions.\n8\n, \n22\n, \n23\n\n\nAfter 4‐to‐6 months the implants were surgically uncovered, healing abutments were placed, and the mucosal flaps readapted around them. After three weeks of soft tissue healing, impressions were taken using a polyether material (3 M ESPE Impregum Impression Material). Definitive single‐crown porcelain or composite restorations were placed within 2 weeks. The choice for restorative materials (porcelain or composite) was based on patients' preference, which was guided by personal economic resources in most of the cases. The technique used for the composite restorations was the Integrated Abutment Crown (IAC), in which crowns are conventionally fabricated but also extra‐orally cemented to the abutment, excess cement is removed and then the one‐piece abutment and crown are inserted.\n28\n More precisely, the IAC is a cementless restoration for single‐tooth implants, where the crown is extra‐orally chemo‐mechanically bonded to the coronal part of a titanium alloy non‐shouldered or shouldered locking‐taper abutment, reduced using carbide burs to provide for smooth surface contours and subgingival margins: in this way, the implant abutment and the crown material constitute one unit. The crown is inserted into place by mean a gentle tapping using a 250‐g mallet, by mean a crown seating tip supplied by the manufacturer and a custom‐made acrylic tapping jig to ensure accurate proper seating. When composite material was preferred for the crown, a micro‐hybrid composite containing 73% by weight micro‐fine ceramic particles, embedded in an organic polymer matrix (Ceramage, Shofu Inc), was used. In case of choice of ceramic material, a bilayer crown was planned using a zirconia framework veneered with feldspar ceramic (Ceramica Natural ZiR, Tressis Italia srl).\n23\n\n\nRecall appointments were established to manage prosthetic complications as needed, and a maintenance program was designed to provide patients a professional oral hygiene session every 4 months.\n23\n\n\nPeri‐implant bone levels were measured through digitally scanned intraoral radiographs, performed with a paralleling technique, using Rinn centering devices (Rinn XCP Posterior Aiming Ring‐Yellow, Dentsply). This was done immediately after implant placement, at healing abutment placement, at prosthetic loading, and after 5 years of loading. Measurements were taken as previously described.\n8\n, \n22\n, \n23\n The implant‐abutment interface (IAI) was taken as a reference for measurements. CBL was measured on mesial and distal sides as the linear distance between the IAI and the highest point of the interproximal bone crest parallel to the lateral sides of the implant body: a positive value was given when the crest was located coronally to the IAI and a negative value when the crest was located apically to the IAI; for every implant, at each examination interval, an average mesial‐distal value was calculated. F‐BIC was defined as the first most coronal bone‐to‐implant relationship visible at the first line of contact, on both mesial and distal sides; if F‐BIC matches with IAI, the measurement was 0; if it is located apically, the measurement was a positive value.\n8\n, \n22\n, \n23\n As described in the literature, implants were divided into two groups on the basis of presenting a CIR less than or greater than two. The crown height was measured on the radiograph immediately after the prosthetic loading, from the most occlusal point to the IAI. Anatomical crown‐to‐implant ratio (in which the fulcrum is positioned at the interface between the implant shoulder and the crown‐abutment complex) was calculated by dividing the digital length of the crown by the digital length of the implant.\n8\n, \n22\n, \n23\n\n\nMeasurements were assessed with the aid of a software program (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA) which uses a measuring tool in conjunction with a magnification tool. To correct the distortion of the radiographic image, the apparent size of each implant (measured directly on the radiograph) was compared with the actual length to determine, with adequate accuracy and precision, the amount of any changes of the crestal bone around each implant. Setting up the implant length as a known initial reference, the measurements were made to the nearest 0.01 mm. One dentist who was not involved in the treatment of the patients completed all the measurements on periapical radiographs; the observation intervals of radiographs were masked to the examiner. Before the start of the study, this investigator was calibrated for adequate intra‐ and inter‐examiner levels of reproducibility in recording the radiographic parameters.\n8\n, \n22\n, \n23\n\n\nThe calibration for intra‐examiner reproducibility was done with double recording of 25 measurements (25 implants), with an interval of 24 hours between the first and second recording. Three basic parameters, directly connected to CBL, F‐BIC and CIR, were measured on three radiographs, utilized for this purpose: mesial CBL, mesial F‐BIC and crown height, all at prosthetic loading. An average value lower than 0.20 mm was considered reliable as threshold limit from a clinically point of view. According to Bland–Altman Method,\n32\n 3 plots were obtained, with respective average values of difference between each pair of measurements, together with confidence intervals (C.I.) of: 0.01 mm (95% C.I.[−0.06;0.09]) for mesial CBL at loading, 0.00 mm (95% C.I.[−0.04;0.04])for mesial F‐BIC at loading, and − 0.01 mm (95% C.I.[−0.12;0.11]) for crown height.\nFurthermore, the abovementioned exercise, according to the same method, was repeated by another dentist (always not involved in the treatments of patients) for inter‐examiner reproducibility. The following respective average values of difference between each pair of measurements, together with C.I., were obtained: 0.00 mm (95% C.I.[−0.05;0.06]) for mesial CBL at loading, 0.00 mm (95% C.I.[−0.05;0.06]) for mesial F‐BIC at loading, and 0.01 mm (95% C.I.[−0.04;0.06]) for crown height.\nFor data collection, a database including all patients evaluated in the study was created with Microsoft Excel. All data analysis was carried out using Stata v.13.0 for Macintosh (StataCorp). The normality assumptions for continuous data were assessed by using the Shapiro–Wilk test; mean and standard deviation were reported for normally distributed data, median and interquartile range (iqr) otherwise. For categorical data, absolute frequencies, percentages and 95% confidence intervals were reported. The association between categorical variables was tested with χ\n2 test; if any of the expected values was less than 5, a Fisher's exact test was performed. The comparison between the means of continuous variables in two different times was performed by using paired Student's “t” test or Wilcoxon matched‐pairs signed‐rank test. The comparison between the means of two different groups was performed using unpaired Student's “t”, or Wilcoxon rank‐sum test. The comparison of the means among more than two groups was done using one‐way analysis of variance (ANOVA), or Kruskal‐Wallis equality‐of‐populations rank test. A multivariate analysis (logistic regression) was carried out to find factors associated with implant success. Significance level was set at 0.05.\n8\n, \n22\n, \n23\n\n\nThe study presents compliance with the STROBE checklist guidelines.\n33\n\n\nThe locking‐taper (Morse taper or Morse cone) dental implant system (Bicon Dental Implants, Bicon LLC) used in this study is characterized by a convergent crest module, platform switching, plateau root‐form design, and an Integra CP surface (hydroxyapatite treated and acid‐etched).\n8\n, \n22\n, \n23\n\n\nA complete clinical and radiographic evaluation (dental and periodontal status; panoramic and periapical radiograph, cone beam computed tomography) and basic periodontal treatment was performed before implant placement. A pre‐operative medication consisting of 2 g of Augmentin (875 mg amoxicillin plus 125 mg clavulanic acid), or 1 g of Klacid (Clarithromycin 500 mg) if allergic to penicillin, was given 1 h before surgery. All surgical procedures were performed under local anesthesia, using only Articain 4% with adrenaline 1:100 000 (Citocartin) or Articain 4% with adrenaline 1:100 000 (Citocartin) associated with oral sedation (Halcion 0.25 mg).\n8\n, \n22\n, \n23\n\n\nA full‐thickness flap was performed, and a high‐speed 2.0 mm‐diameter pilot drill (with a cutting edge at the apical portion and drilling at 1100 rpm) with external saline irrigation was used to perforate the cortical plate. Final pilot drilling length was determined by measuring residual bone height and adding at least 1.0 mm to the selected implant length to allow for a sub‐crestal implant placement. Latch reamers presenting a 0.5 mm progressive increase in diameter were used at 50 rpm, without external irrigation to widen the osteotomy until the final implant diameter was reached. The selected implant was manually inserted into the osteotomy, a healing plug was placed in the implant well, and autogenous bone collected during the slow speed preparation of the osteotomy was used to fill the gap between the implant and the bony walls. The incisions were closed by single polyglycolic acid sutures (Vycril, ACE Surgical Supply Co.). A post‐operative periapical radiograph was taken, post‐operative instructions were given as well as antibiotic and analgesic prescriptions.\n8\n, \n22\n, \n23\n\n\nAfter 4‐to‐6 months the implants were surgically uncovered, healing abutments were placed, and the mucosal flaps readapted around them. After three weeks of soft tissue healing, impressions were taken using a polyether material (3 M ESPE Impregum Impression Material). Definitive single‐crown porcelain or composite restorations were placed within 2 weeks. The choice for restorative materials (porcelain or composite) was based on patients' preference, which was guided by personal economic resources in most of the cases. The technique used for the composite restorations was the Integrated Abutment Crown (IAC), in which crowns are conventionally fabricated but also extra‐orally cemented to the abutment, excess cement is removed and then the one‐piece abutment and crown are inserted.\n28\n More precisely, the IAC is a cementless restoration for single‐tooth implants, where the crown is extra‐orally chemo‐mechanically bonded to the coronal part of a titanium alloy non‐shouldered or shouldered locking‐taper abutment, reduced using carbide burs to provide for smooth surface contours and subgingival margins: in this way, the implant abutment and the crown material constitute one unit. The crown is inserted into place by mean a gentle tapping using a 250‐g mallet, by mean a crown seating tip supplied by the manufacturer and a custom‐made acrylic tapping jig to ensure accurate proper seating. When composite material was preferred for the crown, a micro‐hybrid composite containing 73% by weight micro‐fine ceramic particles, embedded in an organic polymer matrix (Ceramage, Shofu Inc), was used. In case of choice of ceramic material, a bilayer crown was planned using a zirconia framework veneered with feldspar ceramic (Ceramica Natural ZiR, Tressis Italia srl).\n23\n\n\nRecall appointments were established to manage prosthetic complications as needed, and a maintenance program was designed to provide patients a professional oral hygiene session every 4 months.\n23\n\n\nPeri‐implant bone levels were measured through digitally scanned intraoral radiographs, performed with a paralleling technique, using Rinn centering devices (Rinn XCP Posterior Aiming Ring‐Yellow, Dentsply). This was done immediately after implant placement, at healing abutment placement, at prosthetic loading, and after 5 years of loading. Measurements were taken as previously described.\n8\n, \n22\n, \n23\n The implant‐abutment interface (IAI) was taken as a reference for measurements. CBL was measured on mesial and distal sides as the linear distance between the IAI and the highest point of the interproximal bone crest parallel to the lateral sides of the implant body: a positive value was given when the crest was located coronally to the IAI and a negative value when the crest was located apically to the IAI; for every implant, at each examination interval, an average mesial‐distal value was calculated. F‐BIC was defined as the first most coronal bone‐to‐implant relationship visible at the first line of contact, on both mesial and distal sides; if F‐BIC matches with IAI, the measurement was 0; if it is located apically, the measurement was a positive value.\n8\n, \n22\n, \n23\n As described in the literature, implants were divided into two groups on the basis of presenting a CIR less than or greater than two. The crown height was measured on the radiograph immediately after the prosthetic loading, from the most occlusal point to the IAI. Anatomical crown‐to‐implant ratio (in which the fulcrum is positioned at the interface between the implant shoulder and the crown‐abutment complex) was calculated by dividing the digital length of the crown by the digital length of the implant.\n8\n, \n22\n, \n23\n\n\nMeasurements were assessed with the aid of a software program (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA) which uses a measuring tool in conjunction with a magnification tool. To correct the distortion of the radiographic image, the apparent size of each implant (measured directly on the radiograph) was compared with the actual length to determine, with adequate accuracy and precision, the amount of any changes of the crestal bone around each implant. Setting up the implant length as a known initial reference, the measurements were made to the nearest 0.01 mm. One dentist who was not involved in the treatment of the patients completed all the measurements on periapical radiographs; the observation intervals of radiographs were masked to the examiner. Before the start of the study, this investigator was calibrated for adequate intra‐ and inter‐examiner levels of reproducibility in recording the radiographic parameters.\n8\n, \n22\n, \n23\n\n\nThe calibration for intra‐examiner reproducibility was done with double recording of 25 measurements (25 implants), with an interval of 24 hours between the first and second recording. Three basic parameters, directly connected to CBL, F‐BIC and CIR, were measured on three radiographs, utilized for this purpose: mesial CBL, mesial F‐BIC and crown height, all at prosthetic loading. An average value lower than 0.20 mm was considered reliable as threshold limit from a clinically point of view. According to Bland–Altman Method,\n32\n 3 plots were obtained, with respective average values of difference between each pair of measurements, together with confidence intervals (C.I.) of: 0.01 mm (95% C.I.[−0.06;0.09]) for mesial CBL at loading, 0.00 mm (95% C.I.[−0.04;0.04])for mesial F‐BIC at loading, and − 0.01 mm (95% C.I.[−0.12;0.11]) for crown height.\nFurthermore, the abovementioned exercise, according to the same method, was repeated by another dentist (always not involved in the treatments of patients) for inter‐examiner reproducibility. The following respective average values of difference between each pair of measurements, together with C.I., were obtained: 0.00 mm (95% C.I.[−0.05;0.06]) for mesial CBL at loading, 0.00 mm (95% C.I.[−0.05;0.06]) for mesial F‐BIC at loading, and 0.01 mm (95% C.I.[−0.04;0.06]) for crown height.\nFor data collection, a database including all patients evaluated in the study was created with Microsoft Excel. All data analysis was carried out using Stata v.13.0 for Macintosh (StataCorp). The normality assumptions for continuous data were assessed by using the Shapiro–Wilk test; mean and standard deviation were reported for normally distributed data, median and interquartile range (iqr) otherwise. For categorical data, absolute frequencies, percentages and 95% confidence intervals were reported. The association between categorical variables was tested with χ\n2 test; if any of the expected values was less than 5, a Fisher's exact test was performed. The comparison between the means of continuous variables in two different times was performed by using paired Student's “t” test or Wilcoxon matched‐pairs signed‐rank test. The comparison between the means of two different groups was performed using unpaired Student's “t”, or Wilcoxon rank‐sum test. The comparison of the means among more than two groups was done using one‐way analysis of variance (ANOVA), or Kruskal‐Wallis equality‐of‐populations rank test. A multivariate analysis (logistic regression) was carried out to find factors associated with implant success. Significance level was set at 0.05.\n8\n, \n22\n, \n23\n\n\nThe study presents compliance with the STROBE checklist guidelines.\n33\n\n", "The locking‐taper (Morse taper or Morse cone) dental implant system (Bicon Dental Implants, Bicon LLC) used in this study is characterized by a convergent crest module, platform switching, plateau root‐form design, and an Integra CP surface (hydroxyapatite treated and acid‐etched).\n8\n, \n22\n, \n23\n\n\nA complete clinical and radiographic evaluation (dental and periodontal status; panoramic and periapical radiograph, cone beam computed tomography) and basic periodontal treatment was performed before implant placement. A pre‐operative medication consisting of 2 g of Augmentin (875 mg amoxicillin plus 125 mg clavulanic acid), or 1 g of Klacid (Clarithromycin 500 mg) if allergic to penicillin, was given 1 h before surgery. All surgical procedures were performed under local anesthesia, using only Articain 4% with adrenaline 1:100 000 (Citocartin) or Articain 4% with adrenaline 1:100 000 (Citocartin) associated with oral sedation (Halcion 0.25 mg).\n8\n, \n22\n, \n23\n\n\nA full‐thickness flap was performed, and a high‐speed 2.0 mm‐diameter pilot drill (with a cutting edge at the apical portion and drilling at 1100 rpm) with external saline irrigation was used to perforate the cortical plate. Final pilot drilling length was determined by measuring residual bone height and adding at least 1.0 mm to the selected implant length to allow for a sub‐crestal implant placement. Latch reamers presenting a 0.5 mm progressive increase in diameter were used at 50 rpm, without external irrigation to widen the osteotomy until the final implant diameter was reached. The selected implant was manually inserted into the osteotomy, a healing plug was placed in the implant well, and autogenous bone collected during the slow speed preparation of the osteotomy was used to fill the gap between the implant and the bony walls. The incisions were closed by single polyglycolic acid sutures (Vycril, ACE Surgical Supply Co.). A post‐operative periapical radiograph was taken, post‐operative instructions were given as well as antibiotic and analgesic prescriptions.\n8\n, \n22\n, \n23\n\n\nAfter 4‐to‐6 months the implants were surgically uncovered, healing abutments were placed, and the mucosal flaps readapted around them. After three weeks of soft tissue healing, impressions were taken using a polyether material (3 M ESPE Impregum Impression Material). Definitive single‐crown porcelain or composite restorations were placed within 2 weeks. The choice for restorative materials (porcelain or composite) was based on patients' preference, which was guided by personal economic resources in most of the cases. The technique used for the composite restorations was the Integrated Abutment Crown (IAC), in which crowns are conventionally fabricated but also extra‐orally cemented to the abutment, excess cement is removed and then the one‐piece abutment and crown are inserted.\n28\n More precisely, the IAC is a cementless restoration for single‐tooth implants, where the crown is extra‐orally chemo‐mechanically bonded to the coronal part of a titanium alloy non‐shouldered or shouldered locking‐taper abutment, reduced using carbide burs to provide for smooth surface contours and subgingival margins: in this way, the implant abutment and the crown material constitute one unit. The crown is inserted into place by mean a gentle tapping using a 250‐g mallet, by mean a crown seating tip supplied by the manufacturer and a custom‐made acrylic tapping jig to ensure accurate proper seating. When composite material was preferred for the crown, a micro‐hybrid composite containing 73% by weight micro‐fine ceramic particles, embedded in an organic polymer matrix (Ceramage, Shofu Inc), was used. In case of choice of ceramic material, a bilayer crown was planned using a zirconia framework veneered with feldspar ceramic (Ceramica Natural ZiR, Tressis Italia srl).\n23\n\n\nRecall appointments were established to manage prosthetic complications as needed, and a maintenance program was designed to provide patients a professional oral hygiene session every 4 months.\n23\n\n\nPeri‐implant bone levels were measured through digitally scanned intraoral radiographs, performed with a paralleling technique, using Rinn centering devices (Rinn XCP Posterior Aiming Ring‐Yellow, Dentsply). This was done immediately after implant placement, at healing abutment placement, at prosthetic loading, and after 5 years of loading. Measurements were taken as previously described.\n8\n, \n22\n, \n23\n The implant‐abutment interface (IAI) was taken as a reference for measurements. CBL was measured on mesial and distal sides as the linear distance between the IAI and the highest point of the interproximal bone crest parallel to the lateral sides of the implant body: a positive value was given when the crest was located coronally to the IAI and a negative value when the crest was located apically to the IAI; for every implant, at each examination interval, an average mesial‐distal value was calculated. F‐BIC was defined as the first most coronal bone‐to‐implant relationship visible at the first line of contact, on both mesial and distal sides; if F‐BIC matches with IAI, the measurement was 0; if it is located apically, the measurement was a positive value.\n8\n, \n22\n, \n23\n As described in the literature, implants were divided into two groups on the basis of presenting a CIR less than or greater than two. The crown height was measured on the radiograph immediately after the prosthetic loading, from the most occlusal point to the IAI. Anatomical crown‐to‐implant ratio (in which the fulcrum is positioned at the interface between the implant shoulder and the crown‐abutment complex) was calculated by dividing the digital length of the crown by the digital length of the implant.\n8\n, \n22\n, \n23\n\n\nMeasurements were assessed with the aid of a software program (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA) which uses a measuring tool in conjunction with a magnification tool. To correct the distortion of the radiographic image, the apparent size of each implant (measured directly on the radiograph) was compared with the actual length to determine, with adequate accuracy and precision, the amount of any changes of the crestal bone around each implant. Setting up the implant length as a known initial reference, the measurements were made to the nearest 0.01 mm. One dentist who was not involved in the treatment of the patients completed all the measurements on periapical radiographs; the observation intervals of radiographs were masked to the examiner. Before the start of the study, this investigator was calibrated for adequate intra‐ and inter‐examiner levels of reproducibility in recording the radiographic parameters.\n8\n, \n22\n, \n23\n\n\nThe calibration for intra‐examiner reproducibility was done with double recording of 25 measurements (25 implants), with an interval of 24 hours between the first and second recording. Three basic parameters, directly connected to CBL, F‐BIC and CIR, were measured on three radiographs, utilized for this purpose: mesial CBL, mesial F‐BIC and crown height, all at prosthetic loading. An average value lower than 0.20 mm was considered reliable as threshold limit from a clinically point of view. According to Bland–Altman Method,\n32\n 3 plots were obtained, with respective average values of difference between each pair of measurements, together with confidence intervals (C.I.) of: 0.01 mm (95% C.I.[−0.06;0.09]) for mesial CBL at loading, 0.00 mm (95% C.I.[−0.04;0.04])for mesial F‐BIC at loading, and − 0.01 mm (95% C.I.[−0.12;0.11]) for crown height.\nFurthermore, the abovementioned exercise, according to the same method, was repeated by another dentist (always not involved in the treatments of patients) for inter‐examiner reproducibility. The following respective average values of difference between each pair of measurements, together with C.I., were obtained: 0.00 mm (95% C.I.[−0.05;0.06]) for mesial CBL at loading, 0.00 mm (95% C.I.[−0.05;0.06]) for mesial F‐BIC at loading, and 0.01 mm (95% C.I.[−0.04;0.06]) for crown height.\nFor data collection, a database including all patients evaluated in the study was created with Microsoft Excel. All data analysis was carried out using Stata v.13.0 for Macintosh (StataCorp). The normality assumptions for continuous data were assessed by using the Shapiro–Wilk test; mean and standard deviation were reported for normally distributed data, median and interquartile range (iqr) otherwise. For categorical data, absolute frequencies, percentages and 95% confidence intervals were reported. The association between categorical variables was tested with χ\n2 test; if any of the expected values was less than 5, a Fisher's exact test was performed. The comparison between the means of continuous variables in two different times was performed by using paired Student's “t” test or Wilcoxon matched‐pairs signed‐rank test. The comparison between the means of two different groups was performed using unpaired Student's “t”, or Wilcoxon rank‐sum test. The comparison of the means among more than two groups was done using one‐way analysis of variance (ANOVA), or Kruskal‐Wallis equality‐of‐populations rank test. A multivariate analysis (logistic regression) was carried out to find factors associated with implant success. Significance level was set at 0.05.\n8\n, \n22\n, \n23\n\n\nThe study presents compliance with the STROBE checklist guidelines.\n33\n\n", "Of the 333 implants placed in 142 patients (65 men and 77 women), 213 (63.96%) and 120 (36.04%) were respectively positioned in PP and NPP.\n23\n Furthermore, CIR in PP and NPP was respectively 2.01 ± 0.87 (range 0.91–3.81) and 1.78 ± 0.79 (range 1.05–3.52), with statistically significant differences among groups (p = 0.002). The implants distribution, analyzed according to PP and NPP, is presented in Table 1.\nOverall implants and PP/NPP‐groups distribution according to study variables\n\nNote: Age at follow‐up and oral professional hygiene/year are presented as mean ± standard deviation and median (iqr); for all other variables, values are presented as n (%).\nAbbreviations: NS, not statistically significant; d.f., degrees of freedom.\nAs one early failure in one patient was assessed, the overall implant‐based survival at 60‐month follow‐up was 96.1%: 96.67% (116/120) in NPP and 95.77% (204/213) in PP, without significant differences between groups (p = 0.77).\n23\n Peri‐implantitis revealed to be the cause of failure in six out of nine cases (66.7%) of failed implants in PP, but in none of failed implants in NPP. The distribution of different variables related to failed implants is reported in Table 2.\nFailures features and cause of failures\nAbbreviations: ep, error of placement; peri, peri‐implantitis; rp, retrograde peri‐implantitis; fp, fracture of the post of the abutment.\nΔCBL was 0.61 (0.87) mm and 0.74 (1.54) mm respectively in NPP and PP, with statistically significant differences between groups (p = 0.04). ΔF‐BIC was 0.01 (0.43) mm and 0.09 (0.79) mm respectively in NPP and PP, with no statistically significant differences between groups (p = 0.08).\n23\n\n\nTable 3 reports soft tissues conditions (PPD, mBI, mPLI and KT) at 5‐year recall appointment, according to length‐groups, arch‐groups or PP/NPP‐groups. Peri‐implantitis was found in 3 (2.59%) and 16 (7.84%) cases in NPP and PP respectively, with no statistically significant differences between groups (p = 0.08).\n23\n The overall implant‐based success at 60‐month follow‐up was 94.06% (301/320)\n23\n:92.16% for PP (respectively 36.7% in 8‐mm length, 32.45% in 6‐mm length and 30.85% in 5‐mm length);97.41% for NPP (respectively 43.36% in 8‐mm length, 36.28% in 6‐mm length and 20.36% in 5‐mm length).Regarding prevalence of mucositis, no significant differences (p < 0.05) were found between groups regarding PP/NPP, length or arch‐groups (Table 4).\n92.16% for PP (respectively 36.7% in 8‐mm length, 32.45% in 6‐mm length and 30.85% in 5‐mm length);\n97.41% for NPP (respectively 43.36% in 8‐mm length, 36.28% in 6‐mm length and 20.36% in 5‐mm length).\nOverall soft tissues indices (mBI, mPLI, PPD [mm], KT [mm]) according to PP/NPP‐groups, length‐groups and arch‐groups\n\nNote: mBI, mPLI, PPD and KT are presented as median (iqr).\nAbbreviations: NS, not statistically significant; d.f., degrees of freedom.\nPrevalence of peri‐mucositis according to PP/NPP‐groups, length‐groups and arch‐groups\n\nNote: For all variables, values are presented as n (%).\nAbbreviations: NS, not statistically significant; d.f., degrees of freedom; C.I., confidence interval.", "Over the last decades, implant placement has become a widespread and predictable methodology of treatment for partially edentulous posterior regions. As sufficient bone volume available for standard‐length implant rehabilitation is not always present, the use of short implants (<6‐mm) is frequently chosen among clinicians as a minimally invasive treatment modality, for the reduction of number of major surgeries, potential morbidity, times and costs.\n34\n, \n35\n, \n36\n, \n37\n Recent metanalysis reported that short implants have almost comparable outcomes, in terms of implant survival, marginal bone loss and peri‐implant complications, to standard‐sized dental implant (>6‐mm) and may be considered a valid alternative therapy to augmentation procedures for maxillary and mandibular jaw atrophies.\n2\n, \n3\n, \n6\n, \n38\n, \n39\n\n\nNevertheless, while a large amount of research data on long‐term success of standard and short implants in periodontally healthy patients is currently available, only few short‐term studies (3‐year follow‐up) are present in the literature about the influence of periodontitis on the implant survival of short implants in PP, and they do not allow for definitive conclusions.\n20\n, \n21\n, \n40\n, \n41\n, \n42\n Han et al.,\n40\n in a retrospective study in which two or three splinted implants 6 mm‐length and 4 mm‐diameter were placed in each patient, found a one‐year survival rate of 95.8% for 95 short implants; the great majority of participants had chronic periodontitis (77.1%). Correia et al.,\n41\n in a 3‐year retrospective study on 202 patients and 689 implants of different types, design and implant‐abutment connections, of which 45.2% (301 implants) rehabilitated with single crowns, declared an overall implant survival of 97.3% for 116 short implants placed in NPP and of 93.1% for 156 placed in PP; the latter seemed to present a greater, even if not statistically significant, risk of implant failure after 3 years, when compared with implants placed in NPP. The present study, which, to the best of our knowledge, is the first study concerning short and ultra‐short implants supporting single crowns in PP, 333 implants, characterized by a plateau‐design and a locking‐taper implant‐abutment connection, showed an implant survival of 96.1% after 5 years of loading. These results are consistent with the study of Hasanoglu et al.,\n21\n a retrospective evaluation with an average follow‐up of 33.5 months, which reported an overall implant survival of 95.86%, for 460 short implants (4‐to‐9 mm in length) of different brands.\nAfter splitting the sample of placed implants in NPP and PP‐groups (36.04% and 63.96% respectively), significantly different implant survival were not found (96.67% and 95.77% for NPP and PP, p = 0.77). Although some authors in the literature recommend not to use short implants to support single crowns, due to the possible risk of overloading for high CIR,\n7\n, \n43\n, \n44\n in the present study, not only short implants, but also ultra‐short implants, offered a favorable implant survival after 5 years; moreover, outcomes were not different among implant length‐groups and PP/NPP‐groups. These results are consistent with those found in a previous 3‐year retrospective study of the same authors,\n22\n where, with the same type of implant utilized, a survival rate of 98.08% for 208 implants placed in 77 PP and of 96.61% for 118 implants placed in 63 NPP was assessed.\nThe favorable bone levels stability showed in this study by both short and ultra‐short implants may be explained by the specific macro‐design of these implants, characterized by the presence of a screw‐less, 3° locking‐taper implant‐abutment connection. This connection confers an impervious seal to microbial penetration or infiltration,\n45\n and a greater mechanical stability to the implant‐crown assembly, with absence of micro movements and micro gaps at the implant‐abutment interface, which lead to minimal bone resorption.\n6\n, \n7\n The plateau‐design allows from an initial woven bone formation at the healing chambers and further bone morphologic evolution toward a haversian‐like configuration that over time increases significantly in mechanical properties.\n22\n, \n23\n, \n45\n, \n46\n, \n47\n In this way, adjacent bone is hardly loaded at levels that could exceed the minimum effective strain necessary for bone modeling and remodeling.\n48\n, \n49\n Nevertheless, after 5 years of loading, greater values of marginal bone loss and apical shift of F‐BIC were found in PP. Even if these differences can be considered as minimally relevant, ΔCBL was 0.61 and 0.74 mm respectively in NPP and PP, with statistically significant differences between groups (p = 0.04).\nDespite the promising outcomes in term of survival rate and bone levels, in accordance with the general consensus existing in literature,\n50\n indicating that PP are at higher risk for peri‐implantitis and marginal bone loss compared to NPP, the present study showed an overall negative influence of history of periodontitis on implant survival and success. Peri‐implantitis revealed to be the cause of failure in six out of nine cases (66.7%) of failed implants in PP, but in none of failed implants in NPP, which were lost for other causes (error of placement, retrograde peri‐implantitis,\n13\n mechanical fracture of the abutment's component). Authors consider this outcome as clinically relevant, underlying that history of periodontitis seems to be connected to peri‐implantitis in terms of cause of failure. This finding is also consistent with the report of Hasanoglu,\n21\n who found peri‐implantitis as the greater cause of failure in implants placed in PP, but not in NPP. In regard with retrograde peri‐implantitis, it is reported as a periapical radiographic lesion different from peri‐implant infection at sites with deepened PPD, and directly correlated, in most of cases, with existing periapical endodontic lesions at adjacent teeth.\n13\n\n\nWith the threshold limit for defining peri‐implantitis set at 1 mm of bone resorption 5 years after loading, among 320 survived implants, 19 (5.94%) presented peri‐implantitis, 3 (2.59%) and 16 (7.84%) in NPP and PP respectively: this difference between the two groups, even if not statistically significant (p = 0.08), shows to be relevant from a clinical point of view. At this proposal, as the chosen 1‐mm limit for bone loss may appear very stringent, authors underline that 2‐mm limit, currently used in the literature as a 5‐year follow‐up threshold\n51\n cannot be considered acceptable for the definition of a potentially dangerous condition as peri‐implantitis around implants that are only 5 or 6 mm long.\n10\n, \n11\n\n\nOur findings agree with other studies on standard implants with the same follow‐up in PP.\n51\n, \n52\n, \n53\n, \n54\n, \n55\n Martens et al.,\n52\n in a case series with 57‐months of follow‐up on 163 implants in 33 periodontally compromised patients, found a survival rate of 96.3% and a percentage of peri‐implantitis of 6%. Aguirre‐Zorzano et al.,\n53\n in a cross‐sectional study with 786 implants in 239 patients all with history of periodontitis, found a percentage of peri‐implantitis of 9.8%. Canullo et al.,\n54\n in a cross‐sectional study with 1507 implants in 534 patients including also subjects with history of periodontitis, found a percentage of peri‐implantitis of 7.3%, with a higher percentage of healthy periodontal subjects in the group not affected by peri‐implantitis. Konstantinidis et al.,\n55\n in a cross‐sectional study with a mean follow‐up of 5.5 years on 597 implants in 186 patients, including also subjects with history of periodontitis, found a percentage of peri‐implantitis of 6.2%. Dalago et al.,\n51\n in a cross‐sectional study with 916 implants in 183 patients, found a percentage of peri‐implantitis of 7.3%; this study considered factors related to patient's conditions, implant's characteristics and clinical parameters, finally assessing history of periodontitis and total rehabilitations as risk indicators for peri‐implantitis.\nFinally, insurgence of mucositis is strictly related to inadequate plaque control,\n56\n, \n57\n typical of a reversible inflammation which can lead to a non‐reversible disease: in the present study all patients showed a positive compliance to the maintenance program, following a specific protocol of professional oral hygiene recalls, and, according to the literature,\n58\n, \n59\n low inflammatory indexes were registered both in PP and NPP after 60 months of follow‐up, as reported by other studies on short and ultra‐short locking‐taper implants.\n26\n, \n29\n The design and type of the implant supported prosthesis can influence the cleanability of the implant site\n8\n, \n22\n, \n23\n, \n60\n: single‐crown rehabilitation used in the study represent a gold standard in facilitating plaque removal.\n26\n, \n28\n, \n61\nOn the other hand, as reported in another study on the same sample of patients,\n23\n percentages of success, even if not statically different between groups, were respectively lower in 5.0 and 6.00 mm‐length implants (93.10% and 92.73%) compared to 8.0 mm‐length implants (95.93%).\nTo the best of our knowledge, the evidence regarding the possible association between the width of keratinized mucosa and the success of ultra‐short implants is scarce in literature and still inconclusive. However, it is well known that a good level of oral hygiene is also related to an adequate width of keratinized tissue.\n62\n At the same time, the presence of an appropriate width and thickness of KT may facilitate soft tissues flap management during surgical procedures and also represent a favorable feature for clinical improvement after peri‐implantitis treatments.\n63\n It does not therefore seem inappropriate to suggest that ultra‐short implants require continuous monitoring of patient's compliance and extra attention during supportive therapy, particularly in case of implants placed in mandibular sites, which do not easily allow surgical procedures for soft tissues augmentation.\n64\n Comparing the outcomes of this study with the previous similar studies on locking‐taper implants done at 3‐year follow‐up, some issues remain critical. Main limitations\n23\n related to its retrospective nature, even if reduced, still consist in a non‐balanced distribution among implant length‐groups, PP/NPP‐groups and arch‐groups, besides the University setting (single‐centre). However, a proper evaluation of clinical and radiographic conditions at a longer‐term follow‐up (5 years), as well as limiting the present analysis to single crown restorations, suggests predictability of treatments using short and ultra‐short locking‐taper implants even in presence of history of periodontitis.", "Outcomes of the present study revealed that history of periodontitis represents a crucial factor for the success of short and ultra‐short implants, which need to be strictly monitored.\nThese 5‐year results showed that short and ultra‐short locking‐taper implants supporting single‐crown restorations may represent a successful treatment for the rehabilitation of the atrophic posterior jaws both in PP and NPP, provided that patients are enrolled in a specific supportive protocol, carrying out adequate home care procedures and compliance with recall appointments. Regular attendance to a maintenance program may play an important role in determining stable results over time and in preventing peri‐implant diseases, avoiding the worsening of mucositis in potential peri‐implantitis. Further investigations with longer follow‐up and prospective design are of course necessary to validate these conclusions.", "Giorgio Lombardo: Concept and design, critical revision and approval of article. Annarita Signoriello: Data collection and analysis, planning of statistics, drafting article. Alessia Pardo, Xiomara Zilena Serpa Romero, Luisa Arévalo Tovar: Data collection and analysis. Luis Armando Vila Sierra, Pier Francesco Nocini: Critical revision and approval of article. Mauro Marincola: Concept and design, critical revision and approval of article", "All authors declare no conflicts of interests." ]

[ null, "materials-and-methods", null, "results", "discussion", "conclusions", null, "COI-statement" ]

[ "mucositis", "peri‐implantitis", "periodontitis", "short", "single crown", "ultra‐short" ]

[ 802, 1763, 75 ]

[ "implant", "implants", "mm", "bone", "23", "crown", "peri", "length", "study", "patients" ]

[ "tooth implants crown", "peri implantitis study", "short implants periodontally", "peri implantitis compared", "periodontitis implant survival" ]

[ "Adolescent", "Adult", "Aged", "Cross-Sectional Studies", "Europe", "Female", "Health Surveys", "Humans", "Male", "Middle Aged", "Prevalence", "Smokers", "Smoking", "Socioeconomic Factors", "Young Adult" ]

[ "Sample selection", "Questionnaire", "Fieldwork", "Variables and definitions", "Ethical issues", "Statistical analysis" ]

[ "In each country, we considered a sample of around 1,000 individuals aged 15 years and older (participants’ age was ≥16 in England, ≥18 in Ireland, 15–64 in Greece, and 15–74 years in Latvia), representative of the general population in terms of age, sex, habitat (ie, geographic area and/or size of municipality) and, in some countries, socio-economic characteristics. The survey included a total of 11,902 subjects. The whole population domain represented 79.2% of the whole EU population aged 15 years or over (432 million inhabitants).\nSampling methods varied across countries: in Bulgaria, Greece, Italy, Latvia and Romania, a multi-stage sampling was used and respondents were randomly chosen to be representative of the population in terms of sex, age, and geographic area (in Italy, representativeness by socio-economic characteristics was also ensured); in Germany, Ireland, Poland, Portugal, and Spain, stratified random sampling was used, combining also quotas on sex and age and social class in Ireland; in England, United Kingdom, cluster sampling with quotas on age, sex, socio-economic status (SES), region, urban/rural dwelling was used to ensure national representativeness; in France, quotas on age, sex, region, and city size were used for the selection of survey participants.", "A first draft of the survey questionnaire was developed in English language by the researchers from Mario Negri Institute, based on previous tobacco use and secondhand smoke exposure questionnaires.3,19–21 A specific commission with six experts among project partners was created to review the questionnaire and to produce a second version. The final version of the English questionnaire was then developed through the collaboration with all the partners of the TackSHS consortium (eMaterial 1). The questionnaire was translated into Italian language by Mario Negri Institute’s researchers, and into Bulgarian, French, German, Greek, Latvian, Russian, Polish, Portuguese, Romanian, and Spanish by DOXA partners. The translated versions of the questionnaire were revised and validated by bilingual (ie, local language and English language) tobacco control experts.\nThe questionnaire contains four sections: socio-economic and demographic characteristics; cigarette smoking habit and e-cigarette use; exposure to secondhand smoke (SHS) and e-cigarette aerosol, plus cigarette smoking and e-cigarette use, in different indoor and outdoor places; and attitudes and perceptions on smoke-free regulations and awareness of SHS harmful effects.", "Ad hoc trained interviewers conducted a face-to-face, computer-assisted personal interviewing (CAPI) survey in all the 12 European countries. A test fieldwork was conducted by DOXA in Italy in November 2016 among 1,059 participants. The fieldwork in the other 11 countries was conducted between June 2017 (in Romania) and October 2018 (in Latvia). Table 1 provides information on survey characteristics for each country, including fieldwork period, sample size, age range and sampling methods.\nCAPI, computer assisted personal interview; SES, socio-economic status.\naRaw sample size.\nbThis period does not include the time of the survey conducted in Italy (ie, Nov–Dec 2016) since Italy was the pilot country. The participants from Italy are included in all the analyses.", "Demographic characteristics included age (categorized as <25, 25–44, 45–64, ≥65 years) and sex (men and women). Level of education was categorized as country-specific tertiles of schooling years. Self-assessment of household family economic status relative to the country-specific population was classified into three levels (higher than average, average, and lower than average). Never smokers were defined as participants who had never smoked or had smoked less than 100 cigarettes in their lifetime. Smokers were defined as participants who reported smoking at least 100 cigarettes (including roll-your-own cigarettes) during their lifetime. Current smokers were smokers who reported smoking at the time they participated in this survey, while ex-smokers were smokers who stopped smoking by the time they participated in this survey.3,21 Smokers also provided information on age of starting smoking and the number of cigarettes smoked per day. For each country, we calculated the male-to-female smoking prevalence ratio as the current smoking prevalence in men divided by that in women. The 12 countries were classified into Northern (England, Ireland, and Latvia), Western (France and Germany), Southern (Italy, Greece, Portugal, and Spain), and Eastern (Bulgaria, Poland, and Romania), following United Nation M49 standard,22 and halved by their gross domestic product (GDP) per capita23 into <25.000 € (Latvia, Romania, Poland, Portugal, Greece, and Bulgaria) and ≥25.000 € (England, France, Germany, Ireland, Italy, and Spain).", "We obtained study approval from a local Ethics Committee in each of the 12 countries (eTable 1). Details on the survey characteristics were provided to all participants by suitably qualified professionals through a structured information sheet, and all the participants provided their consents by ticking the electronic field in the CAPI questionnaire. The study protocol has been registered in ClinicalTrials.gov (ID: NCT02928536).\nThe procedures for recruitment of subjects, data collection, storage, and protection (based on anonymous identification code) are in accordance with the current country-specific legislation. This was ratified and signed by DOXA and each of its European partners.", "Statistical weights were used to generate representative estimates of the general population of each country (individual weight). To calculate results for the entire sample, we applied “country weights”, which combined individual weights with an additional weighting factor, with each country contributing in proportion to its population aged 15 years or over, obtained by Eurostat.24\nWe considered descriptive statistics including relative frequency (%) and its corresponding 95% confidence intervals (CIs) for categorical variables, and mean and standard deviation (SD) for continuous variables.\nORs and their corresponding 95% CIs were estimated using multilevel logistic random-effects models, to take into account the heterogeneity between the 12 European countries. The study country effects were considered as random intercepts, and sex, age, and level of education as adjusting variables. Country weights were used in all logistic regression models. Analyses were performed with SAS 9.4 (SAS Institute; Cary, NC, USA)." ]

[ null, null, null, null, null, null ]

[ "INTRODUCTION", "METHODS", "Sample selection", "Questionnaire", "Fieldwork", "Variables and definitions", "Ethical issues", "Statistical analysis", "RESULTS", "DISCUSSION" ]

[ "In 2008, the World Health Organization (WHO) introduced the MPOWER policy package, a series of technical measures and resources to assist country-level implementation of the WHO Framework Convention on Tobacco Control (FCTC), developed in response to the globalization of the tobacco epidemic. The first of these technical measures involves the monitoring of prevention policies and tobacco use.1\nSeveral national surveillance systems are available to monitor smoking prevalence and trends in most countries.2 However, data from country-specific populations are poorly comparable, since they are not collected using a standardized study design—including sampling methods, mode of interviewing and questionnaires—which represents a prerequisite for valid comparisons of smoking prevalence and smoking patterns across different countries.3,4\nTo the best of our knowledge, the only standardized and representative investigations that systematically monitor smoking prevalence among adults in more than one European country, are the WHO Global Adult Tobacco Survey (GATS)5,6; the Survey of Health, Ageing and Retirement in Europe (SHARE)7; and the European Commission (EC) Special Eurobarometers.8–10 GATS covers three European Union (EU) member states (MS; ie, Greece, Poland, and Romania)5,6; SHARE is a longitudinal study, conducted in five waves between 2004 and 2013, collecting information on smoking habit among individuals aged 50 years and older from 20 European countries7; Eurobarometer is a representative survey conducted in all the 28 EU MS on approximately 1,000 face-to-face interviews per country, based on selected thematic studies. The most recent Eurobarometer surveys focusing on tobacco smoking indicated that the overall smoking prevalence in the EU had slightly decreased from 29% in 2009 to 26% in 2014, and remained stable until 2017.8,9,11 Among the other tobacco control projects, the International Tobacco Control Policy Evaluation Project (the ITC Project) includes longitudinal studies in 10 European countries. However, all these studies, which include samples not representative of the general population or smokers only, cannot provide smoking prevalence estimates.12 The above-mentioned European surveillance systems are important and useful tools; however, they are not always able to investigate in-depth key tobacco control measures and smoking determinants. Consequently, a few specific studies, mainly supported by the EC, have been conducted to analyse this issue.3,13–15 These include, the Pricing Policies and Control of Tobacco in Europe (PPACTE) survey, conducted in 2010 to investigate the effectiveness of pricing policies on tobacco control in Europe3,16,17; and the present survey, conducted within the TackSHS Project (http://tackshs.eu),18 aimed to improve understanding of exposure to second-hand tobacco smoke (SHS) and e-cigarette aerosol in Europe.\nIn this paper we aimed to describe the TackSHS survey, a multi-country survey conducted in 2017–2018 in 12 EU MS, providing the main results on smoking prevalence and its correlates in Europe.", "The TackSHS survey18 has been conducted in 12 strategically selected EU countries (Bulgaria, England, France, Germany, Greece, Ireland, Italy, Latvia, Poland, Portugal, Romania, and Spain), representing geographical, legislative and cultural variations across the EU.\nThis cross-sectional study was coordinated by Istituto di Ricerche Farmacologiche Mario Negri IRCCS (Mario Negri Institute; Milan, Italy). The fieldwork was conducted by DOXA, the Italian branch of the Worldwide Independent Network/Gallup International Association, and its European partners.\n Sample selection In each country, we considered a sample of around 1,000 individuals aged 15 years and older (participants’ age was ≥16 in England, ≥18 in Ireland, 15–64 in Greece, and 15–74 years in Latvia), representative of the general population in terms of age, sex, habitat (ie, geographic area and/or size of municipality) and, in some countries, socio-economic characteristics. The survey included a total of 11,902 subjects. The whole population domain represented 79.2% of the whole EU population aged 15 years or over (432 million inhabitants).\nSampling methods varied across countries: in Bulgaria, Greece, Italy, Latvia and Romania, a multi-stage sampling was used and respondents were randomly chosen to be representative of the population in terms of sex, age, and geographic area (in Italy, representativeness by socio-economic characteristics was also ensured); in Germany, Ireland, Poland, Portugal, and Spain, stratified random sampling was used, combining also quotas on sex and age and social class in Ireland; in England, United Kingdom, cluster sampling with quotas on age, sex, socio-economic status (SES), region, urban/rural dwelling was used to ensure national representativeness; in France, quotas on age, sex, region, and city size were used for the selection of survey participants.\nIn each country, we considered a sample of around 1,000 individuals aged 15 years and older (participants’ age was ≥16 in England, ≥18 in Ireland, 15–64 in Greece, and 15–74 years in Latvia), representative of the general population in terms of age, sex, habitat (ie, geographic area and/or size of municipality) and, in some countries, socio-economic characteristics. The survey included a total of 11,902 subjects. The whole population domain represented 79.2% of the whole EU population aged 15 years or over (432 million inhabitants).\nSampling methods varied across countries: in Bulgaria, Greece, Italy, Latvia and Romania, a multi-stage sampling was used and respondents were randomly chosen to be representative of the population in terms of sex, age, and geographic area (in Italy, representativeness by socio-economic characteristics was also ensured); in Germany, Ireland, Poland, Portugal, and Spain, stratified random sampling was used, combining also quotas on sex and age and social class in Ireland; in England, United Kingdom, cluster sampling with quotas on age, sex, socio-economic status (SES), region, urban/rural dwelling was used to ensure national representativeness; in France, quotas on age, sex, region, and city size were used for the selection of survey participants.\n Questionnaire A first draft of the survey questionnaire was developed in English language by the researchers from Mario Negri Institute, based on previous tobacco use and secondhand smoke exposure questionnaires.3,19–21 A specific commission with six experts among project partners was created to review the questionnaire and to produce a second version. The final version of the English questionnaire was then developed through the collaboration with all the partners of the TackSHS consortium (eMaterial 1). The questionnaire was translated into Italian language by Mario Negri Institute’s researchers, and into Bulgarian, French, German, Greek, Latvian, Russian, Polish, Portuguese, Romanian, and Spanish by DOXA partners. The translated versions of the questionnaire were revised and validated by bilingual (ie, local language and English language) tobacco control experts.\nThe questionnaire contains four sections: socio-economic and demographic characteristics; cigarette smoking habit and e-cigarette use; exposure to secondhand smoke (SHS) and e-cigarette aerosol, plus cigarette smoking and e-cigarette use, in different indoor and outdoor places; and attitudes and perceptions on smoke-free regulations and awareness of SHS harmful effects.\nA first draft of the survey questionnaire was developed in English language by the researchers from Mario Negri Institute, based on previous tobacco use and secondhand smoke exposure questionnaires.3,19–21 A specific commission with six experts among project partners was created to review the questionnaire and to produce a second version. The final version of the English questionnaire was then developed through the collaboration with all the partners of the TackSHS consortium (eMaterial 1). The questionnaire was translated into Italian language by Mario Negri Institute’s researchers, and into Bulgarian, French, German, Greek, Latvian, Russian, Polish, Portuguese, Romanian, and Spanish by DOXA partners. The translated versions of the questionnaire were revised and validated by bilingual (ie, local language and English language) tobacco control experts.\nThe questionnaire contains four sections: socio-economic and demographic characteristics; cigarette smoking habit and e-cigarette use; exposure to secondhand smoke (SHS) and e-cigarette aerosol, plus cigarette smoking and e-cigarette use, in different indoor and outdoor places; and attitudes and perceptions on smoke-free regulations and awareness of SHS harmful effects.\n Fieldwork Ad hoc trained interviewers conducted a face-to-face, computer-assisted personal interviewing (CAPI) survey in all the 12 European countries. A test fieldwork was conducted by DOXA in Italy in November 2016 among 1,059 participants. The fieldwork in the other 11 countries was conducted between June 2017 (in Romania) and October 2018 (in Latvia). Table 1 provides information on survey characteristics for each country, including fieldwork period, sample size, age range and sampling methods.\nCAPI, computer assisted personal interview; SES, socio-economic status.\naRaw sample size.\nbThis period does not include the time of the survey conducted in Italy (ie, Nov–Dec 2016) since Italy was the pilot country. The participants from Italy are included in all the analyses.\nAd hoc trained interviewers conducted a face-to-face, computer-assisted personal interviewing (CAPI) survey in all the 12 European countries. A test fieldwork was conducted by DOXA in Italy in November 2016 among 1,059 participants. The fieldwork in the other 11 countries was conducted between June 2017 (in Romania) and October 2018 (in Latvia). Table 1 provides information on survey characteristics for each country, including fieldwork period, sample size, age range and sampling methods.\nCAPI, computer assisted personal interview; SES, socio-economic status.\naRaw sample size.\nbThis period does not include the time of the survey conducted in Italy (ie, Nov–Dec 2016) since Italy was the pilot country. The participants from Italy are included in all the analyses.\n Variables and definitions Demographic characteristics included age (categorized as <25, 25–44, 45–64, ≥65 years) and sex (men and women). Level of education was categorized as country-specific tertiles of schooling years. Self-assessment of household family economic status relative to the country-specific population was classified into three levels (higher than average, average, and lower than average). Never smokers were defined as participants who had never smoked or had smoked less than 100 cigarettes in their lifetime. Smokers were defined as participants who reported smoking at least 100 cigarettes (including roll-your-own cigarettes) during their lifetime. Current smokers were smokers who reported smoking at the time they participated in this survey, while ex-smokers were smokers who stopped smoking by the time they participated in this survey.3,21 Smokers also provided information on age of starting smoking and the number of cigarettes smoked per day. For each country, we calculated the male-to-female smoking prevalence ratio as the current smoking prevalence in men divided by that in women. The 12 countries were classified into Northern (England, Ireland, and Latvia), Western (France and Germany), Southern (Italy, Greece, Portugal, and Spain), and Eastern (Bulgaria, Poland, and Romania), following United Nation M49 standard,22 and halved by their gross domestic product (GDP) per capita23 into <25.000 € (Latvia, Romania, Poland, Portugal, Greece, and Bulgaria) and ≥25.000 € (England, France, Germany, Ireland, Italy, and Spain).\nDemographic characteristics included age (categorized as <25, 25–44, 45–64, ≥65 years) and sex (men and women). Level of education was categorized as country-specific tertiles of schooling years. Self-assessment of household family economic status relative to the country-specific population was classified into three levels (higher than average, average, and lower than average). Never smokers were defined as participants who had never smoked or had smoked less than 100 cigarettes in their lifetime. Smokers were defined as participants who reported smoking at least 100 cigarettes (including roll-your-own cigarettes) during their lifetime. Current smokers were smokers who reported smoking at the time they participated in this survey, while ex-smokers were smokers who stopped smoking by the time they participated in this survey.3,21 Smokers also provided information on age of starting smoking and the number of cigarettes smoked per day. For each country, we calculated the male-to-female smoking prevalence ratio as the current smoking prevalence in men divided by that in women. The 12 countries were classified into Northern (England, Ireland, and Latvia), Western (France and Germany), Southern (Italy, Greece, Portugal, and Spain), and Eastern (Bulgaria, Poland, and Romania), following United Nation M49 standard,22 and halved by their gross domestic product (GDP) per capita23 into <25.000 € (Latvia, Romania, Poland, Portugal, Greece, and Bulgaria) and ≥25.000 € (England, France, Germany, Ireland, Italy, and Spain).\n Ethical issues We obtained study approval from a local Ethics Committee in each of the 12 countries (eTable 1). Details on the survey characteristics were provided to all participants by suitably qualified professionals through a structured information sheet, and all the participants provided their consents by ticking the electronic field in the CAPI questionnaire. The study protocol has been registered in ClinicalTrials.gov (ID: NCT02928536).\nThe procedures for recruitment of subjects, data collection, storage, and protection (based on anonymous identification code) are in accordance with the current country-specific legislation. This was ratified and signed by DOXA and each of its European partners.\nWe obtained study approval from a local Ethics Committee in each of the 12 countries (eTable 1). Details on the survey characteristics were provided to all participants by suitably qualified professionals through a structured information sheet, and all the participants provided their consents by ticking the electronic field in the CAPI questionnaire. The study protocol has been registered in ClinicalTrials.gov (ID: NCT02928536).\nThe procedures for recruitment of subjects, data collection, storage, and protection (based on anonymous identification code) are in accordance with the current country-specific legislation. This was ratified and signed by DOXA and each of its European partners.\n Statistical analysis Statistical weights were used to generate representative estimates of the general population of each country (individual weight). To calculate results for the entire sample, we applied “country weights”, which combined individual weights with an additional weighting factor, with each country contributing in proportion to its population aged 15 years or over, obtained by Eurostat.24\nWe considered descriptive statistics including relative frequency (%) and its corresponding 95% confidence intervals (CIs) for categorical variables, and mean and standard deviation (SD) for continuous variables.\nORs and their corresponding 95% CIs were estimated using multilevel logistic random-effects models, to take into account the heterogeneity between the 12 European countries. The study country effects were considered as random intercepts, and sex, age, and level of education as adjusting variables. Country weights were used in all logistic regression models. Analyses were performed with SAS 9.4 (SAS Institute; Cary, NC, USA).\nStatistical weights were used to generate representative estimates of the general population of each country (individual weight). To calculate results for the entire sample, we applied “country weights”, which combined individual weights with an additional weighting factor, with each country contributing in proportion to its population aged 15 years or over, obtained by Eurostat.24\nWe considered descriptive statistics including relative frequency (%) and its corresponding 95% confidence intervals (CIs) for categorical variables, and mean and standard deviation (SD) for continuous variables.\nORs and their corresponding 95% CIs were estimated using multilevel logistic random-effects models, to take into account the heterogeneity between the 12 European countries. The study country effects were considered as random intercepts, and sex, age, and level of education as adjusting variables. Country weights were used in all logistic regression models. Analyses were performed with SAS 9.4 (SAS Institute; Cary, NC, USA).", "In each country, we considered a sample of around 1,000 individuals aged 15 years and older (participants’ age was ≥16 in England, ≥18 in Ireland, 15–64 in Greece, and 15–74 years in Latvia), representative of the general population in terms of age, sex, habitat (ie, geographic area and/or size of municipality) and, in some countries, socio-economic characteristics. The survey included a total of 11,902 subjects. The whole population domain represented 79.2% of the whole EU population aged 15 years or over (432 million inhabitants).\nSampling methods varied across countries: in Bulgaria, Greece, Italy, Latvia and Romania, a multi-stage sampling was used and respondents were randomly chosen to be representative of the population in terms of sex, age, and geographic area (in Italy, representativeness by socio-economic characteristics was also ensured); in Germany, Ireland, Poland, Portugal, and Spain, stratified random sampling was used, combining also quotas on sex and age and social class in Ireland; in England, United Kingdom, cluster sampling with quotas on age, sex, socio-economic status (SES), region, urban/rural dwelling was used to ensure national representativeness; in France, quotas on age, sex, region, and city size were used for the selection of survey participants.", "A first draft of the survey questionnaire was developed in English language by the researchers from Mario Negri Institute, based on previous tobacco use and secondhand smoke exposure questionnaires.3,19–21 A specific commission with six experts among project partners was created to review the questionnaire and to produce a second version. The final version of the English questionnaire was then developed through the collaboration with all the partners of the TackSHS consortium (eMaterial 1). The questionnaire was translated into Italian language by Mario Negri Institute’s researchers, and into Bulgarian, French, German, Greek, Latvian, Russian, Polish, Portuguese, Romanian, and Spanish by DOXA partners. The translated versions of the questionnaire were revised and validated by bilingual (ie, local language and English language) tobacco control experts.\nThe questionnaire contains four sections: socio-economic and demographic characteristics; cigarette smoking habit and e-cigarette use; exposure to secondhand smoke (SHS) and e-cigarette aerosol, plus cigarette smoking and e-cigarette use, in different indoor and outdoor places; and attitudes and perceptions on smoke-free regulations and awareness of SHS harmful effects.", "Ad hoc trained interviewers conducted a face-to-face, computer-assisted personal interviewing (CAPI) survey in all the 12 European countries. A test fieldwork was conducted by DOXA in Italy in November 2016 among 1,059 participants. The fieldwork in the other 11 countries was conducted between June 2017 (in Romania) and October 2018 (in Latvia). Table 1 provides information on survey characteristics for each country, including fieldwork period, sample size, age range and sampling methods.\nCAPI, computer assisted personal interview; SES, socio-economic status.\naRaw sample size.\nbThis period does not include the time of the survey conducted in Italy (ie, Nov–Dec 2016) since Italy was the pilot country. The participants from Italy are included in all the analyses.", "Demographic characteristics included age (categorized as <25, 25–44, 45–64, ≥65 years) and sex (men and women). Level of education was categorized as country-specific tertiles of schooling years. Self-assessment of household family economic status relative to the country-specific population was classified into three levels (higher than average, average, and lower than average). Never smokers were defined as participants who had never smoked or had smoked less than 100 cigarettes in their lifetime. Smokers were defined as participants who reported smoking at least 100 cigarettes (including roll-your-own cigarettes) during their lifetime. Current smokers were smokers who reported smoking at the time they participated in this survey, while ex-smokers were smokers who stopped smoking by the time they participated in this survey.3,21 Smokers also provided information on age of starting smoking and the number of cigarettes smoked per day. For each country, we calculated the male-to-female smoking prevalence ratio as the current smoking prevalence in men divided by that in women. The 12 countries were classified into Northern (England, Ireland, and Latvia), Western (France and Germany), Southern (Italy, Greece, Portugal, and Spain), and Eastern (Bulgaria, Poland, and Romania), following United Nation M49 standard,22 and halved by their gross domestic product (GDP) per capita23 into <25.000 € (Latvia, Romania, Poland, Portugal, Greece, and Bulgaria) and ≥25.000 € (England, France, Germany, Ireland, Italy, and Spain).", "We obtained study approval from a local Ethics Committee in each of the 12 countries (eTable 1). Details on the survey characteristics were provided to all participants by suitably qualified professionals through a structured information sheet, and all the participants provided their consents by ticking the electronic field in the CAPI questionnaire. The study protocol has been registered in ClinicalTrials.gov (ID: NCT02928536).\nThe procedures for recruitment of subjects, data collection, storage, and protection (based on anonymous identification code) are in accordance with the current country-specific legislation. This was ratified and signed by DOXA and each of its European partners.", "Statistical weights were used to generate representative estimates of the general population of each country (individual weight). To calculate results for the entire sample, we applied “country weights”, which combined individual weights with an additional weighting factor, with each country contributing in proportion to its population aged 15 years or over, obtained by Eurostat.24\nWe considered descriptive statistics including relative frequency (%) and its corresponding 95% confidence intervals (CIs) for categorical variables, and mean and standard deviation (SD) for continuous variables.\nORs and their corresponding 95% CIs were estimated using multilevel logistic random-effects models, to take into account the heterogeneity between the 12 European countries. The study country effects were considered as random intercepts, and sex, age, and level of education as adjusting variables. Country weights were used in all logistic regression models. Analyses were performed with SAS 9.4 (SAS Institute; Cary, NC, USA).", "Among 11,902 participants, 57.6% (95% CI, 56.8–58.5%) described themselves as never smokers (49.5% in men and 65.1% in women), 16.5% (95% CI, 15.8–17.2%) as ex-smokers (19.5% in men and 13.7% in women) and 25.9% (95% CI, 25.1–26.7%) as current smokers (31.0% in men and 21.2% in women; Table 2). The smoking prevalence was below 20% in Italy (18.9%), Ireland (19.6%), and England (19.8%), and over 35% in Bulgaria (37.0%) and Portugal (36.8%). Smoking prevalence in men ranged from 20.3% in England to 42.0% in Portugal, and in women from 15.3% in Germany to 34.4% in Bulgaria. Average smoking intensity was 14.7 cigarettes per day (SD, 8.8), ranging from 12.4 (SD, 7.5) in Italy to 17.1 (SD, 15.8) in Greece.\nM/F, male to female; SD, standard deviation.\naSmoking intensity, including both manufactured and roll-your-own cigarettes among current smokers (men and women combined). Questions on cigarette smoking intensity was not asked in Ireland. In the other 11 countries, 2,938 out of 3,050 current smokers provided information on the number of cigarettes smoked per day.\nbCountry weights were applied, which combined individual weights with an additional weighting factor, with each country contributing in proportion to its population aged 15 years or over, obtained by Eurostat.24\nAmong current smokers, 76.1% started smoking before age 18 years and 96.3% before 25 years. Mean age at starting regular smoking was 17.4 (SD, 4.5) overall, 17.1 (SD, 4.1) in males, and 17.8 (SD, 4.8) years in female smokers, and varied between 15.2 (SD, 4.2) in England and 19.1 years (SD, 4.2) in Romania (data not shown in tables).\nTable 3 provides the ORs for current versus non-smokers according to selected individual-level and country-specific characteristics. Smoking prevalence was higher in men than in women (OR 1.67; 95% CI, 1.53–1.82). Compared with subjects aged <45 years, ORs were 0.97 (95% CI, 0.89–1.07) for 45–64 years, and 0.31 (95% CI, 0.27–0.36) for ≥65 years. Compared with high level of education, the ORs were 1.39 (95% CI, 1.25–1.55) for medium and 1.32 (95% CI, 1.17–1.48) for low level of education (P for trend <0.001). Compared to those with a high household economic status, the ORs were 1.33 (95% CI, 1.15–1.54) and 2.05 (95% CI, 1.74–2.42) for those with an average and low economic status, respectively. The patterns observed for all the individual-level characteristics were consistent in men and women. Compared with Northern Europe, the ORs of smoking were 1.58 (95% CI, 0.98–2.56) in Western, 1.52 (95% CI, 0.93–2.49) in Southern and 1.57 (95% CI, 0.92–2.70) in Eastern European countries. Compared to Northern Europe, the OR in men was higher in Western Europe (OR 2.17; 95% CI, 1.35–3.48), Southern Europe (OR 1.77; 95% CI, 1.09–2.87) and Eastern Europe (OR 1.87; 95% CI, 1.10–3.18). Smoking prevalence showed no significant difference between countries with lower and higher GDP per capita, the ORs for less wealthy countries being 1.22 (95% CI, 0.82–1.81).\nCI, confidence interval; GDP, gross domestic product; OR, odds ratio.\naCountry weights were applied, which combined individual weights with an additional weighting factor, with each country contributing in proportion to its population aged 15 years or over, obtained by Eurostat.24\nbRaw sample size.\ncORs and their corresponding 95% CIs were estimated using multilevel logistic random-effects models, to take into account the heterogeneity between the 12 European countries. The study country effects were considered as random intercepts, and sex, age and level of education as adjusting variables. Estimates in bold are statistically significant at 0.05 level.\ndReference category.\neLevel of education was categorized as country-specific tertiles of schooling years. The sum does not add to the total because of a few missing values.\nfSelf-assessment of household (family) economic status relative to the country-specific population. This variable is missing for all German subjects, 79 Latvian and, 35 Romanian subjects.", "Approximately one out of four adults is a current smoker in Europe, including one out of three men and one out of five women. Smoking prevalence showed large differences across European countries, being highest in Eastern Europe (28%) and lowest in Northern Europe (20%).\nIn both men and women, smoking prevalence is highest among young adults aged 25–44 years. Almost all current smokers in Europe started smoking before aged 25 years. Therefore, this study confirms that teenagers and young adults are the most vulnerable group to start smoking3 and thus are the most important target population for tobacco control interventions aimed to decrease nicotine addiction initiation.\nSmoking prevalence among women, exceeding 15% in all the 12 countries, is in an advanced stage of the tobacco epidemic.3,25 However, notable differences were observed in the male-to-female smoking prevalence ratio, ranging from 1.04 in England to 2.58 in Latvia. This suggests that some countries (likely those with a higher male-to-female smoking prevalence ratio) are in an earlier phase compared with others.3,25\nMultilevel logistic analyses indicate that the level of education was inversely related to smoking prevalence in both men and women. Analysing other individual level and country specific indicators of SES, we also found that smoking prevalence was systematically higher in the sub-population with low SES. This finding was independent of sex, age, and education, as reported in a few other investigations.26 Our results are in broad agreement with current evidence,27,28 and underline the need for specific measures specifically focused on low socio-economic groups to reduce health inequalities from tobacco.\nIn general, our findings are consistent with those reported in the Global Health Observatory data repository of the WHO29 and those found in the 2017 Eurobarometer survey.8 The latter study shows a smoking prevalence for the EU of 26% overall, 30% in men, and 20% in women, thus very similar to our estimates. In Italy, the smoking prevalence found in TackSHS survey (19% overall, 23% in men, and 16% in women) appears slightly lower than that found in the 2016 national survey conducted on around 3,000 subjects (ie, 22% overall, 27% in men, and 17% in women).30 Some major discrepancies with current evidence from the scientific literature were noted particularly in Portugal, where we found a smoking prevalence greater than 30% for both men and women. Our estimates are in fact substantially higher than those reported before 2015 for daily smoking (lower than 20%).2,31,32 The different definition of current smoking and the different sampling methodology used are not sufficient to explain the substantial gap found in smoking prevalence. Moreover, the distribution of our Portuguese sample by sex and age group is very similar to the official distribution provided by Eurostat.24 Other adult smoking prevalence estimates for Portugal, including those from the 2010 PPACTE study (32% overall, 36% in men, and 29% in women),3 or the 2015 WHO estimates (32% in men and 14% in women),29 or the 2017 Eurobarometer survey (26% overall)8 are more similar—but still substantially lower—than our estimates.\nOverall, the smoking prevalence remained almost the same (from 27.2% to 26.4%) in the 11 countries that were included in both the TackSHS and PPACTE (2010) surveys, using similar methodologies and definitions. For France, Portugal, Romania, and Spain, we observed increases in smoking prevalence over the last decade but a decrease from 36% to 20% in Ireland.\nThe trends in smoking prevalence found in our data match those from the Eurobarometer surveys: the comparison between the 20178 and 200911 surveys indicated a decrease in smoking prevalence for all countries except for France and Portugal. Smoking prevalence estimates in Europe remain substantially higher as compared to those from other high-income countries, such as the United States, Canada, and Australia, where less than 15% of adults are current smokers.2,33\nThe limitations of the present study include some differences of sampling in the study countries. Notwithstanding, all samples selected were representative of their population in terms of age, sex, and geographic area and/or size of municipality. The age range of the participants was slightly different in some countries. In particular, in Greece the sample was limited to subjects aged 15–64 years, thus likely providing an over-estimation of the adult smoking prevalence. However, even assuming that the smoking prevalence in the elderly (representing in Greece 25% of the adult population24) was 50% that in younger adults—as observed in the other 11 countries—we would estimate a smoking prevalence around 30%, still similar to the provided estimate.\nThe strengths of our survey include the representativeness of the adult population of the 12 selected European countries, the use of the same questionnaire in the 12 countries sampled, developed by a group of experts on tobacco control, the standardized use of a single definition of current smokers, and the use of face-to-face interviews.\nOur data indicate that there are 112 million current smokers in the EU, including 65 million men and 47 million women. We confirm that the large majority of smokers started smoking when they were adolescents, and that lower SES is a major determinant of smoking habit in Europe. Tobacco control measures to decrease smoking initiation and to promote smoking cessation should, therefore, be targeted at young people and the sub-group with lower SES, respectively. Increasing tobacco prices, through the adoption of additional excise taxes, has been shown to be more effective among younger generations and in economically deprived populations.28,34 Therefore, price increase is still likely to be a particularly effective tobacco control strategy, particularly in those European countries where tobacco price is still relatively low and resources for tobacco control are scarce.35" ]

[ "intro", "methods", null, null, null, null, null, null, "results", "discussion" ]

[ "tobacco", "cigarette smoking", "smoking prevalence", "cross-sectional study", "survey", "Europe", "TackSHS" ]

[ 258, 211, 152, 296, 118, 180 ]

[ "smoking", "country", "countries", "survey", "age", "smoking prevalence", "prevalence", "smokers", "years", "population" ]

[ "countries estimate smoking", "tobacco control measures", "european countries tobacco", "current smokers europe", "monitor smoking prevalence" ]

[ "Adolescent", "Aged", "Anti-Citrullinated Protein Antibodies", "Arthritis, Rheumatoid", "Autoantibodies", "Case-Control Studies", "Cohort Studies", "Female", "Genetic Predisposition to Disease", "Humans", "Immunoenzyme Techniques", "Immunoglobulin A", "Immunoglobulin G", "Male", "Middle Aged", "Nephelometry and Turbidimetry", "Predictive Value of Tests", "Prevalence", "Rheumatoid Factor", "Sensitivity and Specificity", "Severity of Illness Index", "Smoking", "South Africa", "Tobacco Use", "Young Adult" ]

[ "Expression of results and statistical analysis", "Demographics", "Disease severity (SDAI scores & disease duration at baseline)", "Risk factors", "Tobacco use", "HLA-SE" ]

[ "Results are expressed as the median values with interquartile ranges. Descriptive and inferential statistics techniques were used in the analyses. Tests for association of contingency tables were performed using two-tailed Chi2 tests. One-way ANOVA was performed using the Dunn and Kruskal-Wallis tests for non-parametric data for more than 2 groups, or the Mann-Whitney test when 2 groups were compared with Bonferroni correction where applicable. Correlation coefficients were derived from non-parametric Spearman's rank correlation test and correlation matrices using the Sidak's method p value correction for multiple testing. Statistical significance was determined by p-value <0.05 and confidence intervals of 95%. The analyses were done using STATA13.\nThe raw data is stored electronically for 15 years as per ethical requirement and is accessible by request to the corresponding author.", "The demographics of the group of RA patients, as well as seropositivity rates for the biomarkers and SE-related data are summarized in Table 1.\nPatient demographics, disease indices, tobacco use, HLA-SE categories and autoantibody positivity rates in the group of RA patients\nThe sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), prevalence rates and Likelihood Ratios for cRF, ACPA-IgG, RF-IgA and ACPA-IgA in the group of RA patients are shown in Table 2. These demonstrate that cRF and ACPA are the best overall predictors of disease. ACPA-IgA antibodies, on the other hand, have the best specificity and PPV of the group of biomarkers, but show lesser sensitivity and NPV in comparison with cRF and ACPA. Likelihood ratios for cRF and ACPA rank the highest with cRF at 9.32. Importantly, RF-IgA and ACPA-IgA were inferior to the traditional cRF and ACPA-IgG sero-diagnostic biomarkers recommended by the ACR 2010.\nSensitivities, Specificities, PPVs, NPVs and Likelihood Ratios for the various auto antibodies\nHighest results achieved are in BOLD\nThe overall combination of reactivities and cross-reactivities of all the auto-antibodies are demonstrated in a Venn diagram as Figure 1.\nVenn diagram of positive reactivity patterns of ACPA-IgG, cRF, ACPA-IgA RF-IgA individually as well as cross-reactivity patterns between these biomarkers.\nThe Venn diagram indicates that 31% (n=23) of RA patients had cRF and ACPA autoantibodies of both serotypes (IgG and IgA), while 48% (n=36) were positive for ACPA-IgG, cRF and RF-IgA, but not for ACPA-IgA.\nThe healthy control subjects had median values (iqr) of 16 (53) IU/ml and 2 (3) IU/ml for RF-IgA and ACPA-IgA, respectively. RF-IgA seropositivity amongst the healthy control subjects was 41% (95% CI: 19% – 67%), while ACPA-IgA demonstrated a positivity rate of 18% (95% CI: 5%–46%).", "Duration since onset of symptoms in months, SDAI scores and the presence of erosions at presentation were used as markers of disease severity. At baseline, 25% (n=19) of patients were scored as MDA and 75% (n=56) as HDA, while no patients were in remission or LDA. Linear regression analysis revealed a statistically significant positive relationship between ACPA-IgA and SDAI at baseline (p=0.030, R2=0.0646, β=0.3, α=6.1, CI: 0.03 – 0.58).", " Tobacco use Tobacco users (serum cotinine >10ng/ml) presented with a higher SDAI score at baseline than non-users (p=0.036) using the Mann-Whitney U-test. ACPA -IgA levels were higher in tobacco users than non-users (p=0.007) in the group of patients with disease duration of <12 months, but not in those with disease duration >12 months.\nTobacco users (serum cotinine >10ng/ml) presented with a higher SDAI score at baseline than non-users (p=0.036) using the Mann-Whitney U-test. ACPA -IgA levels were higher in tobacco users than non-users (p=0.007) in the group of patients with disease duration of <12 months, but not in those with disease duration >12 months.\n HLA-SE Table 1 indicates that 71 (95%) patients had an HLASE allele, of which 30 (40%) were heterozygous (SX) i.e. had 1 of the SE-associated alleles and 55% where homozygous (SS) for HLA-SE. Only 4 (5%) of the patients expressed no HLA-SE phenotype (XX) in keeping with previously described findings in this cohort of patients8. This showed that the 71 patients with HLA-SE alleles could be categorized as High Risk (HR) and Low Risk (LR), according to the amino acids at position 70 and 71. The HR and LR groups consisted of 48 (68%) and 23 (31%) patients respectively. A further subdivision consisting of homozygous, heterozygous, LR and HR HLASE allele permutations revealed the following groupings: i) 23 (31%) patients had both the LR and HR HLA-SE alleles (DUAL), ii) 10 (13%) were homozygous for the HR alleles (HoHR), iii) 17 (23%) were heterozygous for the HR alleles (HeHR), iii) 8 (11%) were homozygous for the LR alleles (HoLR), iv) 13 (17%) were heterozygous for the LR alleles (HeLR), and v) 4(5%) had no HLA-SE alleles.\nAs shown in Figure 2, statistically significant differences in serum levels of ACPA-IgG (p = 0.009) and ACPA-IgA (p=0.032) were noted when comparing the SS vs. SX HLA-SE allele expression groups, with the homozygous (SS) patients having consistently higher median autoantibody titres than their heterozygous (SX) counterparts. This was not apparent in the cases of both cRF and RF-IgA.\nMedian values of ACPA, and ACPA-IgA in patients categorised according to HLA-SE homozygosity (SS) and heterozygosity (SX).\nValues were standardised (z-scores) to display on common y-axis (standardisation: x* = (x-m)/SD\nWhere m is the mean of x, and SD is the standard deviation of x).\nAs shown in Figure 3A, the median titers of ACPA-IgA auto-antibodies in tobacco users show a trend, albeit insignificant (p=0.078), towards higher values, while the data shown in Figure 3B indicate an association between HLA-SE homo- and heterozygosity, tobacco use and ACPA-IgA, which reaches statistical significance only in the heterozygous group ( p=0.028). This may be explained by the presence of the HR modulatory gene present in 57% (n=17) present in the SX group, whereas in the SS group only 24% (n=10) of patients have the HR associated amino acid sequence. The majority 56% (n=23) of the SS patients express the LR modulatory gene in combination with the HR gene, with a further 20% (n=8) homozygous for LR. These findings are in agreement with the involvement of the HR HLA-SE genes in the immunopathogenesis of RA, potentiated by tobacco use.\nMedian ACPA-IgA values in RA tobacco users and non-users showing a trend towards higher ACPA-IgA titres in users of tobacco.\nMedian ACPA-IgA values in tobacco users and non-users categorised according to Shared Epitope homozygosity (SS) and heterozygosity (SX).\nTable 1 indicates that 71 (95%) patients had an HLASE allele, of which 30 (40%) were heterozygous (SX) i.e. had 1 of the SE-associated alleles and 55% where homozygous (SS) for HLA-SE. Only 4 (5%) of the patients expressed no HLA-SE phenotype (XX) in keeping with previously described findings in this cohort of patients8. This showed that the 71 patients with HLA-SE alleles could be categorized as High Risk (HR) and Low Risk (LR), according to the amino acids at position 70 and 71. The HR and LR groups consisted of 48 (68%) and 23 (31%) patients respectively. A further subdivision consisting of homozygous, heterozygous, LR and HR HLASE allele permutations revealed the following groupings: i) 23 (31%) patients had both the LR and HR HLA-SE alleles (DUAL), ii) 10 (13%) were homozygous for the HR alleles (HoHR), iii) 17 (23%) were heterozygous for the HR alleles (HeHR), iii) 8 (11%) were homozygous for the LR alleles (HoLR), iv) 13 (17%) were heterozygous for the LR alleles (HeLR), and v) 4(5%) had no HLA-SE alleles.\nAs shown in Figure 2, statistically significant differences in serum levels of ACPA-IgG (p = 0.009) and ACPA-IgA (p=0.032) were noted when comparing the SS vs. SX HLA-SE allele expression groups, with the homozygous (SS) patients having consistently higher median autoantibody titres than their heterozygous (SX) counterparts. This was not apparent in the cases of both cRF and RF-IgA.\nMedian values of ACPA, and ACPA-IgA in patients categorised according to HLA-SE homozygosity (SS) and heterozygosity (SX).\nValues were standardised (z-scores) to display on common y-axis (standardisation: x* = (x-m)/SD\nWhere m is the mean of x, and SD is the standard deviation of x).\nAs shown in Figure 3A, the median titers of ACPA-IgA auto-antibodies in tobacco users show a trend, albeit insignificant (p=0.078), towards higher values, while the data shown in Figure 3B indicate an association between HLA-SE homo- and heterozygosity, tobacco use and ACPA-IgA, which reaches statistical significance only in the heterozygous group ( p=0.028). This may be explained by the presence of the HR modulatory gene present in 57% (n=17) present in the SX group, whereas in the SS group only 24% (n=10) of patients have the HR associated amino acid sequence. The majority 56% (n=23) of the SS patients express the LR modulatory gene in combination with the HR gene, with a further 20% (n=8) homozygous for LR. These findings are in agreement with the involvement of the HR HLA-SE genes in the immunopathogenesis of RA, potentiated by tobacco use.\nMedian ACPA-IgA values in RA tobacco users and non-users showing a trend towards higher ACPA-IgA titres in users of tobacco.\nMedian ACPA-IgA values in tobacco users and non-users categorised according to Shared Epitope homozygosity (SS) and heterozygosity (SX).", "Tobacco users (serum cotinine >10ng/ml) presented with a higher SDAI score at baseline than non-users (p=0.036) using the Mann-Whitney U-test. ACPA -IgA levels were higher in tobacco users than non-users (p=0.007) in the group of patients with disease duration of <12 months, but not in those with disease duration >12 months.", "Table 1 indicates that 71 (95%) patients had an HLASE allele, of which 30 (40%) were heterozygous (SX) i.e. had 1 of the SE-associated alleles and 55% where homozygous (SS) for HLA-SE. Only 4 (5%) of the patients expressed no HLA-SE phenotype (XX) in keeping with previously described findings in this cohort of patients8. This showed that the 71 patients with HLA-SE alleles could be categorized as High Risk (HR) and Low Risk (LR), according to the amino acids at position 70 and 71. The HR and LR groups consisted of 48 (68%) and 23 (31%) patients respectively. A further subdivision consisting of homozygous, heterozygous, LR and HR HLASE allele permutations revealed the following groupings: i) 23 (31%) patients had both the LR and HR HLA-SE alleles (DUAL), ii) 10 (13%) were homozygous for the HR alleles (HoHR), iii) 17 (23%) were heterozygous for the HR alleles (HeHR), iii) 8 (11%) were homozygous for the LR alleles (HoLR), iv) 13 (17%) were heterozygous for the LR alleles (HeLR), and v) 4(5%) had no HLA-SE alleles.\nAs shown in Figure 2, statistically significant differences in serum levels of ACPA-IgG (p = 0.009) and ACPA-IgA (p=0.032) were noted when comparing the SS vs. SX HLA-SE allele expression groups, with the homozygous (SS) patients having consistently higher median autoantibody titres than their heterozygous (SX) counterparts. This was not apparent in the cases of both cRF and RF-IgA.\nMedian values of ACPA, and ACPA-IgA in patients categorised according to HLA-SE homozygosity (SS) and heterozygosity (SX).\nValues were standardised (z-scores) to display on common y-axis (standardisation: x* = (x-m)/SD\nWhere m is the mean of x, and SD is the standard deviation of x).\nAs shown in Figure 3A, the median titers of ACPA-IgA auto-antibodies in tobacco users show a trend, albeit insignificant (p=0.078), towards higher values, while the data shown in Figure 3B indicate an association between HLA-SE homo- and heterozygosity, tobacco use and ACPA-IgA, which reaches statistical significance only in the heterozygous group ( p=0.028). This may be explained by the presence of the HR modulatory gene present in 57% (n=17) present in the SX group, whereas in the SS group only 24% (n=10) of patients have the HR associated amino acid sequence. The majority 56% (n=23) of the SS patients express the LR modulatory gene in combination with the HR gene, with a further 20% (n=8) homozygous for LR. These findings are in agreement with the involvement of the HR HLA-SE genes in the immunopathogenesis of RA, potentiated by tobacco use.\nMedian ACPA-IgA values in RA tobacco users and non-users showing a trend towards higher ACPA-IgA titres in users of tobacco.\nMedian ACPA-IgA values in tobacco users and non-users categorised according to Shared Epitope homozygosity (SS) and heterozygosity (SX)." ]

[ null, null, null, null, null, null ]

[ "Introduction", "Patients & methods", "Expression of results and statistical analysis", "Results", "Demographics", "Disease severity (SDAI scores & disease duration at baseline)", "Risk factors", "Tobacco use", "HLA-SE", "Discussion" ]

[ "Rheumatoid arthritis (RA) is a crippling disease characterized by chronic inflammation of the articular joints of patients leading to cartilage destruction and disease progression. The incidence is around 1–3% of the general population with a significantly higher prevalence in females. The inclusion of measurement of rheumatoid factor (RF) and anti-citrullinated peptide (ACPA) antibodies, generally composite RF (cRF) and ACPA-IgG, in the 2010 ACR/EULAR RA classification criteria is an indication of the sero-diagnostic utility of measurement of these auto-antibodies in RA1 and is well established worldwide2–5.\nThe profile and complex interplay of various RA-associated auto-antibodies and their different isotypes is influenced by the ethnic, genetic and environmental factors, with RF isotypes having been shown to be associated with a poor prognosis in Chinese and Indian, but not in Malay RF patients6, while in Caucasians, ACPA-IgA have been shown to be associated with severe disease and radiographic progression11,12. However, less is known about the diagnostic and prognostic potential of measurement of ACPA and RF auto-antibodies of the IgA isotype in other ethnicities with RA, as well as their associations with prominent disease risk factors, specifically tobacco use (i.e. cigarettes and snuff) and positivity for the human leucocyte antigen - shared epitope (HLA-SE).\nThese issues have been addressed in the current study in which the levels and prevalence ACPA-IgA and RF-IgA auto-antibodies have been compared with those of ACPA-IgG and cRF in a cohort of disease-modifying anti-rheumatic drug (DMARD)-naïve, black, predominantly female, South African patients with RA, and a control group of healthy individuals, the findings of which may be broadly applicable to other populations.Additional aims included investigation of the relationships of these IgA auto-antibodies with: i) disease severity as measured by the simplified disease activity score (SDAI); and ii) genetic predisposition and tobacco use. As mentioned above these potentially important issues have not been investigated previously in our geographic region.", "The study was approved by the Research Ethics Committees of the Faculties of Health Sciences of the Universities of the Witwatersrand and Pretoria and conformed to good clinical and laboratory practice and the Helsinki declaration. Patients were recruited from the Chris Hani Baragwanath Academic hospital (CHBAH, Soweto, South Africa) and Steve Biko Academic Hospital (SBAH, Pretoria, South Africa) Rheumatology Out-patient clinics. Ethics certificate reference numbers: 09/2017 (UP) and M150513 (WITS).\nInformed consent, signed by each patient, was explained and monitored by the attending physician. Although the informed consent document was available only in English, a translator was present at both the CHBAH and SBAH Rheumatology clinics to ensure that patients fully understood the nature of their participation in the study. No financial compensation was offered, and it was explained to the participants that the results of this study may lead to better understanding of their disease. Healthy controls where anonymously sourced from the South African Blood Transfusion Service.\nThis study was a retrospective, cross-sectional study the primary objective of which was to determine the utility of measurement of immunoglobin A (IgA) rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (ACPA) in sero-diagnosis and prognosis of rheumatoid arthritis (RA), as well as their relationships with known risk factors and roles as predictive biomarkers of disease severity in black South African patients with RA. To our knowledge, the study is the first of its type undertaken in sub-Saharan Africa. The study cohort consisted of a sub-group (n=75) of black African patients that formed part of a previously described cohort7, all diagnosed with RA according to the 1987 ACR RA criteria, all of whom were DMARD-naïve at baseline. Methods for serological measurement cRF and ACPA-IgG have been described previously for this cohort of RA patients7–9.\nTwenty healthy individuals were used as controls, matched in so far as possible for age, ethnicity and gender to determine if commercial stated cut-off values for RF and ACPA IgA are valid, to assist in calculating specificity, sensitivity and positive/ negative predictors and cotinine levels to determine tobacco use cut-offs.\nVenous blood was collected in 2x5ml test tubes, 1 with added clotting factor and 1 containing the anti-coagulant, EDTA. The serum tubes were left to clot and centrifuged thereafter for 10 minutes at 2000 × g. The serum was removed, aliquoted and stored at −20°C until analyzed. The EDTA anti-coagulated blood was used the extract whole genomic DNA using the Maxwell Promega automated DNA extraction instrument and Promega Maxwell® 16 Tissue DNA purification kits (Promega Corporation, Madison, USA).\nClinical disease activity was recorded using the standard classification for SDAI; remission = ≤ 3, low disease activity (LDA) = >3 − ≤11, mild disease activity (MDA) = >11 - ≤26 and high disease activity (HDA) >2612.\nEliA ACPA-IgA and RF-IgA were measured using fluorescence enzyme immunoassay (FEIA) on the automated Phadia 250 platform (Phadia AB, Uppsala, Sweden). Cut-off values to determine positivity for the individual auto-antibodies were as follows: >10 units/millilitre (U/ml) serum for EliA ACPA-IgG and ACPA-IgA, >20 IU/ml for EliA RF IgA, >15 U/ml for cRF. High-sensitivity serum C-reactive protein (CRP) concentrations were measured by latex-enhanced laser nephelometry (Siemens South Africa, Midrand, South Africa) and the results expressed as micrograms (µg)/ml.\nCotinine was determined using an ELISA kit (Calbiotech Inc., Spring Valley, CA, USA). The results are expressed as nanograms (ng/ml). A cut-off above 10 ng/ml was taken as indicative of active tobacco use as recommended by the product insert.\nThe molecular procedures used for HLA-DRB1 typing and HLA-SE categorization have been described previously8. Briefly, purity and concentrations of the extracted DNA were determined, and molecular analysis carried out using a high-resolution rSSO typing technique utilising Luminex® bead technology for HLA-DRB1 alleles (LABType HD Class II DRB1 Typing Test, One-Lambda, Thermo Fisher Scientific, USA) and alleles assigned using One Lambda Fusion software. The HLA-SE has been classified and validated according to the RAA amino acid sequence at positions 72–74 of the third hypervariable region of the HLA-DRB1 gene and assigned SS if both HLA-DRB1 alleles express the RAA motif, or SX if only one HLA-DRB1 allele expresses the RAA motif. Further classification of the SE-sequence beyond the RAA amino acid sequences at positions 72 to 74 have a modulatory effect (positions 70 and 71). Glutamine (Q) or arginine (R) at position 70 of the 3rd hypervariable region of the HLA-DRB1 gene conferred a high risk of severe disease, while aspartic acid (D) is associated with low risk. Lysine (K) at position 71 was indicative of the highest risk, while arginine (R) alanine (A) or glutamic acid (E) were classified as low risk10.\n Expression of results and statistical analysis Results are expressed as the median values with interquartile ranges. Descriptive and inferential statistics techniques were used in the analyses. Tests for association of contingency tables were performed using two-tailed Chi2 tests. One-way ANOVA was performed using the Dunn and Kruskal-Wallis tests for non-parametric data for more than 2 groups, or the Mann-Whitney test when 2 groups were compared with Bonferroni correction where applicable. Correlation coefficients were derived from non-parametric Spearman's rank correlation test and correlation matrices using the Sidak's method p value correction for multiple testing. Statistical significance was determined by p-value <0.05 and confidence intervals of 95%. The analyses were done using STATA13.\nThe raw data is stored electronically for 15 years as per ethical requirement and is accessible by request to the corresponding author.\nResults are expressed as the median values with interquartile ranges. Descriptive and inferential statistics techniques were used in the analyses. Tests for association of contingency tables were performed using two-tailed Chi2 tests. One-way ANOVA was performed using the Dunn and Kruskal-Wallis tests for non-parametric data for more than 2 groups, or the Mann-Whitney test when 2 groups were compared with Bonferroni correction where applicable. Correlation coefficients were derived from non-parametric Spearman's rank correlation test and correlation matrices using the Sidak's method p value correction for multiple testing. Statistical significance was determined by p-value <0.05 and confidence intervals of 95%. The analyses were done using STATA13.\nThe raw data is stored electronically for 15 years as per ethical requirement and is accessible by request to the corresponding author.", "Results are expressed as the median values with interquartile ranges. Descriptive and inferential statistics techniques were used in the analyses. Tests for association of contingency tables were performed using two-tailed Chi2 tests. One-way ANOVA was performed using the Dunn and Kruskal-Wallis tests for non-parametric data for more than 2 groups, or the Mann-Whitney test when 2 groups were compared with Bonferroni correction where applicable. Correlation coefficients were derived from non-parametric Spearman's rank correlation test and correlation matrices using the Sidak's method p value correction for multiple testing. Statistical significance was determined by p-value <0.05 and confidence intervals of 95%. The analyses were done using STATA13.\nThe raw data is stored electronically for 15 years as per ethical requirement and is accessible by request to the corresponding author.", " Demographics The demographics of the group of RA patients, as well as seropositivity rates for the biomarkers and SE-related data are summarized in Table 1.\nPatient demographics, disease indices, tobacco use, HLA-SE categories and autoantibody positivity rates in the group of RA patients\nThe sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), prevalence rates and Likelihood Ratios for cRF, ACPA-IgG, RF-IgA and ACPA-IgA in the group of RA patients are shown in Table 2. These demonstrate that cRF and ACPA are the best overall predictors of disease. ACPA-IgA antibodies, on the other hand, have the best specificity and PPV of the group of biomarkers, but show lesser sensitivity and NPV in comparison with cRF and ACPA. Likelihood ratios for cRF and ACPA rank the highest with cRF at 9.32. Importantly, RF-IgA and ACPA-IgA were inferior to the traditional cRF and ACPA-IgG sero-diagnostic biomarkers recommended by the ACR 2010.\nSensitivities, Specificities, PPVs, NPVs and Likelihood Ratios for the various auto antibodies\nHighest results achieved are in BOLD\nThe overall combination of reactivities and cross-reactivities of all the auto-antibodies are demonstrated in a Venn diagram as Figure 1.\nVenn diagram of positive reactivity patterns of ACPA-IgG, cRF, ACPA-IgA RF-IgA individually as well as cross-reactivity patterns between these biomarkers.\nThe Venn diagram indicates that 31% (n=23) of RA patients had cRF and ACPA autoantibodies of both serotypes (IgG and IgA), while 48% (n=36) were positive for ACPA-IgG, cRF and RF-IgA, but not for ACPA-IgA.\nThe healthy control subjects had median values (iqr) of 16 (53) IU/ml and 2 (3) IU/ml for RF-IgA and ACPA-IgA, respectively. RF-IgA seropositivity amongst the healthy control subjects was 41% (95% CI: 19% – 67%), while ACPA-IgA demonstrated a positivity rate of 18% (95% CI: 5%–46%).\nThe demographics of the group of RA patients, as well as seropositivity rates for the biomarkers and SE-related data are summarized in Table 1.\nPatient demographics, disease indices, tobacco use, HLA-SE categories and autoantibody positivity rates in the group of RA patients\nThe sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), prevalence rates and Likelihood Ratios for cRF, ACPA-IgG, RF-IgA and ACPA-IgA in the group of RA patients are shown in Table 2. These demonstrate that cRF and ACPA are the best overall predictors of disease. ACPA-IgA antibodies, on the other hand, have the best specificity and PPV of the group of biomarkers, but show lesser sensitivity and NPV in comparison with cRF and ACPA. Likelihood ratios for cRF and ACPA rank the highest with cRF at 9.32. Importantly, RF-IgA and ACPA-IgA were inferior to the traditional cRF and ACPA-IgG sero-diagnostic biomarkers recommended by the ACR 2010.\nSensitivities, Specificities, PPVs, NPVs and Likelihood Ratios for the various auto antibodies\nHighest results achieved are in BOLD\nThe overall combination of reactivities and cross-reactivities of all the auto-antibodies are demonstrated in a Venn diagram as Figure 1.\nVenn diagram of positive reactivity patterns of ACPA-IgG, cRF, ACPA-IgA RF-IgA individually as well as cross-reactivity patterns between these biomarkers.\nThe Venn diagram indicates that 31% (n=23) of RA patients had cRF and ACPA autoantibodies of both serotypes (IgG and IgA), while 48% (n=36) were positive for ACPA-IgG, cRF and RF-IgA, but not for ACPA-IgA.\nThe healthy control subjects had median values (iqr) of 16 (53) IU/ml and 2 (3) IU/ml for RF-IgA and ACPA-IgA, respectively. RF-IgA seropositivity amongst the healthy control subjects was 41% (95% CI: 19% – 67%), while ACPA-IgA demonstrated a positivity rate of 18% (95% CI: 5%–46%).\n Disease severity (SDAI scores & disease duration at baseline) Duration since onset of symptoms in months, SDAI scores and the presence of erosions at presentation were used as markers of disease severity. At baseline, 25% (n=19) of patients were scored as MDA and 75% (n=56) as HDA, while no patients were in remission or LDA. Linear regression analysis revealed a statistically significant positive relationship between ACPA-IgA and SDAI at baseline (p=0.030, R2=0.0646, β=0.3, α=6.1, CI: 0.03 – 0.58).\nDuration since onset of symptoms in months, SDAI scores and the presence of erosions at presentation were used as markers of disease severity. At baseline, 25% (n=19) of patients were scored as MDA and 75% (n=56) as HDA, while no patients were in remission or LDA. Linear regression analysis revealed a statistically significant positive relationship between ACPA-IgA and SDAI at baseline (p=0.030, R2=0.0646, β=0.3, α=6.1, CI: 0.03 – 0.58).\n Risk factors Tobacco use Tobacco users (serum cotinine >10ng/ml) presented with a higher SDAI score at baseline than non-users (p=0.036) using the Mann-Whitney U-test. ACPA -IgA levels were higher in tobacco users than non-users (p=0.007) in the group of patients with disease duration of <12 months, but not in those with disease duration >12 months.\nTobacco users (serum cotinine >10ng/ml) presented with a higher SDAI score at baseline than non-users (p=0.036) using the Mann-Whitney U-test. ACPA -IgA levels were higher in tobacco users than non-users (p=0.007) in the group of patients with disease duration of <12 months, but not in those with disease duration >12 months.\n HLA-SE Table 1 indicates that 71 (95%) patients had an HLASE allele, of which 30 (40%) were heterozygous (SX) i.e. had 1 of the SE-associated alleles and 55% where homozygous (SS) for HLA-SE. Only 4 (5%) of the patients expressed no HLA-SE phenotype (XX) in keeping with previously described findings in this cohort of patients8. This showed that the 71 patients with HLA-SE alleles could be categorized as High Risk (HR) and Low Risk (LR), according to the amino acids at position 70 and 71. The HR and LR groups consisted of 48 (68%) and 23 (31%) patients respectively. A further subdivision consisting of homozygous, heterozygous, LR and HR HLASE allele permutations revealed the following groupings: i) 23 (31%) patients had both the LR and HR HLA-SE alleles (DUAL), ii) 10 (13%) were homozygous for the HR alleles (HoHR), iii) 17 (23%) were heterozygous for the HR alleles (HeHR), iii) 8 (11%) were homozygous for the LR alleles (HoLR), iv) 13 (17%) were heterozygous for the LR alleles (HeLR), and v) 4(5%) had no HLA-SE alleles.\nAs shown in Figure 2, statistically significant differences in serum levels of ACPA-IgG (p = 0.009) and ACPA-IgA (p=0.032) were noted when comparing the SS vs. SX HLA-SE allele expression groups, with the homozygous (SS) patients having consistently higher median autoantibody titres than their heterozygous (SX) counterparts. This was not apparent in the cases of both cRF and RF-IgA.\nMedian values of ACPA, and ACPA-IgA in patients categorised according to HLA-SE homozygosity (SS) and heterozygosity (SX).\nValues were standardised (z-scores) to display on common y-axis (standardisation: x* = (x-m)/SD\nWhere m is the mean of x, and SD is the standard deviation of x).\nAs shown in Figure 3A, the median titers of ACPA-IgA auto-antibodies in tobacco users show a trend, albeit insignificant (p=0.078), towards higher values, while the data shown in Figure 3B indicate an association between HLA-SE homo- and heterozygosity, tobacco use and ACPA-IgA, which reaches statistical significance only in the heterozygous group ( p=0.028). This may be explained by the presence of the HR modulatory gene present in 57% (n=17) present in the SX group, whereas in the SS group only 24% (n=10) of patients have the HR associated amino acid sequence. The majority 56% (n=23) of the SS patients express the LR modulatory gene in combination with the HR gene, with a further 20% (n=8) homozygous for LR. These findings are in agreement with the involvement of the HR HLA-SE genes in the immunopathogenesis of RA, potentiated by tobacco use.\nMedian ACPA-IgA values in RA tobacco users and non-users showing a trend towards higher ACPA-IgA titres in users of tobacco.\nMedian ACPA-IgA values in tobacco users and non-users categorised according to Shared Epitope homozygosity (SS) and heterozygosity (SX).\nTable 1 indicates that 71 (95%) patients had an HLASE allele, of which 30 (40%) were heterozygous (SX) i.e. had 1 of the SE-associated alleles and 55% where homozygous (SS) for HLA-SE. Only 4 (5%) of the patients expressed no HLA-SE phenotype (XX) in keeping with previously described findings in this cohort of patients8. This showed that the 71 patients with HLA-SE alleles could be categorized as High Risk (HR) and Low Risk (LR), according to the amino acids at position 70 and 71. The HR and LR groups consisted of 48 (68%) and 23 (31%) patients respectively. A further subdivision consisting of homozygous, heterozygous, LR and HR HLASE allele permutations revealed the following groupings: i) 23 (31%) patients had both the LR and HR HLA-SE alleles (DUAL), ii) 10 (13%) were homozygous for the HR alleles (HoHR), iii) 17 (23%) were heterozygous for the HR alleles (HeHR), iii) 8 (11%) were homozygous for the LR alleles (HoLR), iv) 13 (17%) were heterozygous for the LR alleles (HeLR), and v) 4(5%) had no HLA-SE alleles.\nAs shown in Figure 2, statistically significant differences in serum levels of ACPA-IgG (p = 0.009) and ACPA-IgA (p=0.032) were noted when comparing the SS vs. SX HLA-SE allele expression groups, with the homozygous (SS) patients having consistently higher median autoantibody titres than their heterozygous (SX) counterparts. This was not apparent in the cases of both cRF and RF-IgA.\nMedian values of ACPA, and ACPA-IgA in patients categorised according to HLA-SE homozygosity (SS) and heterozygosity (SX).\nValues were standardised (z-scores) to display on common y-axis (standardisation: x* = (x-m)/SD\nWhere m is the mean of x, and SD is the standard deviation of x).\nAs shown in Figure 3A, the median titers of ACPA-IgA auto-antibodies in tobacco users show a trend, albeit insignificant (p=0.078), towards higher values, while the data shown in Figure 3B indicate an association between HLA-SE homo- and heterozygosity, tobacco use and ACPA-IgA, which reaches statistical significance only in the heterozygous group ( p=0.028). This may be explained by the presence of the HR modulatory gene present in 57% (n=17) present in the SX group, whereas in the SS group only 24% (n=10) of patients have the HR associated amino acid sequence. The majority 56% (n=23) of the SS patients express the LR modulatory gene in combination with the HR gene, with a further 20% (n=8) homozygous for LR. These findings are in agreement with the involvement of the HR HLA-SE genes in the immunopathogenesis of RA, potentiated by tobacco use.\nMedian ACPA-IgA values in RA tobacco users and non-users showing a trend towards higher ACPA-IgA titres in users of tobacco.\nMedian ACPA-IgA values in tobacco users and non-users categorised according to Shared Epitope homozygosity (SS) and heterozygosity (SX).\n Tobacco use Tobacco users (serum cotinine >10ng/ml) presented with a higher SDAI score at baseline than non-users (p=0.036) using the Mann-Whitney U-test. ACPA -IgA levels were higher in tobacco users than non-users (p=0.007) in the group of patients with disease duration of <12 months, but not in those with disease duration >12 months.\nTobacco users (serum cotinine >10ng/ml) presented with a higher SDAI score at baseline than non-users (p=0.036) using the Mann-Whitney U-test. ACPA -IgA levels were higher in tobacco users than non-users (p=0.007) in the group of patients with disease duration of <12 months, but not in those with disease duration >12 months.\n HLA-SE Table 1 indicates that 71 (95%) patients had an HLASE allele, of which 30 (40%) were heterozygous (SX) i.e. had 1 of the SE-associated alleles and 55% where homozygous (SS) for HLA-SE. Only 4 (5%) of the patients expressed no HLA-SE phenotype (XX) in keeping with previously described findings in this cohort of patients8. This showed that the 71 patients with HLA-SE alleles could be categorized as High Risk (HR) and Low Risk (LR), according to the amino acids at position 70 and 71. The HR and LR groups consisted of 48 (68%) and 23 (31%) patients respectively. A further subdivision consisting of homozygous, heterozygous, LR and HR HLASE allele permutations revealed the following groupings: i) 23 (31%) patients had both the LR and HR HLA-SE alleles (DUAL), ii) 10 (13%) were homozygous for the HR alleles (HoHR), iii) 17 (23%) were heterozygous for the HR alleles (HeHR), iii) 8 (11%) were homozygous for the LR alleles (HoLR), iv) 13 (17%) were heterozygous for the LR alleles (HeLR), and v) 4(5%) had no HLA-SE alleles.\nAs shown in Figure 2, statistically significant differences in serum levels of ACPA-IgG (p = 0.009) and ACPA-IgA (p=0.032) were noted when comparing the SS vs. SX HLA-SE allele expression groups, with the homozygous (SS) patients having consistently higher median autoantibody titres than their heterozygous (SX) counterparts. This was not apparent in the cases of both cRF and RF-IgA.\nMedian values of ACPA, and ACPA-IgA in patients categorised according to HLA-SE homozygosity (SS) and heterozygosity (SX).\nValues were standardised (z-scores) to display on common y-axis (standardisation: x* = (x-m)/SD\nWhere m is the mean of x, and SD is the standard deviation of x).\nAs shown in Figure 3A, the median titers of ACPA-IgA auto-antibodies in tobacco users show a trend, albeit insignificant (p=0.078), towards higher values, while the data shown in Figure 3B indicate an association between HLA-SE homo- and heterozygosity, tobacco use and ACPA-IgA, which reaches statistical significance only in the heterozygous group ( p=0.028). This may be explained by the presence of the HR modulatory gene present in 57% (n=17) present in the SX group, whereas in the SS group only 24% (n=10) of patients have the HR associated amino acid sequence. The majority 56% (n=23) of the SS patients express the LR modulatory gene in combination with the HR gene, with a further 20% (n=8) homozygous for LR. These findings are in agreement with the involvement of the HR HLA-SE genes in the immunopathogenesis of RA, potentiated by tobacco use.\nMedian ACPA-IgA values in RA tobacco users and non-users showing a trend towards higher ACPA-IgA titres in users of tobacco.\nMedian ACPA-IgA values in tobacco users and non-users categorised according to Shared Epitope homozygosity (SS) and heterozygosity (SX).\nTable 1 indicates that 71 (95%) patients had an HLASE allele, of which 30 (40%) were heterozygous (SX) i.e. had 1 of the SE-associated alleles and 55% where homozygous (SS) for HLA-SE. Only 4 (5%) of the patients expressed no HLA-SE phenotype (XX) in keeping with previously described findings in this cohort of patients8. This showed that the 71 patients with HLA-SE alleles could be categorized as High Risk (HR) and Low Risk (LR), according to the amino acids at position 70 and 71. The HR and LR groups consisted of 48 (68%) and 23 (31%) patients respectively. A further subdivision consisting of homozygous, heterozygous, LR and HR HLASE allele permutations revealed the following groupings: i) 23 (31%) patients had both the LR and HR HLA-SE alleles (DUAL), ii) 10 (13%) were homozygous for the HR alleles (HoHR), iii) 17 (23%) were heterozygous for the HR alleles (HeHR), iii) 8 (11%) were homozygous for the LR alleles (HoLR), iv) 13 (17%) were heterozygous for the LR alleles (HeLR), and v) 4(5%) had no HLA-SE alleles.\nAs shown in Figure 2, statistically significant differences in serum levels of ACPA-IgG (p = 0.009) and ACPA-IgA (p=0.032) were noted when comparing the SS vs. SX HLA-SE allele expression groups, with the homozygous (SS) patients having consistently higher median autoantibody titres than their heterozygous (SX) counterparts. This was not apparent in the cases of both cRF and RF-IgA.\nMedian values of ACPA, and ACPA-IgA in patients categorised according to HLA-SE homozygosity (SS) and heterozygosity (SX).\nValues were standardised (z-scores) to display on common y-axis (standardisation: x* = (x-m)/SD\nWhere m is the mean of x, and SD is the standard deviation of x).\nAs shown in Figure 3A, the median titers of ACPA-IgA auto-antibodies in tobacco users show a trend, albeit insignificant (p=0.078), towards higher values, while the data shown in Figure 3B indicate an association between HLA-SE homo- and heterozygosity, tobacco use and ACPA-IgA, which reaches statistical significance only in the heterozygous group ( p=0.028). This may be explained by the presence of the HR modulatory gene present in 57% (n=17) present in the SX group, whereas in the SS group only 24% (n=10) of patients have the HR associated amino acid sequence. The majority 56% (n=23) of the SS patients express the LR modulatory gene in combination with the HR gene, with a further 20% (n=8) homozygous for LR. These findings are in agreement with the involvement of the HR HLA-SE genes in the immunopathogenesis of RA, potentiated by tobacco use.\nMedian ACPA-IgA values in RA tobacco users and non-users showing a trend towards higher ACPA-IgA titres in users of tobacco.\nMedian ACPA-IgA values in tobacco users and non-users categorised according to Shared Epitope homozygosity (SS) and heterozygosity (SX).", "The demographics of the group of RA patients, as well as seropositivity rates for the biomarkers and SE-related data are summarized in Table 1.\nPatient demographics, disease indices, tobacco use, HLA-SE categories and autoantibody positivity rates in the group of RA patients\nThe sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), prevalence rates and Likelihood Ratios for cRF, ACPA-IgG, RF-IgA and ACPA-IgA in the group of RA patients are shown in Table 2. These demonstrate that cRF and ACPA are the best overall predictors of disease. ACPA-IgA antibodies, on the other hand, have the best specificity and PPV of the group of biomarkers, but show lesser sensitivity and NPV in comparison with cRF and ACPA. Likelihood ratios for cRF and ACPA rank the highest with cRF at 9.32. Importantly, RF-IgA and ACPA-IgA were inferior to the traditional cRF and ACPA-IgG sero-diagnostic biomarkers recommended by the ACR 2010.\nSensitivities, Specificities, PPVs, NPVs and Likelihood Ratios for the various auto antibodies\nHighest results achieved are in BOLD\nThe overall combination of reactivities and cross-reactivities of all the auto-antibodies are demonstrated in a Venn diagram as Figure 1.\nVenn diagram of positive reactivity patterns of ACPA-IgG, cRF, ACPA-IgA RF-IgA individually as well as cross-reactivity patterns between these biomarkers.\nThe Venn diagram indicates that 31% (n=23) of RA patients had cRF and ACPA autoantibodies of both serotypes (IgG and IgA), while 48% (n=36) were positive for ACPA-IgG, cRF and RF-IgA, but not for ACPA-IgA.\nThe healthy control subjects had median values (iqr) of 16 (53) IU/ml and 2 (3) IU/ml for RF-IgA and ACPA-IgA, respectively. RF-IgA seropositivity amongst the healthy control subjects was 41% (95% CI: 19% – 67%), while ACPA-IgA demonstrated a positivity rate of 18% (95% CI: 5%–46%).", "Duration since onset of symptoms in months, SDAI scores and the presence of erosions at presentation were used as markers of disease severity. At baseline, 25% (n=19) of patients were scored as MDA and 75% (n=56) as HDA, while no patients were in remission or LDA. Linear regression analysis revealed a statistically significant positive relationship between ACPA-IgA and SDAI at baseline (p=0.030, R2=0.0646, β=0.3, α=6.1, CI: 0.03 – 0.58).", " Tobacco use Tobacco users (serum cotinine >10ng/ml) presented with a higher SDAI score at baseline than non-users (p=0.036) using the Mann-Whitney U-test. ACPA -IgA levels were higher in tobacco users than non-users (p=0.007) in the group of patients with disease duration of <12 months, but not in those with disease duration >12 months.\nTobacco users (serum cotinine >10ng/ml) presented with a higher SDAI score at baseline than non-users (p=0.036) using the Mann-Whitney U-test. ACPA -IgA levels were higher in tobacco users than non-users (p=0.007) in the group of patients with disease duration of <12 months, but not in those with disease duration >12 months.\n HLA-SE Table 1 indicates that 71 (95%) patients had an HLASE allele, of which 30 (40%) were heterozygous (SX) i.e. had 1 of the SE-associated alleles and 55% where homozygous (SS) for HLA-SE. Only 4 (5%) of the patients expressed no HLA-SE phenotype (XX) in keeping with previously described findings in this cohort of patients8. This showed that the 71 patients with HLA-SE alleles could be categorized as High Risk (HR) and Low Risk (LR), according to the amino acids at position 70 and 71. The HR and LR groups consisted of 48 (68%) and 23 (31%) patients respectively. A further subdivision consisting of homozygous, heterozygous, LR and HR HLASE allele permutations revealed the following groupings: i) 23 (31%) patients had both the LR and HR HLA-SE alleles (DUAL), ii) 10 (13%) were homozygous for the HR alleles (HoHR), iii) 17 (23%) were heterozygous for the HR alleles (HeHR), iii) 8 (11%) were homozygous for the LR alleles (HoLR), iv) 13 (17%) were heterozygous for the LR alleles (HeLR), and v) 4(5%) had no HLA-SE alleles.\nAs shown in Figure 2, statistically significant differences in serum levels of ACPA-IgG (p = 0.009) and ACPA-IgA (p=0.032) were noted when comparing the SS vs. SX HLA-SE allele expression groups, with the homozygous (SS) patients having consistently higher median autoantibody titres than their heterozygous (SX) counterparts. This was not apparent in the cases of both cRF and RF-IgA.\nMedian values of ACPA, and ACPA-IgA in patients categorised according to HLA-SE homozygosity (SS) and heterozygosity (SX).\nValues were standardised (z-scores) to display on common y-axis (standardisation: x* = (x-m)/SD\nWhere m is the mean of x, and SD is the standard deviation of x).\nAs shown in Figure 3A, the median titers of ACPA-IgA auto-antibodies in tobacco users show a trend, albeit insignificant (p=0.078), towards higher values, while the data shown in Figure 3B indicate an association between HLA-SE homo- and heterozygosity, tobacco use and ACPA-IgA, which reaches statistical significance only in the heterozygous group ( p=0.028). This may be explained by the presence of the HR modulatory gene present in 57% (n=17) present in the SX group, whereas in the SS group only 24% (n=10) of patients have the HR associated amino acid sequence. The majority 56% (n=23) of the SS patients express the LR modulatory gene in combination with the HR gene, with a further 20% (n=8) homozygous for LR. These findings are in agreement with the involvement of the HR HLA-SE genes in the immunopathogenesis of RA, potentiated by tobacco use.\nMedian ACPA-IgA values in RA tobacco users and non-users showing a trend towards higher ACPA-IgA titres in users of tobacco.\nMedian ACPA-IgA values in tobacco users and non-users categorised according to Shared Epitope homozygosity (SS) and heterozygosity (SX).\nTable 1 indicates that 71 (95%) patients had an HLASE allele, of which 30 (40%) were heterozygous (SX) i.e. had 1 of the SE-associated alleles and 55% where homozygous (SS) for HLA-SE. Only 4 (5%) of the patients expressed no HLA-SE phenotype (XX) in keeping with previously described findings in this cohort of patients8. This showed that the 71 patients with HLA-SE alleles could be categorized as High Risk (HR) and Low Risk (LR), according to the amino acids at position 70 and 71. The HR and LR groups consisted of 48 (68%) and 23 (31%) patients respectively. A further subdivision consisting of homozygous, heterozygous, LR and HR HLASE allele permutations revealed the following groupings: i) 23 (31%) patients had both the LR and HR HLA-SE alleles (DUAL), ii) 10 (13%) were homozygous for the HR alleles (HoHR), iii) 17 (23%) were heterozygous for the HR alleles (HeHR), iii) 8 (11%) were homozygous for the LR alleles (HoLR), iv) 13 (17%) were heterozygous for the LR alleles (HeLR), and v) 4(5%) had no HLA-SE alleles.\nAs shown in Figure 2, statistically significant differences in serum levels of ACPA-IgG (p = 0.009) and ACPA-IgA (p=0.032) were noted when comparing the SS vs. SX HLA-SE allele expression groups, with the homozygous (SS) patients having consistently higher median autoantibody titres than their heterozygous (SX) counterparts. This was not apparent in the cases of both cRF and RF-IgA.\nMedian values of ACPA, and ACPA-IgA in patients categorised according to HLA-SE homozygosity (SS) and heterozygosity (SX).\nValues were standardised (z-scores) to display on common y-axis (standardisation: x* = (x-m)/SD\nWhere m is the mean of x, and SD is the standard deviation of x).\nAs shown in Figure 3A, the median titers of ACPA-IgA auto-antibodies in tobacco users show a trend, albeit insignificant (p=0.078), towards higher values, while the data shown in Figure 3B indicate an association between HLA-SE homo- and heterozygosity, tobacco use and ACPA-IgA, which reaches statistical significance only in the heterozygous group ( p=0.028). This may be explained by the presence of the HR modulatory gene present in 57% (n=17) present in the SX group, whereas in the SS group only 24% (n=10) of patients have the HR associated amino acid sequence. The majority 56% (n=23) of the SS patients express the LR modulatory gene in combination with the HR gene, with a further 20% (n=8) homozygous for LR. These findings are in agreement with the involvement of the HR HLA-SE genes in the immunopathogenesis of RA, potentiated by tobacco use.\nMedian ACPA-IgA values in RA tobacco users and non-users showing a trend towards higher ACPA-IgA titres in users of tobacco.\nMedian ACPA-IgA values in tobacco users and non-users categorised according to Shared Epitope homozygosity (SS) and heterozygosity (SX).", "Tobacco users (serum cotinine >10ng/ml) presented with a higher SDAI score at baseline than non-users (p=0.036) using the Mann-Whitney U-test. ACPA -IgA levels were higher in tobacco users than non-users (p=0.007) in the group of patients with disease duration of <12 months, but not in those with disease duration >12 months.", "Table 1 indicates that 71 (95%) patients had an HLASE allele, of which 30 (40%) were heterozygous (SX) i.e. had 1 of the SE-associated alleles and 55% where homozygous (SS) for HLA-SE. Only 4 (5%) of the patients expressed no HLA-SE phenotype (XX) in keeping with previously described findings in this cohort of patients8. This showed that the 71 patients with HLA-SE alleles could be categorized as High Risk (HR) and Low Risk (LR), according to the amino acids at position 70 and 71. The HR and LR groups consisted of 48 (68%) and 23 (31%) patients respectively. A further subdivision consisting of homozygous, heterozygous, LR and HR HLASE allele permutations revealed the following groupings: i) 23 (31%) patients had both the LR and HR HLA-SE alleles (DUAL), ii) 10 (13%) were homozygous for the HR alleles (HoHR), iii) 17 (23%) were heterozygous for the HR alleles (HeHR), iii) 8 (11%) were homozygous for the LR alleles (HoLR), iv) 13 (17%) were heterozygous for the LR alleles (HeLR), and v) 4(5%) had no HLA-SE alleles.\nAs shown in Figure 2, statistically significant differences in serum levels of ACPA-IgG (p = 0.009) and ACPA-IgA (p=0.032) were noted when comparing the SS vs. SX HLA-SE allele expression groups, with the homozygous (SS) patients having consistently higher median autoantibody titres than their heterozygous (SX) counterparts. This was not apparent in the cases of both cRF and RF-IgA.\nMedian values of ACPA, and ACPA-IgA in patients categorised according to HLA-SE homozygosity (SS) and heterozygosity (SX).\nValues were standardised (z-scores) to display on common y-axis (standardisation: x* = (x-m)/SD\nWhere m is the mean of x, and SD is the standard deviation of x).\nAs shown in Figure 3A, the median titers of ACPA-IgA auto-antibodies in tobacco users show a trend, albeit insignificant (p=0.078), towards higher values, while the data shown in Figure 3B indicate an association between HLA-SE homo- and heterozygosity, tobacco use and ACPA-IgA, which reaches statistical significance only in the heterozygous group ( p=0.028). This may be explained by the presence of the HR modulatory gene present in 57% (n=17) present in the SX group, whereas in the SS group only 24% (n=10) of patients have the HR associated amino acid sequence. The majority 56% (n=23) of the SS patients express the LR modulatory gene in combination with the HR gene, with a further 20% (n=8) homozygous for LR. These findings are in agreement with the involvement of the HR HLA-SE genes in the immunopathogenesis of RA, potentiated by tobacco use.\nMedian ACPA-IgA values in RA tobacco users and non-users showing a trend towards higher ACPA-IgA titres in users of tobacco.\nMedian ACPA-IgA values in tobacco users and non-users categorised according to Shared Epitope homozygosity (SS) and heterozygosity (SX).", "The current study demonstrates a lack of diagnostic or predictive value of measurement of RF-IgA relative to that of cRF in this study group of DMARD-naïve, African RA patients. Measurement of ACPA-IgA, on the other hand, demonstrated an extremely high specificity and positive predictive value, as well as a positive association with disease activity, but was notably lacking in both sensitivity and negative predictive value in comparison with both ACPA-IgG and cRF. Assuming that these observations can be extrapolated to other population groups, they appear to argue against the clinical utility of measurement of either RF-IgA or ACPA-IgA as diagnostic adjuncts to cRF and ACPA11,12.\nInterestingly, however, tobacco usage, which was associated with higher disease activity on presentation, was also associated with higher levels of ACPA-IgA, specifically in those RA patients categorized as having disease duration of <12 months. Higher levels of ACPA-IgA prior to disease onset may be a reflection of epitope spreading and isotype expansion, a phenomenon well described in RA13 In addition, when comparing RA tobacco users with RA non-tobacco users, a statistically significant, positive association between smoking, SE genotype and serum concentrations of ACPA-IgA was also evident14–16. These observations are of possible significance in relation to the role of smoking and HLA genotype in the immunopathogenesis of RA, seemingly strengthening the link with smoking-related protein citrullination in the airway mucosa17–19, and possibly the gastrointestinal mucosa20,21, resulting in interactions of citrullinated proteins with SE-expressing T lymphocytes, leading to the production of potentially pathogenic ACPA-IgA auto-antibodies13. In this context, it is, however, noteworthy, that tobacco usage in the current cohort of RA patients is predominantly inhalation of powdered tobacco (snuff), as opposed to active smoking, with both types of tobacco usage resulting in equivalent circulating concentrations of cotinine22, suggesting, that like active smoking, usage of smokeless tobacco products, particularly those that are inhaled, may also predispose to development of RA.\nNotwithstanding the relatively small number of patients, which is a limitation of this study, it is, however, noteworthy that similar sized or smaller cohort studies have been published in a number of peer-reviewed journals as illustrated in the systematic review article by Peter Taylor et al.22 The findings of our study do, however, appear to indicate the lack of superiority of RF-IgA and ACPA-IgA over cRF and ACPA-IgG in the sero-diagnosis of RA. On the other hand, the finding of associations of ACPA-IgA with tobacco usage and HLA genotype indicates that this augmentative combination of risk factors for development and severity of RA in Black South Africans is similar to that described for their counterparts of Caucasian origin. Clearly, usage of any type of tobacco product is to be discouraged in those identified as being at high risk for development of RA, or who are in the early stages of the disease." ]

[ "intro", "subjects|methods", null, "results", null, null, null, null, null, "discussion" ]

[ "Tobacco usage", "genetic predisposition", "immunoglobulin A", "anti-citrullinated peptide", "South Africa", "rheumatoid arthritis patients" ]

[ 155, 411, 90, 1453, 73, 648 ]

[ "acpa", "iga", "patients", "acpa iga", "se", "hla", "tobacco", "hla se", "users", "hr" ]

[ "associated auto antibodies", "peptide acpa antibodies", "rheumatoid arthritis ra", "antibodies compared acpa", "iga rheumatoid factor" ]

[ "Australia", "COVID-19", "COVID-19 Testing", "Humans", "Tertiary Care Centers", "Tomography, X-Ray Computed" ]

[ "Introduction", "Chest radiograph descriptors", "CT descriptors", "V/Q scan descriptors", "Evaluation of readers", "Chest radiograph findings", "CT findings", "V/Q scan findings", "Evaluation of readers" ]

[ "Coronavirus disease 2019 (COVID‐19) was first described in late 2019 where the first cluster of cases was reported in Wuhan, Hubei Province, China.\n1\n COVID‐19 has since spread globally and on 11 March, 2020 the World Health Organisation (WHO) officially characterised COVID‐19 as a pandemic. As of 3rd October 2021, over 215 million individuals have been infected worldwide resulting in significant mortality and morbidity. The first case of COVID‐19 in Australia was confirmed on the 25th January 2020, and by 3rd October 2021 Australia has had more than 110,000 cases which have resulted in over 1000 deaths.\nChest radiographs (CXR) and computed tomography (CT) have frequently been utilised in assistance with diagnosis and assessment of progression of disease. A multinational consensus statement from the Fleischner society describes the role of chest imaging in the management of COVID‐19, recommending that imaging is not indicated in patients suspected of having COVID‐19 with mild clinical features, but is recommended for patients at risk for disease progression and in those with worsening respiratory status.\n2\n Imaging of patients with COVID‐19 infection has raised challenges in ensuring that studies are performed appropriately whilst ensuring the safety of health personnel.\nWhilst the CXR is low in sensitivity, it can be highly suggestive of COVID‐19 in the correct clinical setting when the radiographs exhibit characteristic COVID‐19 findings.\n3\n Typical findings include a peripheral mid to lower zone lung distribution of opacification.\n3\n, \n4\n\n\nCT imaging is not recommended for screening or as a first line test to diagnose COVID‐19 and should be used sparingly, for hospitalised, symptomatic patients with specific clinical indications.\n5\n, \n6\n The Royal Australian and New Zealand College of Radiologists (RANZCR) recommendation for CT technique in the setting of COVID‐19 is a volumetric CT of the entire chest at end inspiration, with reconstruction of contiguous high‐resolution (HR) images at 0.625–1.5 mm for assessment of the lungs. Studies have demonstrated that COVID‐19 can cause microvascular injuries resulting in a procoagulant state.\n5\n The role of computed tomography pulmonary angiography (CTPA) is reserved for the detection of pulmonary emboli in individuals who have clinical deterioration.\n7\n Characteristic CT findings include bilateral, peripheral and basal predominant ground glass opacities and consolidation.\n8\n Consolidation superimposed on ground glass opacity is found in a small number of cases, predominantly in the elderly.\n7\n\n\nThis study describes the imaging findings of 180 patients who presented to The Royal Melbourne Hospital (an Australian tertiary hospital), evaluating the patterns demonstrated on CXR, CT and ventilation perfusion (V/Q) scans.", "Chest radiograph descriptors were divided into zone involvement, distribution and findings. The chest radiograph was divided into six zones, right upper, right middle, right lower, left upper, left middle and left lower (Fig. 1). This method of dividing the lungs into thirds is commonly used in reporting and has been proposed for the reporting of COVID‐19 chest radiographs.\n9\n\n\nThe chest X‐rays were distributed into six zones as illustrated.\nThe distribution of the lung changes were also classified into three categories: peripheral, central and diffuse (Fig. 2). Peripheral distribution was selected when only peripheral opacities were present. Central distribution was selected when only central opacities were present. A diffuse distribution was selected if there were opacities that either crossed both regions or were both peripheral and central. Figure 3 demonstrates a chest radiograph with a peripheral distribution.\nDescriptors for the distribution of opacity.\nChest radiograph with a peripheral distribution.\nOther findings recorded included the presence of pleural effusions or interstitial change.", "CT characteristics were classified according to pattern, distribution, lobe involvement and other findings. The CT descriptors were used for all chest CT imaging including pre‐ and post‐contrast CT chests such as pulmonary angiograms.\nCT patterns reviewed were based on patterns described in the literature.\n10\n Patterns that were included were ground glass opacities, consolidation, ground glass with consolidation, crazy paving pattern, reverse halo, interlobular septal thickening and air bronchograms.\nDistribution on CT was divided into peripheral, central, peribronchovascular or diffuse. Similar to the chest radiograph distribution, if more than one pattern was involved the diffuse descriptor for distribution was selected. Lobar involvement was also recorded based on the normal anatomical division of the right and left lung lobes (right upper, middle and lower and left upper and lower). Each lobe involved in the CT was selected.\nOther findings that were recorded included pleural effusions, lymphadenopathy, pulmonary nodules, scarring and presence of pulmonary embolism on CT pulmonary angiograms.", "V/Q scans were analysed as either with findings or without findings. The V/Q scans recorded in this study were either performed just prior to a positive COVID swab or after the acute episode of COVID as follow‐up imaging.", "Between the five assessors, the senior radiology registrar reviewed all 661 studies. The consultant radiologists each reviewed 20 studies, 20 of which were also reviewed by the radiology registrar and 10 reviewed by another consultant radiologist. Fleiss kappa values were calculated to assess inter‐rater variability between all five assessors for chest radiograph distribution (peripheral, central and diffuse) as well as for each of the six lung zones. A qualitative assessment of the responses of the five assessors was performed with all discrepancies reviewed.", "A total of 103 (17%) of radiographs performed were normal with no findings. Three chest radiographs were considered not suitable for evaluation as they did not have the entire chest in view for evaluation. The most commonly involved zones were the left lower zone (n = 441, 72%) and right lower zone (n = 433, 70%). The least involved zone was the left upper zone (n = 112, 18%). The distribution in each lung zone is shown in Table 1. Findings in both lungs (n = 417, 67%) were demonstrated more often than unilateral findings (n = 239, 39%). The most common pattern of distribution was a diffuse pattern (n = 351, 57%) followed by a peripheral pattern (n = 146, 24%) with the least common pattern of distribution being central (n = 13, 2%). In the other findings evaluated, 3% had pleural effusions and 3% had interstitial change.\nDistribution of changes in each zone on chest radiograph", "Of the 59 CTs, nine were performed as follow‐up after the acute episode. Ten (17%) CTs performed were normal. Of these normal CT studies, two were in follow‐up CT scans. Thirty‐three of the scans were CT pulmonary angiograms, with five positive for pulmonary embolism. The most common patterns demonstrated on CT imaging were ground glass opacity (n = 20, 45%) and ground glass with consolidation (n = 20, 34%). The most common distribution demonstrated on CT was peripheral (n = 29, 49%) and peribronchovascular (n = 16, 27%). The most commonly involved lobe was the right lower lobe (n = 42, 71%) followed by the left lower lobe (n = 41, 69%). Bilateral involvement (n = 45, 76%) was more common than unilateral involvement (n = 3, 6%). In the other findings evaluated, four demonstrated pleural effusion (n = 4, 7%), two cases demonstrated lymphadenopathy and five cases demonstrated pulmonary nodules. Scarring was observed in three cases, two of which were in follow‐up CT scans. An example CT demonstrating peripheral regions of consolidation, ground glass and air bronchograms is shown in Figure 4.\nCT chest performed on a COVID‐19‐positive patient with regions of peripheral consolidation, ground glass opacity and air bronchograms which are lower lobe predominant.", "Of the six V/Q scans performed, two (33%) demonstrated findings and four (67%) were normal. One V/Q scan was performed 7 days prior to the positive COVID test and 4 days prior to the onset of symptoms to investigate a 6 month history of chest pain. The V/Q scan was negative for pulmonary embolism with bronchopulmonary changes seen in the left apex on both ventilation and perfusion images (Fig. 5). Although CT chest two days later demonstrated no correlative changes, bilateral regions of opacification were subsequently demonstrated on CXR 7 days after the V/Q scan. The other positive V/Q scan was requested for follow‐up of pulmonary emboli demonstrated on CT pulmonary angiogram on admission. This V/Q scan demonstrated mild heterogeneous tracer uptake in both lungs.\nV/Q scan (a) in a patient who subsequently became COVID‐19 positive with bilateral regions of opacification on CXR performed 7 days after the V/Q scan (b).", "Moderate agreement was demonstrated between the readers for assessment of distribution on the chest radiograph (peripheral, central, diffuse) with a Fleiss Kappa value of 0.42 (P‐value < 0.05). The Fleiss Kappa values of the inter‐rater agreement for the distribution of the opacities in zones ranged from fair to substantial agreement with Fleiss kappa values ranging from 0.29 to 0.73 (P‐value < 0.05).\nA qualitative assessment performed on the CTs read by all consultant radiologists demonstrated consistency in labelling of the lobe involvement. There was a general consensus for the presence of ground glass opacity, air bronchograms and ground glass with consolidation." ]

[ null, null, null, null, null, null, null, null, null ]

[ "Introduction", "Methods", "Chest radiograph descriptors", "CT descriptors", "V/Q scan descriptors", "Evaluation of readers", "Results", "Chest radiograph findings", "CT findings", "V/Q scan findings", "Evaluation of readers", "Discussion" ]

[ "Coronavirus disease 2019 (COVID‐19) was first described in late 2019 where the first cluster of cases was reported in Wuhan, Hubei Province, China.\n1\n COVID‐19 has since spread globally and on 11 March, 2020 the World Health Organisation (WHO) officially characterised COVID‐19 as a pandemic. As of 3rd October 2021, over 215 million individuals have been infected worldwide resulting in significant mortality and morbidity. The first case of COVID‐19 in Australia was confirmed on the 25th January 2020, and by 3rd October 2021 Australia has had more than 110,000 cases which have resulted in over 1000 deaths.\nChest radiographs (CXR) and computed tomography (CT) have frequently been utilised in assistance with diagnosis and assessment of progression of disease. A multinational consensus statement from the Fleischner society describes the role of chest imaging in the management of COVID‐19, recommending that imaging is not indicated in patients suspected of having COVID‐19 with mild clinical features, but is recommended for patients at risk for disease progression and in those with worsening respiratory status.\n2\n Imaging of patients with COVID‐19 infection has raised challenges in ensuring that studies are performed appropriately whilst ensuring the safety of health personnel.\nWhilst the CXR is low in sensitivity, it can be highly suggestive of COVID‐19 in the correct clinical setting when the radiographs exhibit characteristic COVID‐19 findings.\n3\n Typical findings include a peripheral mid to lower zone lung distribution of opacification.\n3\n, \n4\n\n\nCT imaging is not recommended for screening or as a first line test to diagnose COVID‐19 and should be used sparingly, for hospitalised, symptomatic patients with specific clinical indications.\n5\n, \n6\n The Royal Australian and New Zealand College of Radiologists (RANZCR) recommendation for CT technique in the setting of COVID‐19 is a volumetric CT of the entire chest at end inspiration, with reconstruction of contiguous high‐resolution (HR) images at 0.625–1.5 mm for assessment of the lungs. Studies have demonstrated that COVID‐19 can cause microvascular injuries resulting in a procoagulant state.\n5\n The role of computed tomography pulmonary angiography (CTPA) is reserved for the detection of pulmonary emboli in individuals who have clinical deterioration.\n7\n Characteristic CT findings include bilateral, peripheral and basal predominant ground glass opacities and consolidation.\n8\n Consolidation superimposed on ground glass opacity is found in a small number of cases, predominantly in the elderly.\n7\n\n\nThis study describes the imaging findings of 180 patients who presented to The Royal Melbourne Hospital (an Australian tertiary hospital), evaluating the patterns demonstrated on CXR, CT and ventilation perfusion (V/Q) scans.", "The retrospective review was approved by the ethics committee through the quality assurance pathway. COVID‐PCR‐positive patients who had chest imaging (CXR, CT and V/Q scans) from January 2020 to August 2020 were included. The imaging was evaluated by a senior radiology registrar and four subspecialty chest radiologists including one dual trained radiologist and nuclear medicine specialist. Imaging performed within 5 days prior to the COVID‐PCR‐positive test was also included to account for the potential lag time between onset of infection and a COVID‐PCR‐positive test.\nA set of descriptors were developed for the purposes of labelling based on descriptors in the current literature.\nChest radiograph descriptors Chest radiograph descriptors were divided into zone involvement, distribution and findings. The chest radiograph was divided into six zones, right upper, right middle, right lower, left upper, left middle and left lower (Fig. 1). This method of dividing the lungs into thirds is commonly used in reporting and has been proposed for the reporting of COVID‐19 chest radiographs.\n9\n\n\nThe chest X‐rays were distributed into six zones as illustrated.\nThe distribution of the lung changes were also classified into three categories: peripheral, central and diffuse (Fig. 2). Peripheral distribution was selected when only peripheral opacities were present. Central distribution was selected when only central opacities were present. A diffuse distribution was selected if there were opacities that either crossed both regions or were both peripheral and central. Figure 3 demonstrates a chest radiograph with a peripheral distribution.\nDescriptors for the distribution of opacity.\nChest radiograph with a peripheral distribution.\nOther findings recorded included the presence of pleural effusions or interstitial change.\nChest radiograph descriptors were divided into zone involvement, distribution and findings. The chest radiograph was divided into six zones, right upper, right middle, right lower, left upper, left middle and left lower (Fig. 1). This method of dividing the lungs into thirds is commonly used in reporting and has been proposed for the reporting of COVID‐19 chest radiographs.\n9\n\n\nThe chest X‐rays were distributed into six zones as illustrated.\nThe distribution of the lung changes were also classified into three categories: peripheral, central and diffuse (Fig. 2). Peripheral distribution was selected when only peripheral opacities were present. Central distribution was selected when only central opacities were present. A diffuse distribution was selected if there were opacities that either crossed both regions or were both peripheral and central. Figure 3 demonstrates a chest radiograph with a peripheral distribution.\nDescriptors for the distribution of opacity.\nChest radiograph with a peripheral distribution.\nOther findings recorded included the presence of pleural effusions or interstitial change.\nCT descriptors CT characteristics were classified according to pattern, distribution, lobe involvement and other findings. The CT descriptors were used for all chest CT imaging including pre‐ and post‐contrast CT chests such as pulmonary angiograms.\nCT patterns reviewed were based on patterns described in the literature.\n10\n Patterns that were included were ground glass opacities, consolidation, ground glass with consolidation, crazy paving pattern, reverse halo, interlobular septal thickening and air bronchograms.\nDistribution on CT was divided into peripheral, central, peribronchovascular or diffuse. Similar to the chest radiograph distribution, if more than one pattern was involved the diffuse descriptor for distribution was selected. Lobar involvement was also recorded based on the normal anatomical division of the right and left lung lobes (right upper, middle and lower and left upper and lower). Each lobe involved in the CT was selected.\nOther findings that were recorded included pleural effusions, lymphadenopathy, pulmonary nodules, scarring and presence of pulmonary embolism on CT pulmonary angiograms.\nCT characteristics were classified according to pattern, distribution, lobe involvement and other findings. The CT descriptors were used for all chest CT imaging including pre‐ and post‐contrast CT chests such as pulmonary angiograms.\nCT patterns reviewed were based on patterns described in the literature.\n10\n Patterns that were included were ground glass opacities, consolidation, ground glass with consolidation, crazy paving pattern, reverse halo, interlobular septal thickening and air bronchograms.\nDistribution on CT was divided into peripheral, central, peribronchovascular or diffuse. Similar to the chest radiograph distribution, if more than one pattern was involved the diffuse descriptor for distribution was selected. Lobar involvement was also recorded based on the normal anatomical division of the right and left lung lobes (right upper, middle and lower and left upper and lower). Each lobe involved in the CT was selected.\nOther findings that were recorded included pleural effusions, lymphadenopathy, pulmonary nodules, scarring and presence of pulmonary embolism on CT pulmonary angiograms.\nV/Q scan descriptors V/Q scans were analysed as either with findings or without findings. The V/Q scans recorded in this study were either performed just prior to a positive COVID swab or after the acute episode of COVID as follow‐up imaging.\nV/Q scans were analysed as either with findings or without findings. The V/Q scans recorded in this study were either performed just prior to a positive COVID swab or after the acute episode of COVID as follow‐up imaging.\nEvaluation of readers Between the five assessors, the senior radiology registrar reviewed all 661 studies. The consultant radiologists each reviewed 20 studies, 20 of which were also reviewed by the radiology registrar and 10 reviewed by another consultant radiologist. Fleiss kappa values were calculated to assess inter‐rater variability between all five assessors for chest radiograph distribution (peripheral, central and diffuse) as well as for each of the six lung zones. A qualitative assessment of the responses of the five assessors was performed with all discrepancies reviewed.\nBetween the five assessors, the senior radiology registrar reviewed all 661 studies. The consultant radiologists each reviewed 20 studies, 20 of which were also reviewed by the radiology registrar and 10 reviewed by another consultant radiologist. Fleiss kappa values were calculated to assess inter‐rater variability between all five assessors for chest radiograph distribution (peripheral, central and diffuse) as well as for each of the six lung zones. A qualitative assessment of the responses of the five assessors was performed with all discrepancies reviewed.", "Chest radiograph descriptors were divided into zone involvement, distribution and findings. The chest radiograph was divided into six zones, right upper, right middle, right lower, left upper, left middle and left lower (Fig. 1). This method of dividing the lungs into thirds is commonly used in reporting and has been proposed for the reporting of COVID‐19 chest radiographs.\n9\n\n\nThe chest X‐rays were distributed into six zones as illustrated.\nThe distribution of the lung changes were also classified into three categories: peripheral, central and diffuse (Fig. 2). Peripheral distribution was selected when only peripheral opacities were present. Central distribution was selected when only central opacities were present. A diffuse distribution was selected if there were opacities that either crossed both regions or were both peripheral and central. Figure 3 demonstrates a chest radiograph with a peripheral distribution.\nDescriptors for the distribution of opacity.\nChest radiograph with a peripheral distribution.\nOther findings recorded included the presence of pleural effusions or interstitial change.", "CT characteristics were classified according to pattern, distribution, lobe involvement and other findings. The CT descriptors were used for all chest CT imaging including pre‐ and post‐contrast CT chests such as pulmonary angiograms.\nCT patterns reviewed were based on patterns described in the literature.\n10\n Patterns that were included were ground glass opacities, consolidation, ground glass with consolidation, crazy paving pattern, reverse halo, interlobular septal thickening and air bronchograms.\nDistribution on CT was divided into peripheral, central, peribronchovascular or diffuse. Similar to the chest radiograph distribution, if more than one pattern was involved the diffuse descriptor for distribution was selected. Lobar involvement was also recorded based on the normal anatomical division of the right and left lung lobes (right upper, middle and lower and left upper and lower). Each lobe involved in the CT was selected.\nOther findings that were recorded included pleural effusions, lymphadenopathy, pulmonary nodules, scarring and presence of pulmonary embolism on CT pulmonary angiograms.", "V/Q scans were analysed as either with findings or without findings. The V/Q scans recorded in this study were either performed just prior to a positive COVID swab or after the acute episode of COVID as follow‐up imaging.", "Between the five assessors, the senior radiology registrar reviewed all 661 studies. The consultant radiologists each reviewed 20 studies, 20 of which were also reviewed by the radiology registrar and 10 reviewed by another consultant radiologist. Fleiss kappa values were calculated to assess inter‐rater variability between all five assessors for chest radiograph distribution (peripheral, central and diffuse) as well as for each of the six lung zones. A qualitative assessment of the responses of the five assessors was performed with all discrepancies reviewed.", "A total of 681 studies (616 Chest X‐Rays, 59 CTs, six V/Q scans) from 181 patients were reviewed. Of the 181 patients, there were 92 females and 89 males with a mean age of 61.99 (range 16–101).\nChest radiograph findings A total of 103 (17%) of radiographs performed were normal with no findings. Three chest radiographs were considered not suitable for evaluation as they did not have the entire chest in view for evaluation. The most commonly involved zones were the left lower zone (n = 441, 72%) and right lower zone (n = 433, 70%). The least involved zone was the left upper zone (n = 112, 18%). The distribution in each lung zone is shown in Table 1. Findings in both lungs (n = 417, 67%) were demonstrated more often than unilateral findings (n = 239, 39%). The most common pattern of distribution was a diffuse pattern (n = 351, 57%) followed by a peripheral pattern (n = 146, 24%) with the least common pattern of distribution being central (n = 13, 2%). In the other findings evaluated, 3% had pleural effusions and 3% had interstitial change.\nDistribution of changes in each zone on chest radiograph\nA total of 103 (17%) of radiographs performed were normal with no findings. Three chest radiographs were considered not suitable for evaluation as they did not have the entire chest in view for evaluation. The most commonly involved zones were the left lower zone (n = 441, 72%) and right lower zone (n = 433, 70%). The least involved zone was the left upper zone (n = 112, 18%). The distribution in each lung zone is shown in Table 1. Findings in both lungs (n = 417, 67%) were demonstrated more often than unilateral findings (n = 239, 39%). The most common pattern of distribution was a diffuse pattern (n = 351, 57%) followed by a peripheral pattern (n = 146, 24%) with the least common pattern of distribution being central (n = 13, 2%). In the other findings evaluated, 3% had pleural effusions and 3% had interstitial change.\nDistribution of changes in each zone on chest radiograph\nCT findings Of the 59 CTs, nine were performed as follow‐up after the acute episode. Ten (17%) CTs performed were normal. Of these normal CT studies, two were in follow‐up CT scans. Thirty‐three of the scans were CT pulmonary angiograms, with five positive for pulmonary embolism. The most common patterns demonstrated on CT imaging were ground glass opacity (n = 20, 45%) and ground glass with consolidation (n = 20, 34%). The most common distribution demonstrated on CT was peripheral (n = 29, 49%) and peribronchovascular (n = 16, 27%). The most commonly involved lobe was the right lower lobe (n = 42, 71%) followed by the left lower lobe (n = 41, 69%). Bilateral involvement (n = 45, 76%) was more common than unilateral involvement (n = 3, 6%). In the other findings evaluated, four demonstrated pleural effusion (n = 4, 7%), two cases demonstrated lymphadenopathy and five cases demonstrated pulmonary nodules. Scarring was observed in three cases, two of which were in follow‐up CT scans. An example CT demonstrating peripheral regions of consolidation, ground glass and air bronchograms is shown in Figure 4.\nCT chest performed on a COVID‐19‐positive patient with regions of peripheral consolidation, ground glass opacity and air bronchograms which are lower lobe predominant.\nOf the 59 CTs, nine were performed as follow‐up after the acute episode. Ten (17%) CTs performed were normal. Of these normal CT studies, two were in follow‐up CT scans. Thirty‐three of the scans were CT pulmonary angiograms, with five positive for pulmonary embolism. The most common patterns demonstrated on CT imaging were ground glass opacity (n = 20, 45%) and ground glass with consolidation (n = 20, 34%). The most common distribution demonstrated on CT was peripheral (n = 29, 49%) and peribronchovascular (n = 16, 27%). The most commonly involved lobe was the right lower lobe (n = 42, 71%) followed by the left lower lobe (n = 41, 69%). Bilateral involvement (n = 45, 76%) was more common than unilateral involvement (n = 3, 6%). In the other findings evaluated, four demonstrated pleural effusion (n = 4, 7%), two cases demonstrated lymphadenopathy and five cases demonstrated pulmonary nodules. Scarring was observed in three cases, two of which were in follow‐up CT scans. An example CT demonstrating peripheral regions of consolidation, ground glass and air bronchograms is shown in Figure 4.\nCT chest performed on a COVID‐19‐positive patient with regions of peripheral consolidation, ground glass opacity and air bronchograms which are lower lobe predominant.\nV/Q scan findings Of the six V/Q scans performed, two (33%) demonstrated findings and four (67%) were normal. One V/Q scan was performed 7 days prior to the positive COVID test and 4 days prior to the onset of symptoms to investigate a 6 month history of chest pain. The V/Q scan was negative for pulmonary embolism with bronchopulmonary changes seen in the left apex on both ventilation and perfusion images (Fig. 5). Although CT chest two days later demonstrated no correlative changes, bilateral regions of opacification were subsequently demonstrated on CXR 7 days after the V/Q scan. The other positive V/Q scan was requested for follow‐up of pulmonary emboli demonstrated on CT pulmonary angiogram on admission. This V/Q scan demonstrated mild heterogeneous tracer uptake in both lungs.\nV/Q scan (a) in a patient who subsequently became COVID‐19 positive with bilateral regions of opacification on CXR performed 7 days after the V/Q scan (b).\nOf the six V/Q scans performed, two (33%) demonstrated findings and four (67%) were normal. One V/Q scan was performed 7 days prior to the positive COVID test and 4 days prior to the onset of symptoms to investigate a 6 month history of chest pain. The V/Q scan was negative for pulmonary embolism with bronchopulmonary changes seen in the left apex on both ventilation and perfusion images (Fig. 5). Although CT chest two days later demonstrated no correlative changes, bilateral regions of opacification were subsequently demonstrated on CXR 7 days after the V/Q scan. The other positive V/Q scan was requested for follow‐up of pulmonary emboli demonstrated on CT pulmonary angiogram on admission. This V/Q scan demonstrated mild heterogeneous tracer uptake in both lungs.\nV/Q scan (a) in a patient who subsequently became COVID‐19 positive with bilateral regions of opacification on CXR performed 7 days after the V/Q scan (b).\nEvaluation of readers Moderate agreement was demonstrated between the readers for assessment of distribution on the chest radiograph (peripheral, central, diffuse) with a Fleiss Kappa value of 0.42 (P‐value < 0.05). The Fleiss Kappa values of the inter‐rater agreement for the distribution of the opacities in zones ranged from fair to substantial agreement with Fleiss kappa values ranging from 0.29 to 0.73 (P‐value < 0.05).\nA qualitative assessment performed on the CTs read by all consultant radiologists demonstrated consistency in labelling of the lobe involvement. There was a general consensus for the presence of ground glass opacity, air bronchograms and ground glass with consolidation.\nModerate agreement was demonstrated between the readers for assessment of distribution on the chest radiograph (peripheral, central, diffuse) with a Fleiss Kappa value of 0.42 (P‐value < 0.05). The Fleiss Kappa values of the inter‐rater agreement for the distribution of the opacities in zones ranged from fair to substantial agreement with Fleiss kappa values ranging from 0.29 to 0.73 (P‐value < 0.05).\nA qualitative assessment performed on the CTs read by all consultant radiologists demonstrated consistency in labelling of the lobe involvement. There was a general consensus for the presence of ground glass opacity, air bronchograms and ground glass with consolidation.", "A total of 103 (17%) of radiographs performed were normal with no findings. Three chest radiographs were considered not suitable for evaluation as they did not have the entire chest in view for evaluation. The most commonly involved zones were the left lower zone (n = 441, 72%) and right lower zone (n = 433, 70%). The least involved zone was the left upper zone (n = 112, 18%). The distribution in each lung zone is shown in Table 1. Findings in both lungs (n = 417, 67%) were demonstrated more often than unilateral findings (n = 239, 39%). The most common pattern of distribution was a diffuse pattern (n = 351, 57%) followed by a peripheral pattern (n = 146, 24%) with the least common pattern of distribution being central (n = 13, 2%). In the other findings evaluated, 3% had pleural effusions and 3% had interstitial change.\nDistribution of changes in each zone on chest radiograph", "Of the 59 CTs, nine were performed as follow‐up after the acute episode. Ten (17%) CTs performed were normal. Of these normal CT studies, two were in follow‐up CT scans. Thirty‐three of the scans were CT pulmonary angiograms, with five positive for pulmonary embolism. The most common patterns demonstrated on CT imaging were ground glass opacity (n = 20, 45%) and ground glass with consolidation (n = 20, 34%). The most common distribution demonstrated on CT was peripheral (n = 29, 49%) and peribronchovascular (n = 16, 27%). The most commonly involved lobe was the right lower lobe (n = 42, 71%) followed by the left lower lobe (n = 41, 69%). Bilateral involvement (n = 45, 76%) was more common than unilateral involvement (n = 3, 6%). In the other findings evaluated, four demonstrated pleural effusion (n = 4, 7%), two cases demonstrated lymphadenopathy and five cases demonstrated pulmonary nodules. Scarring was observed in three cases, two of which were in follow‐up CT scans. An example CT demonstrating peripheral regions of consolidation, ground glass and air bronchograms is shown in Figure 4.\nCT chest performed on a COVID‐19‐positive patient with regions of peripheral consolidation, ground glass opacity and air bronchograms which are lower lobe predominant.", "Of the six V/Q scans performed, two (33%) demonstrated findings and four (67%) were normal. One V/Q scan was performed 7 days prior to the positive COVID test and 4 days prior to the onset of symptoms to investigate a 6 month history of chest pain. The V/Q scan was negative for pulmonary embolism with bronchopulmonary changes seen in the left apex on both ventilation and perfusion images (Fig. 5). Although CT chest two days later demonstrated no correlative changes, bilateral regions of opacification were subsequently demonstrated on CXR 7 days after the V/Q scan. The other positive V/Q scan was requested for follow‐up of pulmonary emboli demonstrated on CT pulmonary angiogram on admission. This V/Q scan demonstrated mild heterogeneous tracer uptake in both lungs.\nV/Q scan (a) in a patient who subsequently became COVID‐19 positive with bilateral regions of opacification on CXR performed 7 days after the V/Q scan (b).", "Moderate agreement was demonstrated between the readers for assessment of distribution on the chest radiograph (peripheral, central, diffuse) with a Fleiss Kappa value of 0.42 (P‐value < 0.05). The Fleiss Kappa values of the inter‐rater agreement for the distribution of the opacities in zones ranged from fair to substantial agreement with Fleiss kappa values ranging from 0.29 to 0.73 (P‐value < 0.05).\nA qualitative assessment performed on the CTs read by all consultant radiologists demonstrated consistency in labelling of the lobe involvement. There was a general consensus for the presence of ground glass opacity, air bronchograms and ground glass with consolidation.", "A review of imaging in an Australian tertiary hospital demonstrates similar trends on chest radiographs with those described in the literature of international populations with predominantly bilateral mid to lower zone regions of opacification.\n3\n, \n11\n The population had approximately 17% normal chest radiographs which is slightly lower than what has been reported in other populations but comparable to those reported in hospitalised patients.\n4\n, \n12\n Another factor that would have contributed to a higher percentage of radiographs with findings in this population would be that individuals who were hospitalised received a greater number of radiographs when compared to those managed in the emergency department and discharged for community treatment. Furthermore, this cohort demonstrated a greater number of radiographs with a diffuse distribution of findings instead of a peripheral only distribution which likely reflects the acuity of the patient population who present to hospital.\nLitmanovich et al.\n9\n proposed a suggested reporting language for the review of chest radiograph findings of COVID‐19 pneumonia. The authors proposed a reporting language for grading the severity of lung disease using a modified version of a scoring system used in quantifying the severity of acute respiratory distress syndrome. The authors base their grading on the number of lung zones involved, dividing the lung into six zones, similar to what was performed in this study. Mild with involvement of 1–2 lung zones, moderate with opacities in 3–4 lung zones and severe > 4 lung zones. This standardisation in reporting is thought to be helpful in assessing progression over multiple radiographs in a patient with COVID‐19. Our study demonstrates that there is a fair to substantial inter‐rater agreement in the evaluation of opacities in zones which may indicate that such a proposed reporting system may be useful in this clinical setting.\nCT imaging findings described in this population were similar to the characteristics described on CT of a systematic review performed on 919 patients with coronavirus disease with predominantly bilateral and peripheral involvement demonstrated. A large proportion of CTs in this study also demonstrated multilobar involvement, similar to that in the literature, reflecting the severity of symptoms in patients who receive cross sectional imaging.\nCOVID‐19 can cause microvascular injury and is associated with a procoagulant state increasing the risk of thromboembolic disease.\n8\n, \n13\n Greater than half of the CT chest scans performed on COVID‐19‐positive patients were CT pulmonary angiograms with five positive for pulmonary emboli demonstrating that the symptoms of PE may mimic or overlap with those of COVID‐19 infection.\nAlthough V/Q imaging does play a role in the diagnosis of thromboembolic disease especially in those with impaired renal function, the use of ventilation images in COVID‐19‐positive patients is discouraged due to its potential risk of spread of infection.\n14\n All V/Q imaging included in this study was either performed after a negative COVID‐19 swab during an acute episode, or prior to a subsequent positive COVID‐19 swab, without clinical suspicion of the diagnosis at time of study.\nIn conclusion, a review of imaging in an Australian tertiary hospital demonstrates similar trends on chest X‐ray and CT imaging when compared to the international population. A propensity for a number of radiographs with diffuse findings may reflect the acuity of the patients who present to hospital." ]

[ null, "methods", null, null, null, null, "results", null, null, null, null, "discussion" ]

[ "chest CT", "chest imaging", "chest radiograph", "COVID‐19", "VQ" ]

[ 494, 196, 187, 44, 92, 222, 285, 196, 115 ]

[ "ct", "distribution", "chest", "findings", "demonstrated", "peripheral", "covid", "performed", "pulmonary", "lower" ]

[ "chest performed covid", "findings covid 19", "imaging patients covid", "imaging covid 19", "coronavirus disease 2019" ]

[ "Age Factors", "Animals", "Animals, Outbred Strains", "Cell Proliferation", "Cells, Cultured", "Dental Pulp", "Female", "Humans", "Mice", "NF-E2-Related Factor 2", "Osteogenesis", "Oxidative Stress", "Reactive Oxygen Species", "Resveratrol", "Sirtuin 1", "Stromal Cells", "Superoxide Dismutase" ]

[ "Cell culture", "Oxidative stress cell model and regents", "Intracellular ROS fluorescent staining", "Cell proliferation assay", "ROS activity", "SOD enzyme activity", "Glutathione (GSH) concentration assay", "Quantitative real-time PCR (qRT-PCR)", "ALP staining", "In-Cell Western analysis", "Immunofluorescence labelling of Sirt1", "Animals and tissue sections", "Micro-computed tomography", "Nonlinear optical microscope observation", "Immunohistochemistry", "Statistical analysis", "Sirt1 was decreased in oxidative stress", "RSV decreased oxidative stress of hDPSCs", "RSV promotes the expression of osteogenic genes", "RSV promotes bone mass in mice" ]

[ "A total of eight teeth from different donors obtained with patients’ informed consent according to the current study, which had approval from the Research Ethics Committee (YS2020147) were collected separately, and the hDPSCs were extracted according to the method as described in our previous article (Zhang et al. 2019). The hDPSCs were cultured with growth medium (α-modified minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 mM l-glutamine). All reagents were obtained from Gibco (Carlsbad, CA), and the cells were cultured in an incubator at 37 °C with 5% CO2 (Binder, Tuttlingen, Germany). The medium was changed every three days, and the cells were treated with trypsin when they reached 80% confluence. The hDPSCs of passages 3–5 were used for the following experiments.", "The oxidative stress model was optimized by different concentrations of H2O2 (0.1, 0.2 and 0.3 mM) treated for 24 h. All H2O2 solutions were prepared freshly. The RSV (Sigma-Aldrich, Shanghai, China) was dissolved in dimethyl sulfoxide (10 µL) (DMSO, Sigma-Aldrich, Shanghai, China) and diluted to 10 μM final concentration in a growth medium. The hDPSCs (n = 8) were divided into four groups. Normal control (NC) group refers to the hDPSCs which were cultured with growth medium, RSV group refers to the hDPSCs which were treated with RSV, H2O2 group were the hDPSCs which were cultured with H2O2 (0.2 mM), and H-RSV refers to the hDPSCs which were cultured with 0.2 mM H2O2 and 10 μM RSV.", "The cells at 2.5 × 104 cells/cm2 (n = 6) were seeded and attached to eight-chamber glass coverslips (Corning, Falcon culture slides, Corning, NY). Peroxide-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (50 μg) (Invitrogen™, Carlsbad, CA) was dissolved in DMSO (10 μL) and were diluted with the serum-free medium at a final concentration of 17.4 μM. The cells were incubated at 37 °C in darkness for 30 min and washed with 1 × PBS three times. The images were acquired by inverted fluorescence microscopy (ZEISS, AX-10, Oberkochen, Germany).", "Cell proliferation assay was determined by MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Beijing, China). Briefly, the cells were seeded in 96-well plates at 2.5 × 104 cells/cm2 (n = 6) and cultured in a growth medium for 24 h. MTT reagent (10 μL) and serum-free basal media (90 μL) were added to each well and incubated for 4 h. The supernatant was aspirated; formazan solutions (110 μL) were added to each well to dissolve the MTT formazan. Absorbance was measured at 490 nm using the Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).", "The intracellular ROS activity was examined by the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, Shanghai, China). Briefly, the cells were seeded at the density of 2.5 × 104 cells/cm2 in 96-well plates and cultured for 24 h (n = 3). DCFH-DA was diluted 1:1000 in serum-free medium to 10 μmol/L concentration and added to each well. The cells were incubated at 37 °C for 20 min and washed three times with a serum-free medium. The ROS activity was measured at 488 nm by Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).", "A total superoxide dismutase (SOD) assay kit with water-soluble tetrazolium salt, WST-1 (Beyotime Biotechnology, Shanghai, China), was used to analyse the SOD enzyme activity following the manufacturer's instructions. Briefly, the cells were seeded at a density of 2.5 × 104 cells/cm2 in 96-well plates (n = 3) and treated as described previously. The cells were harvested, lysed in 1 × PBS by ultrasonic pyrolysis, and centrifuged at 2200 rpm for 5 min at 4 °C. The supernatant (100 μL) was mixed with SOD working solution (160 μL) at 4 °C. The reaction mixture was centrifuged and transferred to 96-well plates, and the absorbance values were measured at 450 nm using a Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).", "A total glutathione assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with enzyme labelling was used to analyse GSH following the manufacturer's instructions. Briefly, the cells were seeded at 2.5 × 104 cells/cm2 in 24-well plates (n = 3) and treated as described previously. The cells were harvested, lysed in 1 × PBS by ultrasonic pyrolysis. The supernatant (100 μL) was mixed with precipitant agents (100 μL) and centrifuged at 3500 rpm for 10 min at 25 °C. Following this, the reaction mixture was kept at room temperature for 5 min, and the absorbance values were measured at 405 nm using a Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).", "The total RNA was extracted from the cells using RNAiso Plus reagent (TaKaRa, Kusatsu, Japan). RNAiso Plus (1 mL) was added to each culture dish (6 cm diameter) (n = 4) to lyse the cells. Total RNA (1 µg) was reverse transcribed using a PrimeScript TM RT reagent Kit with gDNA Eraser Kit (TaKaRa, Kusatsu, Japan). To quantify the mRNA expression, an amount of cDNA equivalent to the total RNA reacting system (20 µL) was amplified using SYBR Premix Ex Taq TM (TliRNase H Plus, TaKaRa, Kusatsu, Japan) following the manufacturer's protocol. The transcript levels of genes were evaluated with β-actin serving as the housekeeping internal control. The primer sequences are listed in Table 1. Relative transcript levels were calculated as 2–ΔΔCt, in which ΔΔCt = ΔE – ΔC, ΔE=Ctexp – Ctβ-actin and ΔC=Ctct1 – Ctβ-actin.\nPrimer of targeting genes.", "Following treatment, the cells were cultured in the osteogenic medium for 14 days and fixed with 4% formaldehyde at room temperature for 30 min and washed with 1 × PBS three times. The cells were stained with alkaline phosphatase (ALP, Beyotime Institute of Biotechnology, Shanghai, China) solution for 30 min at room temperature following the manufacturer's instructions. The photos were taken by an optical microscope (Olympus IX71, Tokyo, Japan).", "Immediately after treatment, cells were washed in 1 × PBS and fixed in 10% neutral buffered formalin (NBF, Cellpath, Newtown, UK) in 1 × PBS for 20 min before being stained with the CellTag 700 staining ICW Kit I (Li-Cor Biosciences, Lincoln, NE). The samples were then incubated with the mouse anti-human Sirt1 antibody (1:600, Thermo Fisher, Waltham, MA) at 4 °C overnight with gentle shaking, followed by extensive washing in 1 × PBS containing 0.1% Tween 20 (Sigma-Aldrich, Shanghai, China) five times for 5 min per wash. The IRDye 800 CW goat anti-mouse secondary antibody (1:800) with the CellTag™ 700 stains (1:500) were added for 1 h at room temperature with gentle shaking followed by washing in 1 × PBS containing 0.1% Tween 20 for 5 min per wash. The plate was scanned on the Odyssey SA Imaging System (Li-Cor Biosciences, Lincoln, NE) using both 700 and 800 nm detection channels. Image Studio version 5 (Li-Cor Biosciences, Lincoln, NE) was used for performing the quantitative In-Cell Western (ICW) analysis.", "Cells (2.5 × 104 cells/cm2) were seeded in eight-well multi-chambers (Falcon™ Culture Slides, Corning, Corning, NY) (n = 3) and cultured for 24 h. After treatment, the cells were fixed by 4% paraformaldehyde for 10 min at room temperature. The cells were washed three times with 1 × PBS and permeabilized by 0.2% Triton X-100 for 1 min. The cells were blocked with 10% goat serum (100 µL) (diluted in 1% BSA in 1 × PBS) (Dako, Nowy Sącz, Poland) for 30 min and washed five times with 1 × PBS before incubation in primary antibody (200 µL) (mouse anti-human Sirt1, 1:200 in 1% BSA) (Sigma-Aldrich, Shanghai, China) overnight at 4 °C. After rinsing with 1 × PBS, cells were incubated with Alexa-blue conjugated Goat Anti-Mouse secondary antibody (200 µL) (Alexa Fluor® 488, Invitrogen, Carlsbad, CA, 1:50 in 1% BSA-PBS) for 1 h at room temperature in darkness. The slides were examined with an inverted fluorescent microscope (ZEISS, AX-10, Oberkochen, Germany).", "All animal studies were conducted following international standards on animal welfare and approved by the Animal Research Committee of Guangdong Medical University (Zhanjiang, China). The study was approved by the suggestion of animal research ethics (GDY20023l0) at Guangdong Medical University. Forty female Kunming mice (1-month-old and 12-months-old) were arranged and randomly assigned to four groups: Old (12-months-old), Old-RSV (12-months-old + RSV), Young (1-month-old) and Young-RSV (1-month-old + RSV). Cranium defects (1.0 mm) were created in the parietal bones of mice (n = 5 per group). Mice in the Old-RSV and Young-RSV groups were injected intraperitoneally with 0.2 mL RSV of 20 mg/kg/d for seven days, while the mice in the Old and Young groups were intraperitoneally injected with normal saline 0.2 mL. The mice were euthanized at 4 weeks after the surgery. The parietal bones were harvested and fixed for 24 h in 4% paraformaldehyde for further study.", "Micro-computed tomography (micro-CT) analysis was performed as described previously (Zhang et al. 2019). Briefly, the images were obtained via ex vivo micro-CT systems (Skyscanner 1174; Skyscan, Aartselaar, Belgium). Each sample was placed in a sample holder with the sagittal suture oriented parallel to the image plane and scanned in the air using the aluminium filer (0.25 mm), isotropic voxels (13 μm), 1000 ms integration time and one frame average. The scanner was equipped with an 80 kV, 500 μA X-ray tube, and a-36.9 megapixel Calibrate Centre offset coupled to a scintillator. For three-dimensional reconstruction (NRecon software, Skyscanner, Edinburgh, UK), the greyscale was set from 50 to 140. Standard three-dimensional morphometric parameters were determined in the ROI (100 cuts; 2.5 mm circle). Representative three-dimensional images were created using CT vox software (Skyscan, Edinburgh, UK).", "The excitation source of the SHG microscope was used to be pumped by a mode-locked Ti:sapphire laser oscillator (Spectra-Physics, 80 fs, 80 MHz and average powers up to 2.9 W). The laser beam was coupled into a multiphoton fluorescence scanning microscope (BX61 + FV1200, Olympus, Tokyo, Japan). The femtoseconds laser beam was focussed onto the sample by the objective lens (10× U PlanSApo, 0.40 N.A.; Olympus, Tokyo, Japan) with 10 mW. The SHG signal was isolated from the fundamental and any fluorescence by a band-pass filter (400/10 nm) and detected using a photomultiplier tube in the backscattered light path. Images were 800 × 800 pixels with 2 μs/pixel dwell time.", "The samples were dehydrated through a graded series of ethanol solutions before they were embedded in paraffin, which was cut parallel to the cross into five sections for Sirt1 immunohistochemistry. The slides were incubated with primary antibodies anti-Sirt1 (Abcam, Waltham, MA; 1:150). The two-step plus Poly-HRP Anti Rabbit/Mouse IgG Detection System (Elabscience, Houston, TX) was used to detect immunoreactivity. The slides were counterstained with Mayer's haematoxylin (Hongqiaolexiang Inc., Shanghai, China) and cover-slipped using a permanent mounting medium.", "The statistical analysis was carried out by a two-tailed pair Student's t-test using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA). p Value <0.05 was considered statistically significant.", "The concentration of H2O2 was optimized to induce oxidative stress, which was confirmed by the accumulation of ROS within the cells and cellular proliferation. After the treatment with H2O2 (0.2 mM), positive staining for ROS was located within the nuclei and cytoplasm of the cells (green). The ROS fluorescence intensity in the H2O2 (0.2 mM) group was stronger than the other groups (Figure 1(A,D)). Meanwhile, the fluorescence intensity of Sirt1 decreased in the H2O2 pre-treated group, which was localized within the nuclei and cytoplasm of the cells (Figure 1(A,G)).\nThe effect of H2O2 on cell morphology, ROS production, F-actin and Sirt1 protein in hDPSCs. (A) The morphology of hDPSCs treated with H2O2 detected by immunofluorescence, cell panels: (1) under bright field, (2) ROS, (3) F-actin, (4) Sirt1 (scale bar: 100 μM); (B) cell proliferation of hDPSCs shown on day 1, 2 and 3 of treatment with H2O2; (C) MTT assay showing cell viability at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (D) ROS fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (E) fibre fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (F) fibre length at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (G) Sirt1 fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM) (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. H2O2-0.2 mM group).", "To investigate the effect of RSV against oxidative stress, the concentration of RSV was optimized by cell viability assay. The cell viability increased significantly in the RSV10 group than NC and H-RSV 10 than H2O2 group and the IC50 of RSV in hDPSCs is 67.65 ± 9.86. There were significant differences between the H2O2 group and H-RSV 10 group, and RSV (10 μM) was determined for the following experiments (Figure 2(A)). The ROS activity was declined (Figure 2(B)), the SOD enzyme activities were increased (Figure 2(C)) and the GSH concentration displayed enhancement (Figure 2(D)) in the H-RSV group compared with the H2O2 group. Similarly, further data showed that SOD1 and xCT mRNA expression increased significantly in the H-RSV group compared with the H2O2 group (Figure 2(E,F)).\nRSV promoted the proliferation and reduced the oxidative stress of hDPSCs pre-treated with H2O2. (A) Cell proliferation of hDPSCs pre-treated with/without H2O2 and different concentrations of RSV. (B) The ROS activity of hDPSCs pre-treated with/without H2O2 and RSV. (C) The SOD enzyme activity of hDPSCs pre-treated with/without H2O2 and RSV. (D) The GSH concentration of hDPSCs pre-treated with/without H2O2 and RSV. (E) The fold expression of SOD1 mRNA in hDPSCs. (F) The fold expression of xCT mRNA in hDPSCs. Group: NC (untreated cells), RSV (hDPSCs cultured with 10 μM RSV for 24 hours), H2O2 (hDPSCs treated by 0.2 mM H2O2 for 24 hours) and H-RSV (hDPSCs treated by 0.2 mM H2O2 cultured for 24 hours and then 10 μM RSV cultured for 24 hours) (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).", "The mRNA expression of Runx2, OCN, Sirt1 and Nrf2 increased significantly in the H-RSV group compared with the H2O2 group (Figures 3(A,B) and 4(A,B)). Moreover, The ALP staining was significantly stronger by RSV with/without pre-treated with H2O2 (Figure 3(C)). Immunofluorescent staining and ICW showed that Sirt1 fluorescence intensity increased significantly by RSV compared with/without pre-treated with H2O2 (p < 0.05) (Figure 4(C–E)).\nRSV promoted the osteogenic differentiation of hDPSCs after the treatment of H2O2. (A) The fold expression of Runx2 mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (B) The fold expression of OCN mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (C) The ALP staining of hDPSCs pre-treated with/without H2O2 and RSV (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).\nRSV increased the Sirt1 and Nrf2 mRNA expression in hDPSCs by pre-treated with H2O2. (A) The fold expression of Runx2 mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (B) The fold expression of OCN mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (C) Fluorescence intensity of In-Cell Western assay (ZEISS microscope AX-10). (D) The sirt1 fluorescence of hDPSCs pre-treated with/without H2O2 and RSV by In-cell western. (E) Sirt1 immunofluorescence staining of hDPSCs pre-treated with/without H2O2 and RSV (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).", "The area of the bone defect is significantly reduced in the 1-month-old + RSV group than the 1-month-old group, similarly in the 12-month-old + RSV group than the 12-month-old group. The intensity of collagen (green fluorescence) was stronger in the 12-month-old + RSV group than the 12-month-old group and stronger in the 1-month-old + RSV group than the 1-month-old group. The expression of Sirt1 was higher in the 12-month-old + RSV than the 12-month-old group (Figure 5).\nRSV promotes bone repair and increases bone mass. (A) Representative micro-CT images of bone repair and bone mass in calvarial defects. (B) Representative nonlinear optical microscope images of collagen in calvarial defects. (C) Immunohistochemical staining with sirt1 in hDPSCs pre-treated with/without H2O2 and RSV." ]

[ null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]

[ "Introduction", "Materials and methods", "Cell culture", "Oxidative stress cell model and regents", "Intracellular ROS fluorescent staining", "Cell proliferation assay", "ROS activity", "SOD enzyme activity", "Glutathione (GSH) concentration assay", "Quantitative real-time PCR (qRT-PCR)", "ALP staining", "In-Cell Western analysis", "Immunofluorescence labelling of Sirt1", "Animals and tissue sections", "Micro-computed tomography", "Nonlinear optical microscope observation", "Immunohistochemistry", "Statistical analysis", "Results", "Sirt1 was decreased in oxidative stress", "RSV decreased oxidative stress of hDPSCs", "RSV promotes the expression of osteogenic genes", "RSV promotes bone mass in mice", "Discussion", "Conclusions" ]

[ "Cell therapy has gained significant attention as a novel therapeutic approach to restore/repair tissue/organ functionality after injury (Marvasti et al. 2019). Cellular component represents the key factor in cell-based therapy, particularly in using mesenchymal stromal cells (MSCs), which is considered the gold standard for clinical research (Berebichez-Fridman and Montero-Olvera 2018). Although MSCs can hypothetically be obtained from different and accessible adult tissues, there are practical limitations concerning the invasive procedure, low procurement yield, donor site morbidity, extensive in vitro expansion and various donor characteristics (Berebichez-Fridman and Montero-Olvera 2018). A number of studies showed that the human dental pulp stromal cells (hDPSCs) are easily accessible via discarded medical waste (Gronthos et al. 2000) and have greater proliferative and osteogenic potential than the MSCs derived from bone marrow, making them favourable as an alternative MSC source for cell-based therapies (El-Gendy et al. 2013; Jensen et al. 2016).\nRecent evidence indicated that stem cells might undergo progressive senescence or lose their regenerative capacity and experience gradual exhaustion of their characteristic proliferative function. Oxidative stress refers to a functional inability of the reactive oxygen species (ROS) in performing the detoxification of metabolic reactive intermediates of biological systems (Forrester et al. 2018). Three important categories of ROS, hydrogen peroxide (H2O2), hydroxyl radicals (•OH) and superoxide anion (O2−), generated through different signalling pathways are reported to be beneficial for cellular processes such as cellular proliferation and differentiation (Guan et al. 2019; Li et al. 2019). Several studies have demonstrated that persistent intracellular accumulation of H2O2 and exogenous exposure to H2O2 could induce cellular senescence (Song et al. 2014; Zhu et al. 2015; Gao et al. 2017). Therefore, it is necessary to explore the osteogenic potential of hDPSCs in oxidative stress.\nResveratrol (RSV; trans-3,5,4′-trihydroxystilbene), a naturally occurring nonflavonoid polyphenolic compound, richly presents in many fruits and vegetables, such as peanuts, mulberries and grapes (Gambini et al. 2013; Rauf et al. 2017). It has been reported that RSV can control the differentiation of lineage-specific neural stem cells and mediate the biological functions of terminally differentiated cells (Hu et al. 2014). Besides, RSV is also known as the Sirt1 activator (Wood et al. 2004) and has attained wide usage in dietary supplementation and traditional medicine (Biswas et al. 2020; Wu et al. 2020). Studies have shown that RSV exerts substantial antioxidant effects through scavenging excessive free radicals and enhancing the biosynthesis of intracellular antioxidant enzymes (Shakibaei et al. 2012; Kulkarni and Canto 2015; Sadi et al. 2018). Besides, RSV may prevent metabolic diseases through the activation of Sirt1, which further deacetylate the mitochondrial co-enzyme PGC-1α and improve mitochondrial functions (Lagouge et al. 2006). The mechanism of RSV regulating osteogenic potential in hDPSCs in oxidative stress needs further exploration. Therefore, we hypothesize that RSV could promote the osteogenesis of hDPSCs in oxidative stress via activating the Sirt1–Nrf2 pathway.", "Cell culture A total of eight teeth from different donors obtained with patients’ informed consent according to the current study, which had approval from the Research Ethics Committee (YS2020147) were collected separately, and the hDPSCs were extracted according to the method as described in our previous article (Zhang et al. 2019). The hDPSCs were cultured with growth medium (α-modified minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 mM l-glutamine). All reagents were obtained from Gibco (Carlsbad, CA), and the cells were cultured in an incubator at 37 °C with 5% CO2 (Binder, Tuttlingen, Germany). The medium was changed every three days, and the cells were treated with trypsin when they reached 80% confluence. The hDPSCs of passages 3–5 were used for the following experiments.\nA total of eight teeth from different donors obtained with patients’ informed consent according to the current study, which had approval from the Research Ethics Committee (YS2020147) were collected separately, and the hDPSCs were extracted according to the method as described in our previous article (Zhang et al. 2019). The hDPSCs were cultured with growth medium (α-modified minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 mM l-glutamine). All reagents were obtained from Gibco (Carlsbad, CA), and the cells were cultured in an incubator at 37 °C with 5% CO2 (Binder, Tuttlingen, Germany). The medium was changed every three days, and the cells were treated with trypsin when they reached 80% confluence. The hDPSCs of passages 3–5 were used for the following experiments.\nOxidative stress cell model and regents The oxidative stress model was optimized by different concentrations of H2O2 (0.1, 0.2 and 0.3 mM) treated for 24 h. All H2O2 solutions were prepared freshly. The RSV (Sigma-Aldrich, Shanghai, China) was dissolved in dimethyl sulfoxide (10 µL) (DMSO, Sigma-Aldrich, Shanghai, China) and diluted to 10 μM final concentration in a growth medium. The hDPSCs (n = 8) were divided into four groups. Normal control (NC) group refers to the hDPSCs which were cultured with growth medium, RSV group refers to the hDPSCs which were treated with RSV, H2O2 group were the hDPSCs which were cultured with H2O2 (0.2 mM), and H-RSV refers to the hDPSCs which were cultured with 0.2 mM H2O2 and 10 μM RSV.\nThe oxidative stress model was optimized by different concentrations of H2O2 (0.1, 0.2 and 0.3 mM) treated for 24 h. All H2O2 solutions were prepared freshly. The RSV (Sigma-Aldrich, Shanghai, China) was dissolved in dimethyl sulfoxide (10 µL) (DMSO, Sigma-Aldrich, Shanghai, China) and diluted to 10 μM final concentration in a growth medium. The hDPSCs (n = 8) were divided into four groups. Normal control (NC) group refers to the hDPSCs which were cultured with growth medium, RSV group refers to the hDPSCs which were treated with RSV, H2O2 group were the hDPSCs which were cultured with H2O2 (0.2 mM), and H-RSV refers to the hDPSCs which were cultured with 0.2 mM H2O2 and 10 μM RSV.\nIntracellular ROS fluorescent staining The cells at 2.5 × 104 cells/cm2 (n = 6) were seeded and attached to eight-chamber glass coverslips (Corning, Falcon culture slides, Corning, NY). Peroxide-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (50 μg) (Invitrogen™, Carlsbad, CA) was dissolved in DMSO (10 μL) and were diluted with the serum-free medium at a final concentration of 17.4 μM. The cells were incubated at 37 °C in darkness for 30 min and washed with 1 × PBS three times. The images were acquired by inverted fluorescence microscopy (ZEISS, AX-10, Oberkochen, Germany).\nThe cells at 2.5 × 104 cells/cm2 (n = 6) were seeded and attached to eight-chamber glass coverslips (Corning, Falcon culture slides, Corning, NY). Peroxide-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (50 μg) (Invitrogen™, Carlsbad, CA) was dissolved in DMSO (10 μL) and were diluted with the serum-free medium at a final concentration of 17.4 μM. The cells were incubated at 37 °C in darkness for 30 min and washed with 1 × PBS three times. The images were acquired by inverted fluorescence microscopy (ZEISS, AX-10, Oberkochen, Germany).\nCell proliferation assay Cell proliferation assay was determined by MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Beijing, China). Briefly, the cells were seeded in 96-well plates at 2.5 × 104 cells/cm2 (n = 6) and cultured in a growth medium for 24 h. MTT reagent (10 μL) and serum-free basal media (90 μL) were added to each well and incubated for 4 h. The supernatant was aspirated; formazan solutions (110 μL) were added to each well to dissolve the MTT formazan. Absorbance was measured at 490 nm using the Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).\nCell proliferation assay was determined by MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Beijing, China). Briefly, the cells were seeded in 96-well plates at 2.5 × 104 cells/cm2 (n = 6) and cultured in a growth medium for 24 h. MTT reagent (10 μL) and serum-free basal media (90 μL) were added to each well and incubated for 4 h. The supernatant was aspirated; formazan solutions (110 μL) were added to each well to dissolve the MTT formazan. Absorbance was measured at 490 nm using the Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).\nROS activity The intracellular ROS activity was examined by the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, Shanghai, China). Briefly, the cells were seeded at the density of 2.5 × 104 cells/cm2 in 96-well plates and cultured for 24 h (n = 3). DCFH-DA was diluted 1:1000 in serum-free medium to 10 μmol/L concentration and added to each well. The cells were incubated at 37 °C for 20 min and washed three times with a serum-free medium. The ROS activity was measured at 488 nm by Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).\nThe intracellular ROS activity was examined by the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, Shanghai, China). Briefly, the cells were seeded at the density of 2.5 × 104 cells/cm2 in 96-well plates and cultured for 24 h (n = 3). DCFH-DA was diluted 1:1000 in serum-free medium to 10 μmol/L concentration and added to each well. The cells were incubated at 37 °C for 20 min and washed three times with a serum-free medium. The ROS activity was measured at 488 nm by Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).\nSOD enzyme activity A total superoxide dismutase (SOD) assay kit with water-soluble tetrazolium salt, WST-1 (Beyotime Biotechnology, Shanghai, China), was used to analyse the SOD enzyme activity following the manufacturer's instructions. Briefly, the cells were seeded at a density of 2.5 × 104 cells/cm2 in 96-well plates (n = 3) and treated as described previously. The cells were harvested, lysed in 1 × PBS by ultrasonic pyrolysis, and centrifuged at 2200 rpm for 5 min at 4 °C. The supernatant (100 μL) was mixed with SOD working solution (160 μL) at 4 °C. The reaction mixture was centrifuged and transferred to 96-well plates, and the absorbance values were measured at 450 nm using a Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).\nA total superoxide dismutase (SOD) assay kit with water-soluble tetrazolium salt, WST-1 (Beyotime Biotechnology, Shanghai, China), was used to analyse the SOD enzyme activity following the manufacturer's instructions. Briefly, the cells were seeded at a density of 2.5 × 104 cells/cm2 in 96-well plates (n = 3) and treated as described previously. The cells were harvested, lysed in 1 × PBS by ultrasonic pyrolysis, and centrifuged at 2200 rpm for 5 min at 4 °C. The supernatant (100 μL) was mixed with SOD working solution (160 μL) at 4 °C. The reaction mixture was centrifuged and transferred to 96-well plates, and the absorbance values were measured at 450 nm using a Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).\nGlutathione (GSH) concentration assay A total glutathione assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with enzyme labelling was used to analyse GSH following the manufacturer's instructions. Briefly, the cells were seeded at 2.5 × 104 cells/cm2 in 24-well plates (n = 3) and treated as described previously. The cells were harvested, lysed in 1 × PBS by ultrasonic pyrolysis. The supernatant (100 μL) was mixed with precipitant agents (100 μL) and centrifuged at 3500 rpm for 10 min at 25 °C. Following this, the reaction mixture was kept at room temperature for 5 min, and the absorbance values were measured at 405 nm using a Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).\nA total glutathione assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with enzyme labelling was used to analyse GSH following the manufacturer's instructions. Briefly, the cells were seeded at 2.5 × 104 cells/cm2 in 24-well plates (n = 3) and treated as described previously. The cells were harvested, lysed in 1 × PBS by ultrasonic pyrolysis. The supernatant (100 μL) was mixed with precipitant agents (100 μL) and centrifuged at 3500 rpm for 10 min at 25 °C. Following this, the reaction mixture was kept at room temperature for 5 min, and the absorbance values were measured at 405 nm using a Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).\nQuantitative real-time PCR (qRT-PCR) The total RNA was extracted from the cells using RNAiso Plus reagent (TaKaRa, Kusatsu, Japan). RNAiso Plus (1 mL) was added to each culture dish (6 cm diameter) (n = 4) to lyse the cells. Total RNA (1 µg) was reverse transcribed using a PrimeScript TM RT reagent Kit with gDNA Eraser Kit (TaKaRa, Kusatsu, Japan). To quantify the mRNA expression, an amount of cDNA equivalent to the total RNA reacting system (20 µL) was amplified using SYBR Premix Ex Taq TM (TliRNase H Plus, TaKaRa, Kusatsu, Japan) following the manufacturer's protocol. The transcript levels of genes were evaluated with β-actin serving as the housekeeping internal control. The primer sequences are listed in Table 1. Relative transcript levels were calculated as 2–ΔΔCt, in which ΔΔCt = ΔE – ΔC, ΔE=Ctexp – Ctβ-actin and ΔC=Ctct1 – Ctβ-actin.\nPrimer of targeting genes.\nThe total RNA was extracted from the cells using RNAiso Plus reagent (TaKaRa, Kusatsu, Japan). RNAiso Plus (1 mL) was added to each culture dish (6 cm diameter) (n = 4) to lyse the cells. Total RNA (1 µg) was reverse transcribed using a PrimeScript TM RT reagent Kit with gDNA Eraser Kit (TaKaRa, Kusatsu, Japan). To quantify the mRNA expression, an amount of cDNA equivalent to the total RNA reacting system (20 µL) was amplified using SYBR Premix Ex Taq TM (TliRNase H Plus, TaKaRa, Kusatsu, Japan) following the manufacturer's protocol. The transcript levels of genes were evaluated with β-actin serving as the housekeeping internal control. The primer sequences are listed in Table 1. Relative transcript levels were calculated as 2–ΔΔCt, in which ΔΔCt = ΔE – ΔC, ΔE=Ctexp – Ctβ-actin and ΔC=Ctct1 – Ctβ-actin.\nPrimer of targeting genes.\nALP staining Following treatment, the cells were cultured in the osteogenic medium for 14 days and fixed with 4% formaldehyde at room temperature for 30 min and washed with 1 × PBS three times. The cells were stained with alkaline phosphatase (ALP, Beyotime Institute of Biotechnology, Shanghai, China) solution for 30 min at room temperature following the manufacturer's instructions. The photos were taken by an optical microscope (Olympus IX71, Tokyo, Japan).\nFollowing treatment, the cells were cultured in the osteogenic medium for 14 days and fixed with 4% formaldehyde at room temperature for 30 min and washed with 1 × PBS three times. The cells were stained with alkaline phosphatase (ALP, Beyotime Institute of Biotechnology, Shanghai, China) solution for 30 min at room temperature following the manufacturer's instructions. The photos were taken by an optical microscope (Olympus IX71, Tokyo, Japan).\nIn-Cell Western analysis Immediately after treatment, cells were washed in 1 × PBS and fixed in 10% neutral buffered formalin (NBF, Cellpath, Newtown, UK) in 1 × PBS for 20 min before being stained with the CellTag 700 staining ICW Kit I (Li-Cor Biosciences, Lincoln, NE). The samples were then incubated with the mouse anti-human Sirt1 antibody (1:600, Thermo Fisher, Waltham, MA) at 4 °C overnight with gentle shaking, followed by extensive washing in 1 × PBS containing 0.1% Tween 20 (Sigma-Aldrich, Shanghai, China) five times for 5 min per wash. The IRDye 800 CW goat anti-mouse secondary antibody (1:800) with the CellTag™ 700 stains (1:500) were added for 1 h at room temperature with gentle shaking followed by washing in 1 × PBS containing 0.1% Tween 20 for 5 min per wash. The plate was scanned on the Odyssey SA Imaging System (Li-Cor Biosciences, Lincoln, NE) using both 700 and 800 nm detection channels. Image Studio version 5 (Li-Cor Biosciences, Lincoln, NE) was used for performing the quantitative In-Cell Western (ICW) analysis.\nImmediately after treatment, cells were washed in 1 × PBS and fixed in 10% neutral buffered formalin (NBF, Cellpath, Newtown, UK) in 1 × PBS for 20 min before being stained with the CellTag 700 staining ICW Kit I (Li-Cor Biosciences, Lincoln, NE). The samples were then incubated with the mouse anti-human Sirt1 antibody (1:600, Thermo Fisher, Waltham, MA) at 4 °C overnight with gentle shaking, followed by extensive washing in 1 × PBS containing 0.1% Tween 20 (Sigma-Aldrich, Shanghai, China) five times for 5 min per wash. The IRDye 800 CW goat anti-mouse secondary antibody (1:800) with the CellTag™ 700 stains (1:500) were added for 1 h at room temperature with gentle shaking followed by washing in 1 × PBS containing 0.1% Tween 20 for 5 min per wash. The plate was scanned on the Odyssey SA Imaging System (Li-Cor Biosciences, Lincoln, NE) using both 700 and 800 nm detection channels. Image Studio version 5 (Li-Cor Biosciences, Lincoln, NE) was used for performing the quantitative In-Cell Western (ICW) analysis.\nImmunofluorescence labelling of Sirt1 Cells (2.5 × 104 cells/cm2) were seeded in eight-well multi-chambers (Falcon™ Culture Slides, Corning, Corning, NY) (n = 3) and cultured for 24 h. After treatment, the cells were fixed by 4% paraformaldehyde for 10 min at room temperature. The cells were washed three times with 1 × PBS and permeabilized by 0.2% Triton X-100 for 1 min. The cells were blocked with 10% goat serum (100 µL) (diluted in 1% BSA in 1 × PBS) (Dako, Nowy Sącz, Poland) for 30 min and washed five times with 1 × PBS before incubation in primary antibody (200 µL) (mouse anti-human Sirt1, 1:200 in 1% BSA) (Sigma-Aldrich, Shanghai, China) overnight at 4 °C. After rinsing with 1 × PBS, cells were incubated with Alexa-blue conjugated Goat Anti-Mouse secondary antibody (200 µL) (Alexa Fluor® 488, Invitrogen, Carlsbad, CA, 1:50 in 1% BSA-PBS) for 1 h at room temperature in darkness. The slides were examined with an inverted fluorescent microscope (ZEISS, AX-10, Oberkochen, Germany).\nCells (2.5 × 104 cells/cm2) were seeded in eight-well multi-chambers (Falcon™ Culture Slides, Corning, Corning, NY) (n = 3) and cultured for 24 h. After treatment, the cells were fixed by 4% paraformaldehyde for 10 min at room temperature. The cells were washed three times with 1 × PBS and permeabilized by 0.2% Triton X-100 for 1 min. The cells were blocked with 10% goat serum (100 µL) (diluted in 1% BSA in 1 × PBS) (Dako, Nowy Sącz, Poland) for 30 min and washed five times with 1 × PBS before incubation in primary antibody (200 µL) (mouse anti-human Sirt1, 1:200 in 1% BSA) (Sigma-Aldrich, Shanghai, China) overnight at 4 °C. After rinsing with 1 × PBS, cells were incubated with Alexa-blue conjugated Goat Anti-Mouse secondary antibody (200 µL) (Alexa Fluor® 488, Invitrogen, Carlsbad, CA, 1:50 in 1% BSA-PBS) for 1 h at room temperature in darkness. The slides were examined with an inverted fluorescent microscope (ZEISS, AX-10, Oberkochen, Germany).\nAnimals and tissue sections All animal studies were conducted following international standards on animal welfare and approved by the Animal Research Committee of Guangdong Medical University (Zhanjiang, China). The study was approved by the suggestion of animal research ethics (GDY20023l0) at Guangdong Medical University. Forty female Kunming mice (1-month-old and 12-months-old) were arranged and randomly assigned to four groups: Old (12-months-old), Old-RSV (12-months-old + RSV), Young (1-month-old) and Young-RSV (1-month-old + RSV). Cranium defects (1.0 mm) were created in the parietal bones of mice (n = 5 per group). Mice in the Old-RSV and Young-RSV groups were injected intraperitoneally with 0.2 mL RSV of 20 mg/kg/d for seven days, while the mice in the Old and Young groups were intraperitoneally injected with normal saline 0.2 mL. The mice were euthanized at 4 weeks after the surgery. The parietal bones were harvested and fixed for 24 h in 4% paraformaldehyde for further study.\nAll animal studies were conducted following international standards on animal welfare and approved by the Animal Research Committee of Guangdong Medical University (Zhanjiang, China). The study was approved by the suggestion of animal research ethics (GDY20023l0) at Guangdong Medical University. Forty female Kunming mice (1-month-old and 12-months-old) were arranged and randomly assigned to four groups: Old (12-months-old), Old-RSV (12-months-old + RSV), Young (1-month-old) and Young-RSV (1-month-old + RSV). Cranium defects (1.0 mm) were created in the parietal bones of mice (n = 5 per group). Mice in the Old-RSV and Young-RSV groups were injected intraperitoneally with 0.2 mL RSV of 20 mg/kg/d for seven days, while the mice in the Old and Young groups were intraperitoneally injected with normal saline 0.2 mL. The mice were euthanized at 4 weeks after the surgery. The parietal bones were harvested and fixed for 24 h in 4% paraformaldehyde for further study.\nMicro-computed tomography Micro-computed tomography (micro-CT) analysis was performed as described previously (Zhang et al. 2019). Briefly, the images were obtained via ex vivo micro-CT systems (Skyscanner 1174; Skyscan, Aartselaar, Belgium). Each sample was placed in a sample holder with the sagittal suture oriented parallel to the image plane and scanned in the air using the aluminium filer (0.25 mm), isotropic voxels (13 μm), 1000 ms integration time and one frame average. The scanner was equipped with an 80 kV, 500 μA X-ray tube, and a-36.9 megapixel Calibrate Centre offset coupled to a scintillator. For three-dimensional reconstruction (NRecon software, Skyscanner, Edinburgh, UK), the greyscale was set from 50 to 140. Standard three-dimensional morphometric parameters were determined in the ROI (100 cuts; 2.5 mm circle). Representative three-dimensional images were created using CT vox software (Skyscan, Edinburgh, UK).\nMicro-computed tomography (micro-CT) analysis was performed as described previously (Zhang et al. 2019). Briefly, the images were obtained via ex vivo micro-CT systems (Skyscanner 1174; Skyscan, Aartselaar, Belgium). Each sample was placed in a sample holder with the sagittal suture oriented parallel to the image plane and scanned in the air using the aluminium filer (0.25 mm), isotropic voxels (13 μm), 1000 ms integration time and one frame average. The scanner was equipped with an 80 kV, 500 μA X-ray tube, and a-36.9 megapixel Calibrate Centre offset coupled to a scintillator. For three-dimensional reconstruction (NRecon software, Skyscanner, Edinburgh, UK), the greyscale was set from 50 to 140. Standard three-dimensional morphometric parameters were determined in the ROI (100 cuts; 2.5 mm circle). Representative three-dimensional images were created using CT vox software (Skyscan, Edinburgh, UK).\nNonlinear optical microscope observation The excitation source of the SHG microscope was used to be pumped by a mode-locked Ti:sapphire laser oscillator (Spectra-Physics, 80 fs, 80 MHz and average powers up to 2.9 W). The laser beam was coupled into a multiphoton fluorescence scanning microscope (BX61 + FV1200, Olympus, Tokyo, Japan). The femtoseconds laser beam was focussed onto the sample by the objective lens (10× U PlanSApo, 0.40 N.A.; Olympus, Tokyo, Japan) with 10 mW. The SHG signal was isolated from the fundamental and any fluorescence by a band-pass filter (400/10 nm) and detected using a photomultiplier tube in the backscattered light path. Images were 800 × 800 pixels with 2 μs/pixel dwell time.\nThe excitation source of the SHG microscope was used to be pumped by a mode-locked Ti:sapphire laser oscillator (Spectra-Physics, 80 fs, 80 MHz and average powers up to 2.9 W). The laser beam was coupled into a multiphoton fluorescence scanning microscope (BX61 + FV1200, Olympus, Tokyo, Japan). The femtoseconds laser beam was focussed onto the sample by the objective lens (10× U PlanSApo, 0.40 N.A.; Olympus, Tokyo, Japan) with 10 mW. The SHG signal was isolated from the fundamental and any fluorescence by a band-pass filter (400/10 nm) and detected using a photomultiplier tube in the backscattered light path. Images were 800 × 800 pixels with 2 μs/pixel dwell time.\nImmunohistochemistry The samples were dehydrated through a graded series of ethanol solutions before they were embedded in paraffin, which was cut parallel to the cross into five sections for Sirt1 immunohistochemistry. The slides were incubated with primary antibodies anti-Sirt1 (Abcam, Waltham, MA; 1:150). The two-step plus Poly-HRP Anti Rabbit/Mouse IgG Detection System (Elabscience, Houston, TX) was used to detect immunoreactivity. The slides were counterstained with Mayer's haematoxylin (Hongqiaolexiang Inc., Shanghai, China) and cover-slipped using a permanent mounting medium.\nThe samples were dehydrated through a graded series of ethanol solutions before they were embedded in paraffin, which was cut parallel to the cross into five sections for Sirt1 immunohistochemistry. The slides were incubated with primary antibodies anti-Sirt1 (Abcam, Waltham, MA; 1:150). The two-step plus Poly-HRP Anti Rabbit/Mouse IgG Detection System (Elabscience, Houston, TX) was used to detect immunoreactivity. The slides were counterstained with Mayer's haematoxylin (Hongqiaolexiang Inc., Shanghai, China) and cover-slipped using a permanent mounting medium.\nStatistical analysis The statistical analysis was carried out by a two-tailed pair Student's t-test using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA). p Value <0.05 was considered statistically significant.\nThe statistical analysis was carried out by a two-tailed pair Student's t-test using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA). p Value <0.05 was considered statistically significant.", "A total of eight teeth from different donors obtained with patients’ informed consent according to the current study, which had approval from the Research Ethics Committee (YS2020147) were collected separately, and the hDPSCs were extracted according to the method as described in our previous article (Zhang et al. 2019). The hDPSCs were cultured with growth medium (α-modified minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 mM l-glutamine). All reagents were obtained from Gibco (Carlsbad, CA), and the cells were cultured in an incubator at 37 °C with 5% CO2 (Binder, Tuttlingen, Germany). The medium was changed every three days, and the cells were treated with trypsin when they reached 80% confluence. The hDPSCs of passages 3–5 were used for the following experiments.", "The oxidative stress model was optimized by different concentrations of H2O2 (0.1, 0.2 and 0.3 mM) treated for 24 h. All H2O2 solutions were prepared freshly. The RSV (Sigma-Aldrich, Shanghai, China) was dissolved in dimethyl sulfoxide (10 µL) (DMSO, Sigma-Aldrich, Shanghai, China) and diluted to 10 μM final concentration in a growth medium. The hDPSCs (n = 8) were divided into four groups. Normal control (NC) group refers to the hDPSCs which were cultured with growth medium, RSV group refers to the hDPSCs which were treated with RSV, H2O2 group were the hDPSCs which were cultured with H2O2 (0.2 mM), and H-RSV refers to the hDPSCs which were cultured with 0.2 mM H2O2 and 10 μM RSV.", "The cells at 2.5 × 104 cells/cm2 (n = 6) were seeded and attached to eight-chamber glass coverslips (Corning, Falcon culture slides, Corning, NY). Peroxide-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (50 μg) (Invitrogen™, Carlsbad, CA) was dissolved in DMSO (10 μL) and were diluted with the serum-free medium at a final concentration of 17.4 μM. The cells were incubated at 37 °C in darkness for 30 min and washed with 1 × PBS three times. The images were acquired by inverted fluorescence microscopy (ZEISS, AX-10, Oberkochen, Germany).", "Cell proliferation assay was determined by MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Beijing, China). Briefly, the cells were seeded in 96-well plates at 2.5 × 104 cells/cm2 (n = 6) and cultured in a growth medium for 24 h. MTT reagent (10 μL) and serum-free basal media (90 μL) were added to each well and incubated for 4 h. The supernatant was aspirated; formazan solutions (110 μL) were added to each well to dissolve the MTT formazan. Absorbance was measured at 490 nm using the Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).", "The intracellular ROS activity was examined by the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, Shanghai, China). Briefly, the cells were seeded at the density of 2.5 × 104 cells/cm2 in 96-well plates and cultured for 24 h (n = 3). DCFH-DA was diluted 1:1000 in serum-free medium to 10 μmol/L concentration and added to each well. The cells were incubated at 37 °C for 20 min and washed three times with a serum-free medium. The ROS activity was measured at 488 nm by Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).", "A total superoxide dismutase (SOD) assay kit with water-soluble tetrazolium salt, WST-1 (Beyotime Biotechnology, Shanghai, China), was used to analyse the SOD enzyme activity following the manufacturer's instructions. Briefly, the cells were seeded at a density of 2.5 × 104 cells/cm2 in 96-well plates (n = 3) and treated as described previously. The cells were harvested, lysed in 1 × PBS by ultrasonic pyrolysis, and centrifuged at 2200 rpm for 5 min at 4 °C. The supernatant (100 μL) was mixed with SOD working solution (160 μL) at 4 °C. The reaction mixture was centrifuged and transferred to 96-well plates, and the absorbance values were measured at 450 nm using a Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).", "A total glutathione assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with enzyme labelling was used to analyse GSH following the manufacturer's instructions. Briefly, the cells were seeded at 2.5 × 104 cells/cm2 in 24-well plates (n = 3) and treated as described previously. The cells were harvested, lysed in 1 × PBS by ultrasonic pyrolysis. The supernatant (100 μL) was mixed with precipitant agents (100 μL) and centrifuged at 3500 rpm for 10 min at 25 °C. Following this, the reaction mixture was kept at room temperature for 5 min, and the absorbance values were measured at 405 nm using a Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).", "The total RNA was extracted from the cells using RNAiso Plus reagent (TaKaRa, Kusatsu, Japan). RNAiso Plus (1 mL) was added to each culture dish (6 cm diameter) (n = 4) to lyse the cells. Total RNA (1 µg) was reverse transcribed using a PrimeScript TM RT reagent Kit with gDNA Eraser Kit (TaKaRa, Kusatsu, Japan). To quantify the mRNA expression, an amount of cDNA equivalent to the total RNA reacting system (20 µL) was amplified using SYBR Premix Ex Taq TM (TliRNase H Plus, TaKaRa, Kusatsu, Japan) following the manufacturer's protocol. The transcript levels of genes were evaluated with β-actin serving as the housekeeping internal control. The primer sequences are listed in Table 1. Relative transcript levels were calculated as 2–ΔΔCt, in which ΔΔCt = ΔE – ΔC, ΔE=Ctexp – Ctβ-actin and ΔC=Ctct1 – Ctβ-actin.\nPrimer of targeting genes.", "Following treatment, the cells were cultured in the osteogenic medium for 14 days and fixed with 4% formaldehyde at room temperature for 30 min and washed with 1 × PBS three times. The cells were stained with alkaline phosphatase (ALP, Beyotime Institute of Biotechnology, Shanghai, China) solution for 30 min at room temperature following the manufacturer's instructions. The photos were taken by an optical microscope (Olympus IX71, Tokyo, Japan).", "Immediately after treatment, cells were washed in 1 × PBS and fixed in 10% neutral buffered formalin (NBF, Cellpath, Newtown, UK) in 1 × PBS for 20 min before being stained with the CellTag 700 staining ICW Kit I (Li-Cor Biosciences, Lincoln, NE). The samples were then incubated with the mouse anti-human Sirt1 antibody (1:600, Thermo Fisher, Waltham, MA) at 4 °C overnight with gentle shaking, followed by extensive washing in 1 × PBS containing 0.1% Tween 20 (Sigma-Aldrich, Shanghai, China) five times for 5 min per wash. The IRDye 800 CW goat anti-mouse secondary antibody (1:800) with the CellTag™ 700 stains (1:500) were added for 1 h at room temperature with gentle shaking followed by washing in 1 × PBS containing 0.1% Tween 20 for 5 min per wash. The plate was scanned on the Odyssey SA Imaging System (Li-Cor Biosciences, Lincoln, NE) using both 700 and 800 nm detection channels. Image Studio version 5 (Li-Cor Biosciences, Lincoln, NE) was used for performing the quantitative In-Cell Western (ICW) analysis.", "Cells (2.5 × 104 cells/cm2) were seeded in eight-well multi-chambers (Falcon™ Culture Slides, Corning, Corning, NY) (n = 3) and cultured for 24 h. After treatment, the cells were fixed by 4% paraformaldehyde for 10 min at room temperature. The cells were washed three times with 1 × PBS and permeabilized by 0.2% Triton X-100 for 1 min. The cells were blocked with 10% goat serum (100 µL) (diluted in 1% BSA in 1 × PBS) (Dako, Nowy Sącz, Poland) for 30 min and washed five times with 1 × PBS before incubation in primary antibody (200 µL) (mouse anti-human Sirt1, 1:200 in 1% BSA) (Sigma-Aldrich, Shanghai, China) overnight at 4 °C. After rinsing with 1 × PBS, cells were incubated with Alexa-blue conjugated Goat Anti-Mouse secondary antibody (200 µL) (Alexa Fluor® 488, Invitrogen, Carlsbad, CA, 1:50 in 1% BSA-PBS) for 1 h at room temperature in darkness. The slides were examined with an inverted fluorescent microscope (ZEISS, AX-10, Oberkochen, Germany).", "All animal studies were conducted following international standards on animal welfare and approved by the Animal Research Committee of Guangdong Medical University (Zhanjiang, China). The study was approved by the suggestion of animal research ethics (GDY20023l0) at Guangdong Medical University. Forty female Kunming mice (1-month-old and 12-months-old) were arranged and randomly assigned to four groups: Old (12-months-old), Old-RSV (12-months-old + RSV), Young (1-month-old) and Young-RSV (1-month-old + RSV). Cranium defects (1.0 mm) were created in the parietal bones of mice (n = 5 per group). Mice in the Old-RSV and Young-RSV groups were injected intraperitoneally with 0.2 mL RSV of 20 mg/kg/d for seven days, while the mice in the Old and Young groups were intraperitoneally injected with normal saline 0.2 mL. The mice were euthanized at 4 weeks after the surgery. The parietal bones were harvested and fixed for 24 h in 4% paraformaldehyde for further study.", "Micro-computed tomography (micro-CT) analysis was performed as described previously (Zhang et al. 2019). Briefly, the images were obtained via ex vivo micro-CT systems (Skyscanner 1174; Skyscan, Aartselaar, Belgium). Each sample was placed in a sample holder with the sagittal suture oriented parallel to the image plane and scanned in the air using the aluminium filer (0.25 mm), isotropic voxels (13 μm), 1000 ms integration time and one frame average. The scanner was equipped with an 80 kV, 500 μA X-ray tube, and a-36.9 megapixel Calibrate Centre offset coupled to a scintillator. For three-dimensional reconstruction (NRecon software, Skyscanner, Edinburgh, UK), the greyscale was set from 50 to 140. Standard three-dimensional morphometric parameters were determined in the ROI (100 cuts; 2.5 mm circle). Representative three-dimensional images were created using CT vox software (Skyscan, Edinburgh, UK).", "The excitation source of the SHG microscope was used to be pumped by a mode-locked Ti:sapphire laser oscillator (Spectra-Physics, 80 fs, 80 MHz and average powers up to 2.9 W). The laser beam was coupled into a multiphoton fluorescence scanning microscope (BX61 + FV1200, Olympus, Tokyo, Japan). The femtoseconds laser beam was focussed onto the sample by the objective lens (10× U PlanSApo, 0.40 N.A.; Olympus, Tokyo, Japan) with 10 mW. The SHG signal was isolated from the fundamental and any fluorescence by a band-pass filter (400/10 nm) and detected using a photomultiplier tube in the backscattered light path. Images were 800 × 800 pixels with 2 μs/pixel dwell time.", "The samples were dehydrated through a graded series of ethanol solutions before they were embedded in paraffin, which was cut parallel to the cross into five sections for Sirt1 immunohistochemistry. The slides were incubated with primary antibodies anti-Sirt1 (Abcam, Waltham, MA; 1:150). The two-step plus Poly-HRP Anti Rabbit/Mouse IgG Detection System (Elabscience, Houston, TX) was used to detect immunoreactivity. The slides were counterstained with Mayer's haematoxylin (Hongqiaolexiang Inc., Shanghai, China) and cover-slipped using a permanent mounting medium.", "The statistical analysis was carried out by a two-tailed pair Student's t-test using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA). p Value <0.05 was considered statistically significant.", "Sirt1 was decreased in oxidative stress The concentration of H2O2 was optimized to induce oxidative stress, which was confirmed by the accumulation of ROS within the cells and cellular proliferation. After the treatment with H2O2 (0.2 mM), positive staining for ROS was located within the nuclei and cytoplasm of the cells (green). The ROS fluorescence intensity in the H2O2 (0.2 mM) group was stronger than the other groups (Figure 1(A,D)). Meanwhile, the fluorescence intensity of Sirt1 decreased in the H2O2 pre-treated group, which was localized within the nuclei and cytoplasm of the cells (Figure 1(A,G)).\nThe effect of H2O2 on cell morphology, ROS production, F-actin and Sirt1 protein in hDPSCs. (A) The morphology of hDPSCs treated with H2O2 detected by immunofluorescence, cell panels: (1) under bright field, (2) ROS, (3) F-actin, (4) Sirt1 (scale bar: 100 μM); (B) cell proliferation of hDPSCs shown on day 1, 2 and 3 of treatment with H2O2; (C) MTT assay showing cell viability at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (D) ROS fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (E) fibre fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (F) fibre length at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (G) Sirt1 fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM) (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. H2O2-0.2 mM group).\nThe concentration of H2O2 was optimized to induce oxidative stress, which was confirmed by the accumulation of ROS within the cells and cellular proliferation. After the treatment with H2O2 (0.2 mM), positive staining for ROS was located within the nuclei and cytoplasm of the cells (green). The ROS fluorescence intensity in the H2O2 (0.2 mM) group was stronger than the other groups (Figure 1(A,D)). Meanwhile, the fluorescence intensity of Sirt1 decreased in the H2O2 pre-treated group, which was localized within the nuclei and cytoplasm of the cells (Figure 1(A,G)).\nThe effect of H2O2 on cell morphology, ROS production, F-actin and Sirt1 protein in hDPSCs. (A) The morphology of hDPSCs treated with H2O2 detected by immunofluorescence, cell panels: (1) under bright field, (2) ROS, (3) F-actin, (4) Sirt1 (scale bar: 100 μM); (B) cell proliferation of hDPSCs shown on day 1, 2 and 3 of treatment with H2O2; (C) MTT assay showing cell viability at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (D) ROS fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (E) fibre fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (F) fibre length at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (G) Sirt1 fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM) (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. H2O2-0.2 mM group).\nRSV decreased oxidative stress of hDPSCs To investigate the effect of RSV against oxidative stress, the concentration of RSV was optimized by cell viability assay. The cell viability increased significantly in the RSV10 group than NC and H-RSV 10 than H2O2 group and the IC50 of RSV in hDPSCs is 67.65 ± 9.86. There were significant differences between the H2O2 group and H-RSV 10 group, and RSV (10 μM) was determined for the following experiments (Figure 2(A)). The ROS activity was declined (Figure 2(B)), the SOD enzyme activities were increased (Figure 2(C)) and the GSH concentration displayed enhancement (Figure 2(D)) in the H-RSV group compared with the H2O2 group. Similarly, further data showed that SOD1 and xCT mRNA expression increased significantly in the H-RSV group compared with the H2O2 group (Figure 2(E,F)).\nRSV promoted the proliferation and reduced the oxidative stress of hDPSCs pre-treated with H2O2. (A) Cell proliferation of hDPSCs pre-treated with/without H2O2 and different concentrations of RSV. (B) The ROS activity of hDPSCs pre-treated with/without H2O2 and RSV. (C) The SOD enzyme activity of hDPSCs pre-treated with/without H2O2 and RSV. (D) The GSH concentration of hDPSCs pre-treated with/without H2O2 and RSV. (E) The fold expression of SOD1 mRNA in hDPSCs. (F) The fold expression of xCT mRNA in hDPSCs. Group: NC (untreated cells), RSV (hDPSCs cultured with 10 μM RSV for 24 hours), H2O2 (hDPSCs treated by 0.2 mM H2O2 for 24 hours) and H-RSV (hDPSCs treated by 0.2 mM H2O2 cultured for 24 hours and then 10 μM RSV cultured for 24 hours) (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).\nTo investigate the effect of RSV against oxidative stress, the concentration of RSV was optimized by cell viability assay. The cell viability increased significantly in the RSV10 group than NC and H-RSV 10 than H2O2 group and the IC50 of RSV in hDPSCs is 67.65 ± 9.86. There were significant differences between the H2O2 group and H-RSV 10 group, and RSV (10 μM) was determined for the following experiments (Figure 2(A)). The ROS activity was declined (Figure 2(B)), the SOD enzyme activities were increased (Figure 2(C)) and the GSH concentration displayed enhancement (Figure 2(D)) in the H-RSV group compared with the H2O2 group. Similarly, further data showed that SOD1 and xCT mRNA expression increased significantly in the H-RSV group compared with the H2O2 group (Figure 2(E,F)).\nRSV promoted the proliferation and reduced the oxidative stress of hDPSCs pre-treated with H2O2. (A) Cell proliferation of hDPSCs pre-treated with/without H2O2 and different concentrations of RSV. (B) The ROS activity of hDPSCs pre-treated with/without H2O2 and RSV. (C) The SOD enzyme activity of hDPSCs pre-treated with/without H2O2 and RSV. (D) The GSH concentration of hDPSCs pre-treated with/without H2O2 and RSV. (E) The fold expression of SOD1 mRNA in hDPSCs. (F) The fold expression of xCT mRNA in hDPSCs. Group: NC (untreated cells), RSV (hDPSCs cultured with 10 μM RSV for 24 hours), H2O2 (hDPSCs treated by 0.2 mM H2O2 for 24 hours) and H-RSV (hDPSCs treated by 0.2 mM H2O2 cultured for 24 hours and then 10 μM RSV cultured for 24 hours) (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).\nRSV promotes the expression of osteogenic genes The mRNA expression of Runx2, OCN, Sirt1 and Nrf2 increased significantly in the H-RSV group compared with the H2O2 group (Figures 3(A,B) and 4(A,B)). Moreover, The ALP staining was significantly stronger by RSV with/without pre-treated with H2O2 (Figure 3(C)). Immunofluorescent staining and ICW showed that Sirt1 fluorescence intensity increased significantly by RSV compared with/without pre-treated with H2O2 (p < 0.05) (Figure 4(C–E)).\nRSV promoted the osteogenic differentiation of hDPSCs after the treatment of H2O2. (A) The fold expression of Runx2 mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (B) The fold expression of OCN mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (C) The ALP staining of hDPSCs pre-treated with/without H2O2 and RSV (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).\nRSV increased the Sirt1 and Nrf2 mRNA expression in hDPSCs by pre-treated with H2O2. (A) The fold expression of Runx2 mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (B) The fold expression of OCN mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (C) Fluorescence intensity of In-Cell Western assay (ZEISS microscope AX-10). (D) The sirt1 fluorescence of hDPSCs pre-treated with/without H2O2 and RSV by In-cell western. (E) Sirt1 immunofluorescence staining of hDPSCs pre-treated with/without H2O2 and RSV (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).\nThe mRNA expression of Runx2, OCN, Sirt1 and Nrf2 increased significantly in the H-RSV group compared with the H2O2 group (Figures 3(A,B) and 4(A,B)). Moreover, The ALP staining was significantly stronger by RSV with/without pre-treated with H2O2 (Figure 3(C)). Immunofluorescent staining and ICW showed that Sirt1 fluorescence intensity increased significantly by RSV compared with/without pre-treated with H2O2 (p < 0.05) (Figure 4(C–E)).\nRSV promoted the osteogenic differentiation of hDPSCs after the treatment of H2O2. (A) The fold expression of Runx2 mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (B) The fold expression of OCN mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (C) The ALP staining of hDPSCs pre-treated with/without H2O2 and RSV (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).\nRSV increased the Sirt1 and Nrf2 mRNA expression in hDPSCs by pre-treated with H2O2. (A) The fold expression of Runx2 mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (B) The fold expression of OCN mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (C) Fluorescence intensity of In-Cell Western assay (ZEISS microscope AX-10). (D) The sirt1 fluorescence of hDPSCs pre-treated with/without H2O2 and RSV by In-cell western. (E) Sirt1 immunofluorescence staining of hDPSCs pre-treated with/without H2O2 and RSV (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).\nRSV promotes bone mass in mice The area of the bone defect is significantly reduced in the 1-month-old + RSV group than the 1-month-old group, similarly in the 12-month-old + RSV group than the 12-month-old group. The intensity of collagen (green fluorescence) was stronger in the 12-month-old + RSV group than the 12-month-old group and stronger in the 1-month-old + RSV group than the 1-month-old group. The expression of Sirt1 was higher in the 12-month-old + RSV than the 12-month-old group (Figure 5).\nRSV promotes bone repair and increases bone mass. (A) Representative micro-CT images of bone repair and bone mass in calvarial defects. (B) Representative nonlinear optical microscope images of collagen in calvarial defects. (C) Immunohistochemical staining with sirt1 in hDPSCs pre-treated with/without H2O2 and RSV.\nThe area of the bone defect is significantly reduced in the 1-month-old + RSV group than the 1-month-old group, similarly in the 12-month-old + RSV group than the 12-month-old group. The intensity of collagen (green fluorescence) was stronger in the 12-month-old + RSV group than the 12-month-old group and stronger in the 1-month-old + RSV group than the 1-month-old group. The expression of Sirt1 was higher in the 12-month-old + RSV than the 12-month-old group (Figure 5).\nRSV promotes bone repair and increases bone mass. (A) Representative micro-CT images of bone repair and bone mass in calvarial defects. (B) Representative nonlinear optical microscope images of collagen in calvarial defects. (C) Immunohistochemical staining with sirt1 in hDPSCs pre-treated with/without H2O2 and RSV.", "The concentration of H2O2 was optimized to induce oxidative stress, which was confirmed by the accumulation of ROS within the cells and cellular proliferation. After the treatment with H2O2 (0.2 mM), positive staining for ROS was located within the nuclei and cytoplasm of the cells (green). The ROS fluorescence intensity in the H2O2 (0.2 mM) group was stronger than the other groups (Figure 1(A,D)). Meanwhile, the fluorescence intensity of Sirt1 decreased in the H2O2 pre-treated group, which was localized within the nuclei and cytoplasm of the cells (Figure 1(A,G)).\nThe effect of H2O2 on cell morphology, ROS production, F-actin and Sirt1 protein in hDPSCs. (A) The morphology of hDPSCs treated with H2O2 detected by immunofluorescence, cell panels: (1) under bright field, (2) ROS, (3) F-actin, (4) Sirt1 (scale bar: 100 μM); (B) cell proliferation of hDPSCs shown on day 1, 2 and 3 of treatment with H2O2; (C) MTT assay showing cell viability at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (D) ROS fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (E) fibre fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (F) fibre length at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM); (G) Sirt1 fluorescence intensity at different concentrations of H2O2 (0.1, 0.2 and 0.3 mM) (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. H2O2-0.2 mM group).", "To investigate the effect of RSV against oxidative stress, the concentration of RSV was optimized by cell viability assay. The cell viability increased significantly in the RSV10 group than NC and H-RSV 10 than H2O2 group and the IC50 of RSV in hDPSCs is 67.65 ± 9.86. There were significant differences between the H2O2 group and H-RSV 10 group, and RSV (10 μM) was determined for the following experiments (Figure 2(A)). The ROS activity was declined (Figure 2(B)), the SOD enzyme activities were increased (Figure 2(C)) and the GSH concentration displayed enhancement (Figure 2(D)) in the H-RSV group compared with the H2O2 group. Similarly, further data showed that SOD1 and xCT mRNA expression increased significantly in the H-RSV group compared with the H2O2 group (Figure 2(E,F)).\nRSV promoted the proliferation and reduced the oxidative stress of hDPSCs pre-treated with H2O2. (A) Cell proliferation of hDPSCs pre-treated with/without H2O2 and different concentrations of RSV. (B) The ROS activity of hDPSCs pre-treated with/without H2O2 and RSV. (C) The SOD enzyme activity of hDPSCs pre-treated with/without H2O2 and RSV. (D) The GSH concentration of hDPSCs pre-treated with/without H2O2 and RSV. (E) The fold expression of SOD1 mRNA in hDPSCs. (F) The fold expression of xCT mRNA in hDPSCs. Group: NC (untreated cells), RSV (hDPSCs cultured with 10 μM RSV for 24 hours), H2O2 (hDPSCs treated by 0.2 mM H2O2 for 24 hours) and H-RSV (hDPSCs treated by 0.2 mM H2O2 cultured for 24 hours and then 10 μM RSV cultured for 24 hours) (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).", "The mRNA expression of Runx2, OCN, Sirt1 and Nrf2 increased significantly in the H-RSV group compared with the H2O2 group (Figures 3(A,B) and 4(A,B)). Moreover, The ALP staining was significantly stronger by RSV with/without pre-treated with H2O2 (Figure 3(C)). Immunofluorescent staining and ICW showed that Sirt1 fluorescence intensity increased significantly by RSV compared with/without pre-treated with H2O2 (p < 0.05) (Figure 4(C–E)).\nRSV promoted the osteogenic differentiation of hDPSCs after the treatment of H2O2. (A) The fold expression of Runx2 mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (B) The fold expression of OCN mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (C) The ALP staining of hDPSCs pre-treated with/without H2O2 and RSV (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).\nRSV increased the Sirt1 and Nrf2 mRNA expression in hDPSCs by pre-treated with H2O2. (A) The fold expression of Runx2 mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (B) The fold expression of OCN mRNA of hDPSCs pre-treated with/without H2O2 and RSV. (C) Fluorescence intensity of In-Cell Western assay (ZEISS microscope AX-10). (D) The sirt1 fluorescence of hDPSCs pre-treated with/without H2O2 and RSV by In-cell western. (E) Sirt1 immunofluorescence staining of hDPSCs pre-treated with/without H2O2 and RSV (*p < 0.05, **p < 0.01, ***p < 0.001 vs. NC group).", "The area of the bone defect is significantly reduced in the 1-month-old + RSV group than the 1-month-old group, similarly in the 12-month-old + RSV group than the 12-month-old group. The intensity of collagen (green fluorescence) was stronger in the 12-month-old + RSV group than the 12-month-old group and stronger in the 1-month-old + RSV group than the 1-month-old group. The expression of Sirt1 was higher in the 12-month-old + RSV than the 12-month-old group (Figure 5).\nRSV promotes bone repair and increases bone mass. (A) Representative micro-CT images of bone repair and bone mass in calvarial defects. (B) Representative nonlinear optical microscope images of collagen in calvarial defects. (C) Immunohistochemical staining with sirt1 in hDPSCs pre-treated with/without H2O2 and RSV.", "Our present studies showed that hDPSCs pre-treated with H2O2 significantly reduced proliferation activity and increased oxidative stress. This finding was consistent with H2O2 induced oxidative stress in MC3T3-E1 cells (Choi et al. 2018). Previous studies also demonstrated that MSCs, when subjected to oxidative stress, significantly reduced proliferation activity (Mahmoudinia et al. 2019). The antioxidant activities of RSV were confirmed through the SOD enzyme activity and GSH concentration assay both to the normal cells and the oxidative-stressed cells, as well as the mRNA expressions of SOD1 and xCT, which represent the redox state of the cells.\nFurthermore, the up-regulated mRNA expression of RUNX2 and OCN, and the stronger ALP staining in hDPSCs treated by RSV with or without H2O2 pre-treatment, suggest that the RSV promoted osteogenesis of hDPSCs. In vivo, the collagen and bone matrix of the mouse calvarial defect area was significantly increased after RSV treatment, indicating that RSV could promote bone formation in young and ageing mice. This result is consistent with the previous study about RSV preventing bone loss (Su et al. 2017).\nOur results showed that the mRNA and protein expression of Sirt1 decreased in oxidative stress. However, these were increased by the treatment of RSV both in vitro and in vivo. Other studies also found that RSV could increase bone density via activating Sirt1 in mice (Liu et al. 2012). Besides, the Nrf2 mRNA expression was also enhanced by the treatment of RSV. Recent studies confirmed that Sirt1 regulates Nrf2 activation in the process of oxidative stress (Shah et al. 2017). Studies have confirmed that Nrf2 is a downstream target gene of Sirt1 (Wang et al. 2020). Taken together, these findings suggested that RSV could enhance osteogenesis via the Sirt1–Nrf2 pathway, indicating its therapeutic implication in anti-oxidative stress or other bone-related diseases.", "These findings shed light on the potential applications of RSV in stem cells-based therapy, with a higher proliferation rate and greater osteogenic potential of hDPSCs in regenerative medicine. Moreover, RSV might have the effect of promoting bone formation for the elderly or patients with oxidative stress physiological states such as hypertension, heart disease, diabetes, etc., as a potential agent." ]

[ "intro", "materials", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "results", null, null, null, null, "discussion", "conclusions" ]

[ "Dental pulp stromal cell", "ROS", "SOD", "RUNX1", "OCN", "xCT", "skull defect" ]

[ 174, 158, 134, 135, 133, 173, 155, 202, 89, 242, 253, 226, 193, 151, 106, 40, 372, 377, 346, 196 ]

[ "rsv", "h2o2", "hdpscs", "cells", "group", "treated", "10", "old", "sirt1", "pre" ]

[ "cells cultured osteogenic", "cells mscs considered", "stromal cells hdpscs", "restore repair tissue", "dental pulp stromal" ]

[ "Animals", "Environmental Monitoring", "Epidemiological Monitoring", "Female", "Fishes", "Food Contamination", "Food Preservation", "Hair", "Humans", "Maternal Exposure", "Mercury", "Michigan", "Pregnancy", "Premature Birth", "Water Pollutants, Chemical" ]

[ "Population", "Gestational age", "Mercury levels in hair", "Analytic strategy" ]

[ "The POUCH Study recruited women from 52 participating prenatal clinics located in five Michigan communities, each of which include urban, suburban, and rural areas (Holzman et al. 2001) (Figure 1). Women were enrolled in the 15th to 27th week of pregnancy, with approximately 70% before the 24th week. Eligibility criteria included screening for maternal serum alpha-fetoprotein (MSAFP) levels between 15 and 22 weeks of pregnancy, > 14 years of age, competency in English, singleton pregnancy with no known congenital or chromosomal anomalies at the time of recruitment, and no prepregnancy diabetes mellitus. A total of 1,226 women were enrolled in the POUCH cohort during the first part of the study (8 September 1998 through 31 July 2001). All were included in these analyses, except for five because of loss to follow-up and an additional 197 because hair samples were not available (69 declined hair sampling and 129 had hair that was < 7.6 cm long or in a woven hairstyle). Mid-pregnancy hair mercury levels were assessed in the remaining 1,024 cohort women. The study protocol was approved by human subjects review boards at participating institutions. Before enrollment, all participants provided written consent.\nDue to Health Insurance Portability and Accountability Act (HIPAA) regulations, it was not possible to determine an exact response rate or compare characteristics of participants with those of women who declined enrollment in the POUCH study. However, we were able to compare POUCH study data with data recorded on birth certificates of women who delivered in the five study communities in 2000. Ethnic-specific analyses (white non-Hispanic, African American), weighted by the proportion of women enrolled from each community, showed that the POUCH sample was very similar to community mothers on most factors measured—age, parity, education levels, and the proportions of women with Medicaid insurance, preterm delivery, previous stillbirth, previous preterm infant, and previous low birth weight infant. The one exception was the percentage of African Americans > 30 years of age, which was lower in POUCH (14%) than in community birth certificates (21%).", "Gestational age at delivery was determined by date of first day of the last menstrual period (LMP), or a gestational age estimate from an early ultrasound (≤ 25 weeks), the latter used when the two estimates disagreed by > 2 weeks. Early ultrasound data were available for 93% of women.", "At enrollment, approximately ≥ 100 strands of hair were cut close to the scalp from the posterior vertex region of women with hair at least 7.6 cm in length. A segment of hair closest to the scalp, approximating exposure during pregnancy, was assessed for total mercury levels. The length of segment used varied by gestational week at enrollment, assuming average hair growth of approximately 1.3 cm per month (Saitoh et al. 1967). Before analysis, hair was washed with acetone and water to remove mercury deposited from external sources. Cold vapor atomic absorption spectrometry (CVAAS; model M6000; Cetac Technology, Omaha, NE) was used to quantify total mercury levels in hair. It is estimated that around 70–90% of mercury in hair is methylmercury (Chen et al. 2002). Because of this and other factors, researchers consider total mercury levels in hair to be a useful biomarker of exposure to methylmercury (Berglund et al. 2005; Chen et al. 2002).", "We used generalized linear models (GLM) to assess the relationships between mercury levels in hair, maternal characteristics, and fish consumption. We used multicovariate logistic regression to evaluate the association between maternal mercury levels and risk of preterm delivery (< 37 weeks’ gestation), moderate preterm delivery (35–36 weeks’ gestation), and very preterm delivery (< 35 weeks’ gestation). Threshold levels for mercury effects were tested at quintile cutoffs and at the 90th percentile. To meet the underlying assumptions of the GLM, mercury levels were transformed to natural log scale (micrograms per gram) for analyses and then transformed back to mercury levels (micrograms per gram) for display in tables. All analyses were conducted using SAS 9.0 software (SAS Institute Inc., Cary, NC). On examining self-reports of total fish consumption, we found three women who had consumed > 300 fish meals in the 6 months corresponding to the first half of pregnancy. These outliers could not be verified and were removed from the analyses." ]

[ null, null, null, null ]

[ "Materials and Methods", "Population", "Gestational age", "Maternal characteristics and fish consumption", "Mercury levels in hair", "Analytic strategy", "Results", "Discussion" ]

[ " Population The POUCH Study recruited women from 52 participating prenatal clinics located in five Michigan communities, each of which include urban, suburban, and rural areas (Holzman et al. 2001) (Figure 1). Women were enrolled in the 15th to 27th week of pregnancy, with approximately 70% before the 24th week. Eligibility criteria included screening for maternal serum alpha-fetoprotein (MSAFP) levels between 15 and 22 weeks of pregnancy, > 14 years of age, competency in English, singleton pregnancy with no known congenital or chromosomal anomalies at the time of recruitment, and no prepregnancy diabetes mellitus. A total of 1,226 women were enrolled in the POUCH cohort during the first part of the study (8 September 1998 through 31 July 2001). All were included in these analyses, except for five because of loss to follow-up and an additional 197 because hair samples were not available (69 declined hair sampling and 129 had hair that was < 7.6 cm long or in a woven hairstyle). Mid-pregnancy hair mercury levels were assessed in the remaining 1,024 cohort women. The study protocol was approved by human subjects review boards at participating institutions. Before enrollment, all participants provided written consent.\nDue to Health Insurance Portability and Accountability Act (HIPAA) regulations, it was not possible to determine an exact response rate or compare characteristics of participants with those of women who declined enrollment in the POUCH study. However, we were able to compare POUCH study data with data recorded on birth certificates of women who delivered in the five study communities in 2000. Ethnic-specific analyses (white non-Hispanic, African American), weighted by the proportion of women enrolled from each community, showed that the POUCH sample was very similar to community mothers on most factors measured—age, parity, education levels, and the proportions of women with Medicaid insurance, preterm delivery, previous stillbirth, previous preterm infant, and previous low birth weight infant. The one exception was the percentage of African Americans > 30 years of age, which was lower in POUCH (14%) than in community birth certificates (21%).\nThe POUCH Study recruited women from 52 participating prenatal clinics located in five Michigan communities, each of which include urban, suburban, and rural areas (Holzman et al. 2001) (Figure 1). Women were enrolled in the 15th to 27th week of pregnancy, with approximately 70% before the 24th week. Eligibility criteria included screening for maternal serum alpha-fetoprotein (MSAFP) levels between 15 and 22 weeks of pregnancy, > 14 years of age, competency in English, singleton pregnancy with no known congenital or chromosomal anomalies at the time of recruitment, and no prepregnancy diabetes mellitus. A total of 1,226 women were enrolled in the POUCH cohort during the first part of the study (8 September 1998 through 31 July 2001). All were included in these analyses, except for five because of loss to follow-up and an additional 197 because hair samples were not available (69 declined hair sampling and 129 had hair that was < 7.6 cm long or in a woven hairstyle). Mid-pregnancy hair mercury levels were assessed in the remaining 1,024 cohort women. The study protocol was approved by human subjects review boards at participating institutions. Before enrollment, all participants provided written consent.\nDue to Health Insurance Portability and Accountability Act (HIPAA) regulations, it was not possible to determine an exact response rate or compare characteristics of participants with those of women who declined enrollment in the POUCH study. However, we were able to compare POUCH study data with data recorded on birth certificates of women who delivered in the five study communities in 2000. Ethnic-specific analyses (white non-Hispanic, African American), weighted by the proportion of women enrolled from each community, showed that the POUCH sample was very similar to community mothers on most factors measured—age, parity, education levels, and the proportions of women with Medicaid insurance, preterm delivery, previous stillbirth, previous preterm infant, and previous low birth weight infant. The one exception was the percentage of African Americans > 30 years of age, which was lower in POUCH (14%) than in community birth certificates (21%).\n Gestational age Gestational age at delivery was determined by date of first day of the last menstrual period (LMP), or a gestational age estimate from an early ultrasound (≤ 25 weeks), the latter used when the two estimates disagreed by > 2 weeks. Early ultrasound data were available for 93% of women.\nGestational age at delivery was determined by date of first day of the last menstrual period (LMP), or a gestational age estimate from an early ultrasound (≤ 25 weeks), the latter used when the two estimates disagreed by > 2 weeks. Early ultrasound data were available for 93% of women.\n Maternal characteristics and fish consumption Information on maternal characteristics including age, ethnicity, education, Medicaid insurance status, and smoking was collected through in-person interviews and self-administered questionnaires at enrollment. As part of the interview, women were asked “During this pregnancy how often have you eaten any of the following fish: shellfish, canned fish, other fish you purchased at a store or restaurant [referred to as bought fish], sport-caught fish in Michigan waters, and some other fish?” For each fish category, respondents were asked about their number of meals per day, week, month, or previous 6 months. Six months was used as an option for infrequent consumers of fish and was thought to capture the period from conception to study interview for most women in the study. The data were then scaled so that all fish consumption—fish categories and total—could be expressed as meals per 6 months, thereby describing levels of fish intake in approximately the first 6 months of pregnancy. Total fish consumption was calculated by summing consumption of all categories of fish and shellfish.\nInformation on maternal characteristics including age, ethnicity, education, Medicaid insurance status, and smoking was collected through in-person interviews and self-administered questionnaires at enrollment. As part of the interview, women were asked “During this pregnancy how often have you eaten any of the following fish: shellfish, canned fish, other fish you purchased at a store or restaurant [referred to as bought fish], sport-caught fish in Michigan waters, and some other fish?” For each fish category, respondents were asked about their number of meals per day, week, month, or previous 6 months. Six months was used as an option for infrequent consumers of fish and was thought to capture the period from conception to study interview for most women in the study. The data were then scaled so that all fish consumption—fish categories and total—could be expressed as meals per 6 months, thereby describing levels of fish intake in approximately the first 6 months of pregnancy. Total fish consumption was calculated by summing consumption of all categories of fish and shellfish.\n Mercury levels in hair At enrollment, approximately ≥ 100 strands of hair were cut close to the scalp from the posterior vertex region of women with hair at least 7.6 cm in length. A segment of hair closest to the scalp, approximating exposure during pregnancy, was assessed for total mercury levels. The length of segment used varied by gestational week at enrollment, assuming average hair growth of approximately 1.3 cm per month (Saitoh et al. 1967). Before analysis, hair was washed with acetone and water to remove mercury deposited from external sources. Cold vapor atomic absorption spectrometry (CVAAS; model M6000; Cetac Technology, Omaha, NE) was used to quantify total mercury levels in hair. It is estimated that around 70–90% of mercury in hair is methylmercury (Chen et al. 2002). Because of this and other factors, researchers consider total mercury levels in hair to be a useful biomarker of exposure to methylmercury (Berglund et al. 2005; Chen et al. 2002).\nAt enrollment, approximately ≥ 100 strands of hair were cut close to the scalp from the posterior vertex region of women with hair at least 7.6 cm in length. A segment of hair closest to the scalp, approximating exposure during pregnancy, was assessed for total mercury levels. The length of segment used varied by gestational week at enrollment, assuming average hair growth of approximately 1.3 cm per month (Saitoh et al. 1967). Before analysis, hair was washed with acetone and water to remove mercury deposited from external sources. Cold vapor atomic absorption spectrometry (CVAAS; model M6000; Cetac Technology, Omaha, NE) was used to quantify total mercury levels in hair. It is estimated that around 70–90% of mercury in hair is methylmercury (Chen et al. 2002). Because of this and other factors, researchers consider total mercury levels in hair to be a useful biomarker of exposure to methylmercury (Berglund et al. 2005; Chen et al. 2002).\n Analytic strategy We used generalized linear models (GLM) to assess the relationships between mercury levels in hair, maternal characteristics, and fish consumption. We used multicovariate logistic regression to evaluate the association between maternal mercury levels and risk of preterm delivery (< 37 weeks’ gestation), moderate preterm delivery (35–36 weeks’ gestation), and very preterm delivery (< 35 weeks’ gestation). Threshold levels for mercury effects were tested at quintile cutoffs and at the 90th percentile. To meet the underlying assumptions of the GLM, mercury levels were transformed to natural log scale (micrograms per gram) for analyses and then transformed back to mercury levels (micrograms per gram) for display in tables. All analyses were conducted using SAS 9.0 software (SAS Institute Inc., Cary, NC). On examining self-reports of total fish consumption, we found three women who had consumed > 300 fish meals in the 6 months corresponding to the first half of pregnancy. These outliers could not be verified and were removed from the analyses.\nWe used generalized linear models (GLM) to assess the relationships between mercury levels in hair, maternal characteristics, and fish consumption. We used multicovariate logistic regression to evaluate the association between maternal mercury levels and risk of preterm delivery (< 37 weeks’ gestation), moderate preterm delivery (35–36 weeks’ gestation), and very preterm delivery (< 35 weeks’ gestation). Threshold levels for mercury effects were tested at quintile cutoffs and at the 90th percentile. To meet the underlying assumptions of the GLM, mercury levels were transformed to natural log scale (micrograms per gram) for analyses and then transformed back to mercury levels (micrograms per gram) for display in tables. All analyses were conducted using SAS 9.0 software (SAS Institute Inc., Cary, NC). On examining self-reports of total fish consumption, we found three women who had consumed > 300 fish meals in the 6 months corresponding to the first half of pregnancy. These outliers could not be verified and were removed from the analyses.", "The POUCH Study recruited women from 52 participating prenatal clinics located in five Michigan communities, each of which include urban, suburban, and rural areas (Holzman et al. 2001) (Figure 1). Women were enrolled in the 15th to 27th week of pregnancy, with approximately 70% before the 24th week. Eligibility criteria included screening for maternal serum alpha-fetoprotein (MSAFP) levels between 15 and 22 weeks of pregnancy, > 14 years of age, competency in English, singleton pregnancy with no known congenital or chromosomal anomalies at the time of recruitment, and no prepregnancy diabetes mellitus. A total of 1,226 women were enrolled in the POUCH cohort during the first part of the study (8 September 1998 through 31 July 2001). All were included in these analyses, except for five because of loss to follow-up and an additional 197 because hair samples were not available (69 declined hair sampling and 129 had hair that was < 7.6 cm long or in a woven hairstyle). Mid-pregnancy hair mercury levels were assessed in the remaining 1,024 cohort women. The study protocol was approved by human subjects review boards at participating institutions. Before enrollment, all participants provided written consent.\nDue to Health Insurance Portability and Accountability Act (HIPAA) regulations, it was not possible to determine an exact response rate or compare characteristics of participants with those of women who declined enrollment in the POUCH study. However, we were able to compare POUCH study data with data recorded on birth certificates of women who delivered in the five study communities in 2000. Ethnic-specific analyses (white non-Hispanic, African American), weighted by the proportion of women enrolled from each community, showed that the POUCH sample was very similar to community mothers on most factors measured—age, parity, education levels, and the proportions of women with Medicaid insurance, preterm delivery, previous stillbirth, previous preterm infant, and previous low birth weight infant. The one exception was the percentage of African Americans > 30 years of age, which was lower in POUCH (14%) than in community birth certificates (21%).", "Gestational age at delivery was determined by date of first day of the last menstrual period (LMP), or a gestational age estimate from an early ultrasound (≤ 25 weeks), the latter used when the two estimates disagreed by > 2 weeks. Early ultrasound data were available for 93% of women.", "Information on maternal characteristics including age, ethnicity, education, Medicaid insurance status, and smoking was collected through in-person interviews and self-administered questionnaires at enrollment. As part of the interview, women were asked “During this pregnancy how often have you eaten any of the following fish: shellfish, canned fish, other fish you purchased at a store or restaurant [referred to as bought fish], sport-caught fish in Michigan waters, and some other fish?” For each fish category, respondents were asked about their number of meals per day, week, month, or previous 6 months. Six months was used as an option for infrequent consumers of fish and was thought to capture the period from conception to study interview for most women in the study. The data were then scaled so that all fish consumption—fish categories and total—could be expressed as meals per 6 months, thereby describing levels of fish intake in approximately the first 6 months of pregnancy. Total fish consumption was calculated by summing consumption of all categories of fish and shellfish.", "At enrollment, approximately ≥ 100 strands of hair were cut close to the scalp from the posterior vertex region of women with hair at least 7.6 cm in length. A segment of hair closest to the scalp, approximating exposure during pregnancy, was assessed for total mercury levels. The length of segment used varied by gestational week at enrollment, assuming average hair growth of approximately 1.3 cm per month (Saitoh et al. 1967). Before analysis, hair was washed with acetone and water to remove mercury deposited from external sources. Cold vapor atomic absorption spectrometry (CVAAS; model M6000; Cetac Technology, Omaha, NE) was used to quantify total mercury levels in hair. It is estimated that around 70–90% of mercury in hair is methylmercury (Chen et al. 2002). Because of this and other factors, researchers consider total mercury levels in hair to be a useful biomarker of exposure to methylmercury (Berglund et al. 2005; Chen et al. 2002).", "We used generalized linear models (GLM) to assess the relationships between mercury levels in hair, maternal characteristics, and fish consumption. We used multicovariate logistic regression to evaluate the association between maternal mercury levels and risk of preterm delivery (< 37 weeks’ gestation), moderate preterm delivery (35–36 weeks’ gestation), and very preterm delivery (< 35 weeks’ gestation). Threshold levels for mercury effects were tested at quintile cutoffs and at the 90th percentile. To meet the underlying assumptions of the GLM, mercury levels were transformed to natural log scale (micrograms per gram) for analyses and then transformed back to mercury levels (micrograms per gram) for display in tables. All analyses were conducted using SAS 9.0 software (SAS Institute Inc., Cary, NC). On examining self-reports of total fish consumption, we found three women who had consumed > 300 fish meals in the 6 months corresponding to the first half of pregnancy. These outliers could not be verified and were removed from the analyses.", "At the time of enrollment, 41% of women were < 25 years of age, 42% had ≤ 12 years of education, and 43% were insured by Medicaid, a health-related public assistance program (Table 1). The percentage of women from racial/ethnic backgrounds other than non-Hispanic white and African American was only 9%. The “other” ethnic groups were included with non-Hispanic whites in the final models. Alternative analyses excluding these other groups showed similar results to that of the more inclusive final models.\nIn approximately the first 6 months of pregnancy, 11% of women in this sample did not eat any fish, 25% ate two fish meals or fewer, and only 50% ate more than nine fish meals. The mean level of total fish consumption was 19.6 meals/6 months, considerably higher than the median (50th percentile = 9.0 meals/6 months), suggesting a right skewing of the distribution (Table 2). Canned fish was the most frequently consumed fish category, with 25% of women eating ≥ 12 meals/6 months, followed by bought fish, with 25% eating ≥ 6 meals/6 months. Only 9.2% of women reported consumption of sport-caught fish during the first 6 months of pregnancy.\nMercury levels in maternal hair ranged from 0.01 to 2.50 μg/g, with a mean of 0.29 μg/g and a median of 0.23 μg/g. Approximately 20% of women had levels > 0.38 μg/g (Table 3). Mercury levels were divided into quintiles and total fish consumption and consumption of each fish category were divided into four levels: 0 meals/6 months (reference group), 1–5 meals/6 months, 6–23 meals/6 months, and ≥ 24 meals/6 months. Women with higher levels of total fish consumption were more likely to have mercury levels in the upper quintiles (Table 3). The mean and median hair mercury levels for the 109 women who did not consume fish during this period in pregnancy were 0.15 μg/g and 0.13 μg/g, respectively. Interestingly, 10% of women who reported not eating fish during pregnancy had hair mercury levels in the 4th and 5th quintiles.\nIn multicovariate analyses, higher mercury levels were significantly associated with older maternal age (≥ 25 years), white and “other” ethnicity, not being insured by Medicaid, and residing in communities 3, 4, and 5 (Figure 2), even after adjusting for total fish consumption. Maternal mercury levels were not significantly related to gestational week at enrollment or smoking before or during pregnancy. We reevaluated the association between total fish consumption and mercury levels in a model that included maternal covariates related to mercury levels in hair (Table 4). The adjusted mean mercury continued to be significantly higher in women who consumed fish compared with that in women who did not consume fish in the first 6 months of pregnancy, and mean mercury levels increased as levels of fish consumption increased. In another model that included consumption of each fish category along with the other maternal covariates related to mercury levels in this sample, consumption of canned fish, bought fish, and sport-caught fish were each positively associated with mercury levels in maternal hair. Adjustment for gestational week at enrollment did not appreciably alter these associations.\nIn the final analyses, we assessed maternal mercury levels at mid-pregnancy in relation to gestational week at delivery. Women who delivered very preterm (< 35 weeks) were more likely to have had hair mercury levels at or above the 90th percentile (0.55–2.5 μg/g) than were women who delivered at term (≥ 37 weeks), even after adjusting for maternal characteristics and total fish consumption [odds ratio (OR) = 3.0; 95% confidence interval (CI), 1.3–6.7] (Table 5). These results remained relatively unchanged in models that adjusted for gestational week at enrollment and fish categories. The association between maternal mercury levels and very preterm delivery was not evident at lower threshold levels of mercury (i.e., quintile cut-points), and mercury levels were not associated with delivery of a moderately preterm infant (35–36 weeks).", "Women enrolled in the POUCH Study resided in communities surrounded by the Great Lakes, but their levels of fish consumption in the first 6 months of pregnancy would be considered moderate to low relative to populations that subsist on fish. Despite the modest levels of fish consumption, there was strong evidence that mercury levels in maternal hair were higher when fish consumption levels were higher.\nA positive correlation between fish consumption and mercury levels in pregnant women has been reported in studies conducted in European countries (Bjornberg et al. 2003; Daniels et al. 2004; Oskarsson et al. 1994) and the Amazon basin of South America (Bruhn et al. 1995; Hacon et al. 2000). Fewer data are available from pregnant populations in North America. Two studies from Canada in areas close to the Great Lakes showed a positive association between fish consumption and mercury levels in maternal hair (Morrissette et al. 2004; Muckle et al. 2001). Other studies from the Great Lakes area have observed a positive relationship between fish consumption and mercury levels in nonpregnant women of child-bearing age (Nadon et al. 2002), anglers and fish eaters (Cole et al. 2004), Native Americans (Gerstenberger et al. 1997), Montreal sportfishers of Asian origin (Kosatsky et al. 1999b), and the general population (Kosatsky et al. 2000; Mahaffey and Mergler 1998). The concordance of the information provided from these and other studies indicates that in diverse regions of the globe, consumption of fish is a major source of methylmercury exposure in humans.\nIn one study, methylmercury was the most commonly identified pollutant in sport-caught fish (Anderson et al. 2004). Canned tuna has been implicated as one of the main foods contributing to total mercury intake (Legrand et al. 2005; U.S. Department of Health and Human Services and U.S. Environmental Protection Agency 2006). Because of variation in methylmercury levels by fish type and location, studies in different settings have tried, as we tried in this study, to link mercury levels to consumption of particular types of fish. In anglers and Native Americans residing in the Great Lakes region, levels of mercury have been shown to be positively associated with consumption of sport-caught fish (Cole et al. 2004; Gerstenberger et al. 1997; Kosatsky et al. 1999a; Nadon et al. 2002). A study by the Wisconsin Division of Public Health reported that women of child-bearing age who ate sport-caught fish had higher mercury levels in hair than those of women who did not eat sport-caught fish, but the difference was not statistically significant (Knobeloch et al. 2005). Morressette et al. (2004) reported that bought fish, including canned fish, were important sources of mercury exposure during pregnancy. These studies are consistent with our findings of higher hair mercury levels in pregnant women who consumed greater amounts of canned fish, sport-caught fish, and bought fish.\nAs anticipated, hair mercury levels in POUCH Study mothers were lower than levels found in heavily exposed, fish-eating communities. Across studies the unit of measure used to report hair mercury levels varies—parts per million, milligrams per kilogram, or micrograms per gram—but these units are equivalent and can be directly compared. The median hair mercury level in the Faroe Islands sample was 4.5 ppm, and in the Seychelles 5.9 ppm, almost double the highest mercury level observed in the POUCH Study (Davidson et al. 1999; Grandjean et al. 1992). In a study of Swedish women of child-bearing age, 127 women reported consuming, on average, four fish meals/week and had a median hair mercury level of 0.70 mg/kg (Bjornberg et al. 2005). A study of 150 pregnant women from varied locations in Alaska found somewhat lower levels: The median hair mercury was 0.47 mg/kg and the mean was 0.72 mg/kg (Arnold et al. 2005). Two studies—one from Massachusetts, the other from southwest Québec—reported mercury levels more similar to those in our findings. The first study included 135 pregnant women recruited from an HMO in eastern Massachusetts (Oken et al. 2005). On average, women in that study consumed slightly more fish (1.2 meals/week) than did women in the POUCH Study (0.82 meals/week), and had slightly higher hair mercury levels (mean = 0.55 ppm; 10% exceeded 1.2 ppm) than did those in the POUCH Study (mean = 0.29 μg/g; 10 % exceeded 0.54 μg/g). In the Québec study of 159 pregnant women, mercury was assessed in sequential centimeters of hair to represent levels during different months of pregnancy (Morrissette et al. 2004). Among women who consumed two or more fish meals/month, mean hair mercury levels were about 0.20 μg/g at the 5th–6th month of pregnancy. Differences in maternal mercury levels across these studies may be explained by differences in both amount and types of fish consumed.\nIn the POUCH Study, maternal characteristics of older age, ethnicity of white and other, not being insured by Medicaid, and residing in three of the five communities were significantly associated with higher maternal mercury levels after adjusting for fish consumption. The reasons for these associations are unclear and may include other lifestyle factors, mercury exposures not related to fish, and residual confounding from incomplete adjustment for fish types, portion size, and method of preparation. The observed higher mean mercury level among women without Medicaid insurance—a marker of higher socioeconomic status—is consistent with a recently released report by the Wisconsin Division of Public Health, which also found higher mercury levels in association with higher socioeconomic status (i.e., college education and annual household income > $75,000) (Knobeloch et al. 2005). It is unlikely that the community differences in mercury levels within our study reflect variations in pollutant levels in local fish, because most of the fish consumed are not from local waters. About 10% of women in the POUCH Study reported not consuming fish during pregnancy but had hair mercury levels in the top two quintiles. This suggests that their diet histories may have been inaccurate or there may be sources of exposure, other than fish, that are uncommon but deserve consideration.\nOur study is the first to report an association between delivery at < 35 weeks’ gestation and maternal hair mercury levels ≥ 0.55 μg/g (upper 10th percentile). The biologic mechanism supporting this finding requires further investigation. It has been shown that methylmercury produces oxidative stress at the cellular level (Sanfeliu et al. 2003), which may be a contributing factor. In addition, methylmercury can influence shape, aggregation, and levels of platelets (Hornberger and Patscheke 1989; Macfarlane 1981) and thromboxane (Caprino et al. 1983). These effects may potentiate underlying pathology related to maternal vascular diseases in pregnancy. Within the POUCH Study we plan to examine maternal biomarkers of endothelial dysfunction and will explore whether they are related to maternal mercury levels.\nSeveral methodologic differences could account for the inconsistency between our study results and those of previous studies that did not detect an association between mercury levels and gestational age at delivery (Foldspang and Hansen 1990; Fu 1993; Grandjean et al. 2001; Lucas et al. 2004). Our study correlating fish consumption and maternal mercury levels in hair is the largest in the United States to date, providing greater statistical power to detect moderate associations (OR = 3.0) with an outcome that is less frequent, such as delivery at < 35 weeks. Studies using blood levels of mercury (maternal, cord) reflect variations in recent exposure (Foldspang and Hansen 1990; Grandjean et al. 2001; Lucas et al. 2004), whereas mercury in hair is relatively stable and mid-pregnancy hair levels better represent average exposure across the first half of pregnancy. The POUCH Study women had considerably lower mercury levels relative to those of women in other studies, allowing for the testing of lower thresholds. Only one study assessed preterm delivery as an outcome (Fu 1993), and the other three considered mean gestational age at delivery which may have obscured mercury effects in the tail of the distribution (i.e., very preterm) (Foldspang and Hansen 1990; Grandjean et al. 2001; Lucas et al. 2004). In addition, other studies did not adjust for fish consumption, a potential confounder in populations with high levels of fish consumption. Researchers have hypothesized that omega-3 fatty acids in fish can prolong gestation by down-regulating the synthesis of prostaglandin (PG) E2 and PGF2a, and promoting the synthesis of PGI2 and PGI3, thus leading to a more relaxed myometrium (Ferretti et al. 1988; Hansen and Olsen 1988).\nMajor strengths of the present study include the large number of pregnant women participating, the prospective design, and the use of hair as an index of methylmercury exposure. Hair levels of total mercury represent a longer window of exposure than those of blood levels. This study also had several limitations: First, information on fish consumption was based on recall, which can lead to inaccurate estimates. However, the recall period was focused on eating habits during pregnancy, a time when women are often more aware of their dietary patterns. Because the reports of fish consumption were obtained well before the outcome of the pregnancy, there is little concern about differential recall bias. Second, maternal interviews did not include information on portion size of fish meals and methods of preparation. Factoring in these details might further strengthen correlations between levels of fish consumption and levels of mercury in hair. Third, women were not asked about number of dental amalgam fillings. Total mercury measured in maternal hair may include 10–30% inorganic mercury, the major form of mercury in amalgam fillings (Spencer 2000). However, it is widely accepted that in populations similar to those we have studied, food—particularly fish—is the major exposure source of methylmercury, the primary component of total mercury found in hair. In addition, several studies have reported little or no relationship between hair total mercury and exposure to amalgams (Berglund et. al. 2005; Bjornberg et al. 2003; Hansen et al. 2004; Pesch et al. 2002; Tulinius 1995).\nAn important limitation is our lack of information on maternal levels of other pollutants commonly found in fish (e.g., organo-chlorines). Women with higher mercury levels in hair may have been exposed to higher levels of other contaminants, such as polychlorinated biphenyls (PCBs) and dichlorodiphenyl-dichloroethylene (DDE), a metabolite of DDT. Hair mercury levels and blood PCB and DDE levels have all been found to be elevated in high-level fish consumers compared with people who eat less fish (Kosatsky et al. 1999a, 1999b). Based on the estimate of average daily dietary exposure to contaminants for approximately 120,000 adults from the Nurses’ Health Study and the Health Professional Follow-up Study, intraindividual exposures to mercury and DDE were found to be moderately correlated among women (r = 0.27) and men (r = 0.17) (MacIntosh et al. 1996). We tried to adjust for other pollutants in fish and for the potentially beneficial aspects of fish (e.g., omega-3 fatty acids) by including total fish consumption in our final analyses. After adjustment, the positive association between maternal mercury levels in hair and risk of very preterm delivery remained, but we cannot rule out residual confounding by other pollutants. Previous studies have failed to find a link between preterm delivery risk and levels of PCBs (Berkowitz et al. 1996; Longnecker et al. 2005) or DDE (Farhang et al. 2005; Fenster et al. 2006; Torres-Arreola et al. 2003). The one exception is a report of excess preterm delivery among women with high blood levels of DDE (Longnecker et al. 2001). This study used stored blood samples from the Collaborative Perinatal Project, a U.S. cohort assembled in 1959 through 1966. The DDE levels associated with preterm birth were considerably higher than DDE levels found in U.S. women of reproductive age today.\nOur study reinforced previous findings suggesting that fish consumption is a major source of mercury exposure for pregnant women. Although much attention has been focused on pollutants in locally caught fish, and fish advisories are not uncommon, we found that only a small percentage of pregnant women, < 10%, consumed sport-caught fish during pregnancy. The greatest fish source for mercury exposure appeared to be canned fish, both because it was consumed more and, per meal, it was among the fish categories associated with the highest levels of mercury in maternal hair. The observed relationship between elevated mercury levels and increased risk of very preterm delivery is a new finding and requires caution in interpretation. Although the study sample was large, the number of women who delivered before 35 weeks of pregnancy was small (n = 44), and more studies are needed to test this association." ]

[ "materials|methods", null, null, "intro", null, null, "results", "discussion" ]

[ "fish consumption", "pregnancy", "preterm delivery", "mercury" ]

[ 405, 59, 186, 194 ]

[ "fish", "levels", "mercury", "women", "hair", "mercury levels", "study", "consumption", "pregnancy", "maternal" ]

[ "included screening maternal", "pregnancy hair levels", "diseases pregnancy pouch", "pregnancy hair mercury", "pouch cohort study" ]

[ "Female", "Humans", "Breast Neoplasms", "Cancer Survivors", "Mastectomy", "Lymphedema", "Survivors", "Upper Extremity" ]

[ "Conclusion" ]

[ "This study of women who have undergone surgical intervention for breast cancer concludes that there was significant amount of neural tissue impairment noted to mechanical provocation test post operatively after 6 months of surgery. The study suggests that severity of lymphedema was directly related to the nerves affected due to neural tissue impairment. Neural tissue mobility impairments should be assessed in women with lymphedema following the treatment of breast cancer. It should be assessed bilaterally for correlation of the symptoms. Therapists need to integrate the findings of neural tissue mobility impairments into the rehabilitation program for breast cancer survivors with pain and lymphedema for better quality of life." ]

[ null ]

[ "Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion" ]

[ "Breast cancer is most commonly diagnosed and is the leading cause of cancer death among women worldwide (Blecher et al., 2011). It is common in Asian population and with an estimation of 1 in 7 women will develop breast cancer at some time in her life (Loh and Quek, 2011). The incidence of breast cancer (BC) among women has continued to increase within the last decade in spite of screening mammography and the reduction of mortality (Babasaheb et al., 2021). Breast cancer patients are managed with various surgical procedures such as radical mastectomy (RC), modified radical mastectomy (MRM) and breast conserving surgery (BCS) (De GRoef A et al., 2016). Axillary lymph node dissection (ALND), another method is commonly employed as a procedure for diagnosing and treating positive lymph nodes.\nHowever, several consequences may develop after breast surgery, ALND, radiation and chemotherapy, such as axillary web syndrome, frozen shoulder, numbness, shoulder pain and range of motion (ROM) restriction, lymphostasis, and lymphedema (Díaz I et al., 2017). Up to 77% report sensory disturbance in the breast or arm (Smoot B et al., 2014). These short- and long-term consequences have dramatic impact on physical function and quality of life in this population (Norman et al., 2009; Shinde and Patil , 2020; Smoot B et al., 2010).\nThe lymphatic system has vessels which transport fluid and plasma proteins from interstitial tissue to the blood circulation. When the lymphatic drainage system is impaired draining of lymphatic fluid ceases to work and fluids accumulate in the tissue and therefore lymphedema occurs which may further lead to swelling (Johansson and Branje, 2010). This condition may be reversible and effective treatment includes compression bandaging, wearing a sleeve/glove, manual lymphatic drainage and pneumatic pumping (Johansson and Branje, 2010; Jaju and Shinde, 2019). If the oedema is allowed to progress without treatment the volume will increase, and the arm will get heavy and cause discomfort and pain(Johansson and Branje 2010; Casley-Smith JR, 1995). Breast cancer-related lymphedema results from impaired lymph transport due to surgical removal of or radiation-induced damage to axillary lymph nodes and lymphatic channels, which leads to accumulation of lymph in the UE, chest, or trunk (Smoot B et al., 2014).\nNeural tissue mobility also called as neuro dynamics which refers to the communication between the different parts of the nervous system and to the nervous system relationship to the musculoskeletal system. Neurodynamic in the sense implied here is the mobilisation of the nervous system as an approach to physical treatment of pain. This mobilisation activates a range of mechanical and physiological responses in nervous tissue such as neural sliding, pressurisation, elongation, tension and changes in intraneural microcirculation, axonal transport, and nervous impulse movements (Shacklock, 1995).\nInjuries to the nerve have been reported with axillary dissection which maybe a result of positional tractioning, forceful retraction, direct laceration or contusion of neural tissue during surgery. Nerve injury can also be due to entrapment or compression related to post-operative or radiation-induced fibrosis and scarring (Jare et al., 2019; Shinde 2020; Macdonald et al., 2005). Peripheral nerves when they are subjected to trauma they become “sensitized” less tolerant to the physical stresses (compression and stretch), imposed upon them during movement. The mechanisms responsible for development of neuropathic pain from cancer treatment may also affect the tolerance of the nervous system to movement. Additionally, peripheral nerves at risk during surgery or radiation may be subjected to higher physical stresses during movement due to compression or restrictions from adhesions and fibrosis (Smoot et al., 2014).\nThere is no such study which shows the impact of lymphedema on neural tissue mobility. So, it is necessary to analyse whether the presence of lymphedema will have an effect on neural tissue mobility which may result in pain, numbness, tingling sensations, restrictions in range of motion of the upper limb while assessing the patient. Adverse neural tension may arise due to inflammation, nerve compression, or impaired blood supply to the area. This study will help add evidences on neural tissue mobility impairment that may happen due to varying severity of lymphedema. Various studies have suggested that neural structures sensitivity is increased due to provocative sequences of upper quadrant movements in in various upper limb pain syndromes. But there are no studies till the date which have assessed for whether the neural tissues become sensitive to such movements following surgery for breast cancer. Soft tissue structures mainly the contractile tissues noncontractile tissues and inert tissues were restricted and compressed during shortening and lengthening. The restrictive and compressive effect of lymphedema resulted in pain, limited mobility of joints as well as restricted neural tissue mobility.\nThis study was conducted to estimate the changes in pain, upper limb mobility and strength in response to altered upper limb neural tissue mobility and mechano-sensitivity in breast cancer patients with lymphedema. ", "This study was done in a breast cancer survivor support group of Tertiary care hospital in India. The patients who were included in the study had undergone lumpectomy, quadrantectomy and unilateral mastectomy 6 months before entering into this study and suffering from lymphedema. Age group was ranged from 30 to 80 years old women. The subjects excluded from the study were male cancer survivors and patients who have undergone bilateral breast cancer, breast surgery for cosmetic reasons or prophylactic mastectomy, other medical conditions (e.g., arthritis and fibromyalgia syndrome) and recurrent cancer. Before starting the study, all the participants were informed about the study procedure with a written informed consent obtained from each participant. The study was ethically cleared by the Ethical Committee of Krishna Institute of Medical Sciences. Total 72 breast cancer survivor women suffering from lymphedema, who had undergone either lumpectomy, quadrantectomy, unilateral mastectomy 6 months ago and those who are undergoing either radiation therapy, chemotherapy or both were included in this study. The demographic data and past surgical history were taken from subjects prior to the assessment of the condition.\n\nData Collection Tools \n\nPain Intensity: An 11-point numerical rating scale (0 = no pain; 10 = maximum pain) was used to assess the intensity of spontaneous neck and shoulder/axillary pain. The patients were asked to not take any analgesics or muscle relaxants 24 hours prior to the assessment. \nRange of motion for shoulder was measured by using goniometer. Shoulder movements including flexion, extension, abduction and adduction were assessed.\nMeasurement of lymphedema was done on the affected upper extremity (usually the one which was operated) and also comparison was done with non-affected side. The lymphedema was measured using an inch tape for segregation of the subjects into mild, moderate, severe lymphedema.\nNeural tissue mobility was assessed using the neurodynamic tests for upper limb. ULNTs for the median (ULNT1MEDIAN), Median bias (ULNT2 MEDIAN BIAS), radial (ULNT3 RADIAL), and ulnar nerves (ULNT 4 ULNAR) were performed according to the process described by Butler. (Butler MW, Karagiannopoulos C, Galantino ML, Mastrangelo MA 2019) The patients were in a supine position without a pillow under the head or knees and the legs were uncrossed. The head was in neutral rotation, and the hand of the unaffected arm rested on the side. All the tests were performed on both sides in standardized sequence until the end of range or until the symptoms were reproduced. Before performing the tests, the patients were instructed about communicating the onset of any sensation such as pain or stretch in the areas of the arm and neck. The subject’s pain and other symptoms were taken into consideration. The tests were first carried on the unaffected side and then on the affected side. If the patient did not experience pain or any other sensation, the test was further continued with the elbow extension or the shoulder abduction to the normal end of range. The tests were ceased when the patient experienced, at least some symptoms such as the shoulder girdle attempted to elevate, or when muscular resistance to movement was found.\n\nStatistical analysis\n\nThe data collected was statistically analysed using descriptive statistics as mean, percentage and standard deviation. The shoulder mobility of the affected side was analysed and calculated by range of motion. Pain distribution was analysed and calculated using the standard deviation. For lymphedema, girth was measured using an inch tape and analysed by calculating the percentage. The results for upper limb tension test were analysed for radial nerve, median nerve and ulnar nerve.", "Participant Characteristics of the Study: The total 72 Women aged between 30-80 years of age had undergone breast surgery and are undergoing either chemotherapy or radiation therapy. Out of 72, 28 (38%) of women underwent lumpectomy, 12 (16%) underwent quadrantectomy and 32 (44%) underwent unilateral mastectomy. The number of women who were taking a combination of radiation and chemotherapy was more 48 (66%) as compared to radiation and chemotherapy alone of 12 (16%) and 16 (22%) respectively. Women between the ages of 41-70 years were the more frequent cases. The participants were the members of breast cancer support group of tertiary care hospital in India.\n\nPain\n\nThe pain in upper limb during activities of daily living was consistent finding in all the participants. The pain assessment was carried out by using VAS scale during rest and during activity. The mean of pain on rest was 0.888+1.056 and during activity was 3.166+2.307. This finding indicate the mild intensity of pain was experienced by the participants during activities of daily living such as bathing or showering, dressing, getting in and out of bed or a chair, walking, using the toilet, and eating. This indicates soft tissue structures mainly the contractile tissues were restricted and compressed during shortening and lengthening. The restrictive and compressive effect of lymphedema resulted in pain.\n\nShoulder Range of Motion\n\nThe shoulder abduction range of motion was limited significantly followed by shoulder external rotation and shoulder flexion. Table 1 shows that Out of 72 women about 57.8% (SD= ±10.055) women has flexion limitation, 30% (SD= 5.756) women have extension limitation, 31.5% (SD= 9.936) women have abduction limitation and 20% (SD= 5.828) have adduction limitation. So more number of study participants showed limitations in shoulder flexion, extension and abduction. The shoulder joint movements are limited due primarily by the surgical complications of breast cancer surgeries and secondarily due to the restrictions imposed by lymphedema.\n\nNeural tissue mobility impairments\n\nThe neural tissue mobility impairments were directly proportional with the severity of the lymphedema. Table 2 shows neural tissue mobility impairment in breast cancer survivors suffering from lymphedema. Subjects with mild lymphedema, had 48% of neural tissue mobility impairment, with moderate lymphedema, had 32% of neural tissue mobility impairment and with severe lymphedema, had 20% of neural tissue mobility impairment. Neural tissue mobility impairments of the three major nerves of the upper limb were assessed with ULTT and results are showed in Table 4. It showed the involvement of median, ulnar and radial nerve in breast cancer survivors. The result shows that median nerve was more affected in patients suffering from lymphedema 52%, followed by axillary nerve, musculo-cutaneous nerve, ulnar nerve and radial nerve. The lymphedema had resulted in significant neural tissue impairments of all the major nerves of the upper limb. The dependent position of upper limb in the erect standing position resulted in the neural tissue impairments of the distal most structures. Mainly the hand and distal forearm were sites for symptoms of compressive neuropathy. The major portion of hand is supplied by the median and ulnar nerves. Table 3. shows that women with mild lymphedema had median nerve affected more on the involved side i.e. 54.1% then ulnar nerve was affected i.e.13% , subjects with moderate lymphedema had 75% of median and 25% of ulnar nerve affected with median nerve affected in majority of women. Also, women with severe lymphedema had all the three nerves affected i.e., median (50%), radial (20%) and ulnar (10%)\nRange OF Motion of Shoulder Joint\nPatients with Neural Tissue Mobility Impairments Suffering from Lymphedema\nNeural Tissue Mobility Involvement According to Severity of Lymphedema\nNeural Tissue Mobility Involvement According to Severity of Lymphedema\nUpper Limb Tension Test", "In this study was focused on the effect of lymphedema in breast cancer survivors on neural tissue mobility. Breast cancer survivors after breast surgery, ALND, radiation and chemotherapy may develop axillary web syndrome, frozen shoulder, numbness, shoulder pain and range of motion restriction, lymphostasis and lymphedema. Up to 77% report sensory disturbance in breast or arm (Smoot et al., 2014).\nMany studies report that most cases of lymphedema develop during the first 1-2 years after primary treatment (Schunemann and Willich 1997; Clark, et al., 2005; Kiel and Rademacker 1996). The conducted study interpreted whether lymphedema was present or not, neural tissue mobility persists in most of the breast cancer survivors. Measurement of lymphedema was done on the affected upper extremity (usually the one which was operated) and also comparison was done with non-affected side. Lymphedema was assessed with the help of inch tape and the results were analysed on the basis of mild, moderate and severe. Eleven women had not removed the arm sleeve prior to examination, but eight of them still complied with the definition of lymphedema, so that the procedure violation hardly affected the assessment of lymphedema and also while performing the upper limb tension test. \nThe result of this study showed that women had more of mild lymphedema (32%). Norman et al., (2009) in their study of lymphedema in breast cancer survivors stated that after breast cancer lymphedema is common but mostly mild type. On self-examination differences observed in hand/arm size and symptoms can be early signs of progressing lymphedema. The results also showed that women who had mild lymphedema were three times more likely to develop moderate or severe lymphedema than women with no lymphedema.\nPain was assessed using Visual Analogue Scale (VAS) for assessing intensity of the neck pain or shoulder or axillary pain. The results showed that pain was experienced more on activity of shoulder and there was relief at rest irrespective of the presence of lymphedema. In Caro-Morán et al., (2014) study of Nerve pressure pain hypersensitivity and upper limb mechanosensitivity in breast cancer survivors it was found bilaterally and widespread nervous hypersensitivity in breast cancer survivors as compared to healthy controls, which were expressed as significantly decreased pressure pain threshold level over the median, radial and ulnar nerve trunks. All the participants in the patient group showed decrease range of flexion and abduction of shoulder. Patient showed mechano-sensitivity in the upper extremity of the affected side as compared to the healthy controls, as demonstrated by a statistically significant reduced range of motion in elbow extension and shoulder abduction during ULNTs for the median, radial and ulnar nerves.\nThe results of our study showed decrease in range of motion of shoulder flexion and abduction as compared to other movements. Range of motion was assessed using goniometer. Using the VAS pain assessment was taken and it was observed that patients with pain also had restricted range of motion. In some studies, it was stated that the chemotherapy or radiation therapy can cause peripheral neuropathy due to sensory axonal damage, with reduced amplitude of sensory nerve action potentials and changes in afferent activity, leading to widespread pain sensitivity (Wampler, 2006; Wolf, 2008; Argryriou, 2012; Partridge 2004). Smoot et al., in their study of upper extremity impairments in women with or without lymphedema following breast cancer treatment stated that approx. 5-42% of breast cancer survivors develop lymphedema and 47% of have persistent pain.\nThere are no studies which show the impact of lymphedema on neural tissue mobility. So, it was necessary to analyse whether the presence of lymphedema had an effect on neural tissue mobility which may result in pain, numbness, tingling sensations, restrictions in range of motion of the upper limb while assessing the patient. The result of this study showed that women who were breast cancer survivors had mild lymphedema and had more neural tissue impairment then moderate or severe lymphedema women. The observational studies described an increased neural mechano-sensitivity using the upper limb neurodynamic test 1 (ULNT1) in Breast cancer survivors. This increased neural mechano-sensitivity, may arise after nerve damage and lead to shoulder ROM restriction as a protective neural response to movement or traction. Our study witnessed the reduced range of motion in patients with lymphedema. This states that lymphedema also has an effect on range of motion along with the pain factor. Kelley and Jull (1998), who assessed mechanosensitivity of Upper extremity neural tissue in 20 women with one side breast cancer, before and 6 weeks after Breast cancer surgery found that shoulder abduction ROM during the Upper Limb Tension Test 2 median nerve was reduced on the surgical side.\nThe upper limb tension test was performed for all the three nerves median, radial and ulnar for assessing the neural tissue mobility in breast cancer survivors. It was observed in our study that the extent of neural tissue mobility impairment was based on the severity of lymphedema and the nerves affected. None of the previous studies shows the extent of neural tissue mobility in relation to lymphedema. One study shows the mechano-sensitivity in women after breast cancer treatment and found significant reductions in elbow extension ROM during ULNTMEDIAN in patients with pain and lymphedema, though the results were not significant in patients with pain but without lymphedema. This study specifically supports the information about which nerve is affected more in patients with lymphedema (Caro-Moran E, et al., 2014). \nThere were some limitations recognised in this study during the period of study. Range of motion for elbow and wrist are not assessed along with the internal and external rotation of the shoulder joint which can be assessed and noted for future studies. In addition, the study design does not provide information about the possible cause-effect relationship between the lymphedema and the neural tissue mobility impairment. The findings of the current study show the need for rehabilitation programs for the specific treatment of neural tissue mobility impairment in breast cancer survivors and not only targeted to muscular tissue. In view of our results, the health care providers should also look for neural tissue mobility impairments in breast cancer survivors to design rehabilitation programs that take into account the neural component of pain along with lymphedema post-surgical interventions and chemotherapies in this population.\n Conclusion This study of women who have undergone surgical intervention for breast cancer concludes that there was significant amount of neural tissue impairment noted to mechanical provocation test post operatively after 6 months of surgery. The study suggests that severity of lymphedema was directly related to the nerves affected due to neural tissue impairment. Neural tissue mobility impairments should be assessed in women with lymphedema following the treatment of breast cancer. It should be assessed bilaterally for correlation of the symptoms. Therapists need to integrate the findings of neural tissue mobility impairments into the rehabilitation program for breast cancer survivors with pain and lymphedema for better quality of life.\nThis study of women who have undergone surgical intervention for breast cancer concludes that there was significant amount of neural tissue impairment noted to mechanical provocation test post operatively after 6 months of surgery. The study suggests that severity of lymphedema was directly related to the nerves affected due to neural tissue impairment. Neural tissue mobility impairments should be assessed in women with lymphedema following the treatment of breast cancer. It should be assessed bilaterally for correlation of the symptoms. Therapists need to integrate the findings of neural tissue mobility impairments into the rehabilitation program for breast cancer survivors with pain and lymphedema for better quality of life.\n Abbreviations ALND: Axillary Lymph Node Dissection\nROM: Range of Motion\nBC: Breast cancer\nULNT: Upper Limb Neurodynamic Test\nALND: Axillary Lymph Node Dissection\nROM: Range of Motion\nBC: Breast cancer\nULNT: Upper Limb Neurodynamic Test", "This study of women who have undergone surgical intervention for breast cancer concludes that there was significant amount of neural tissue impairment noted to mechanical provocation test post operatively after 6 months of surgery. The study suggests that severity of lymphedema was directly related to the nerves affected due to neural tissue impairment. Neural tissue mobility impairments should be assessed in women with lymphedema following the treatment of breast cancer. It should be assessed bilaterally for correlation of the symptoms. Therapists need to integrate the findings of neural tissue mobility impairments into the rehabilitation program for breast cancer survivors with pain and lymphedema for better quality of life." ]

[ "intro", "materials|methods", "results", "discussion", null ]

[ "Lymphedema", "neural tissue impairment", "visual Analogue scale", "upper limb tension test" ]

[ 113 ]

[ "lymphedema", "neural", "tissue", "breast", "cancer", "neural tissue", "breast cancer", "pain", "mobility", "study" ]

[ "impairment breast cancer", "breast cancer assessed", "following treatment breast", "complications breast cancer", "breast cancer surgery" ]

[ "Animals", "Anopheles", "Anthracenes", "Asteraceae", "Carbohydrates", "Feeding Behavior", "Female", "Insect Vectors", "Malaria", "Male", "Metarhizium", "Mosquito Control", "Ricinus", "Survival Analysis" ]

[ "Mosquitoes", "Detection of plant sugars in mosquitoes using the anthrone test", "Fungal isolate", "Fungal infection process", "Plant species", "Survival of M. anisopliae-infected An. gambiae mosquitoes fed on plant sugars", "Quantity of sugar imbibed by M. anisopliae-infected An. gambiae mosquitoes", "Sugar digestion rate of M. anisopliae-infected An. gambiae mosquitoes", "Ethical approval", "Statistics", "Survival of M. anisopliae-infected An. gambiae mosquitoes fed on plant sugars", "Quantity of sugar imbibed by M. anisopliae-infected An. gambiae mosquitoes", "Sugar digestion rate of M. anisopliae-infected An. gambiae mosquitoes" ]

[ "Experiments were carried out using laboratory-reared Anopheles gambiae Giles sensu stricto (hereafter termed An. gambiae) mosquitoes obtained from a colony established from wild gravid females collected at Mbita Point (000 25’S, 340 13’E), western Kenya in 1999. All mosquito life stages were maintained under ambient conditions in a mosquito insectary present at the Thomas Odhiambo Campus (TOC) of the International Centre of Insect Physiology and Ecology (icipe) located near Mbita Point Township in western Kenya. Larval and adult stages of the mosquitoes were raised using procedures described previously [23]. Both sexes were separated at emergence and held under ambient conditions in 30 × 30 × 30 cm cages inside a screenhouse. Before experiments, the insects were maintained either on an aqueous 6% glucose solution presented on filter paper wicks or on stem cuttings of Ricinus communis (Castor oil plant) and Parthenium hysterophorus (Santa Maria feverfew weed).", "The anthrone test was used to determine the presence of sugars in the mosquitoes. To do so standard sucrose solutions of different strengths in the series 1, 2, 4, 8, 16, 32, 64, 128 and 256 μg/μl were prepared. Initially, 25.6 g of reagent grade sucrose was dissolved in 50 ml of distilled water. More water was added gradually while mixing to make a 100 ml solution from which the serial dilutions were prepared. Distilled water served as the neutral liquid. These solutions were stored at −4°C. Diluted sulphuric acid was then prepared by mixing 380 ml concentrated sulphuric acid with 150 ml distilled water in a fume hood. The whole solution was cooled for 5 hours at room temperature and further for 12 hours in the refrigerator at 5°C before use. Anthrone reagent was then prepared by mixing 0.15 g of anthrone powder per 100 ml of the diluted sulphuric acid.\nTwo test tube racks were used with each rack holding one hundred 5-ml test tubes. A third rack was used to hold 10 test tubes for the standard sucrose solutions. The standards were prepared by pipetting 1 μl from each of the nine standard sucrose solutions into the nine separate test tubes. The tenth tube contained 1 μl of distilled water. The other two racks were used to hold both sexes of uninfected and M. anisopliae-exposed An. gambiae mosquitoes. Each tube held one mosquito. One drop each of chloroform and methanol in the ratio 1:1 was added to each tube containing mosquitoes to dissolve the cuticle. The racks were held in a fume hood where 0.5 ml of anthrone reagent was added to the standards and the tubes containing mosquitoes. The racks were then transferred into a water bath at room temperature for one hr. In the presence of sugar, the colour of the solutions changed from green to green-blue and further dark-blue depending on the amount of sugar. In absence of sugar, the colour of the sample was transparent yellow. After one hour the results were read by comparing the colour change in tubes containing mosquitoes and those with standard solutions.", "The entomopathogenic fungus Metarhizium anisopliae isolate ICIPE 30 was used (courtesy Dr. N.K. Maniania). This fungus was originally isolated from the stem borer Busseola fusca (Lepidoptera, Noctuidea) in Kendu Bay, western Kenya, in 1999 and has been maintained at the icipe’s Germplasm Centre. Conidia were produced on long rice as substrate [24]. Harvested spores were dried for 48 hours in a desiccator containing active silica gel and stored in a refrigerator (4-6°C) until required. The viability of spores was determined before being used in the experiments. Germination rates >85% after 24 hours on Sabouraud dextrose agar was considered adequate for use in the experiments.", "Transparent plastic cylinders (9 cm diameter; 15 cm height) were used to inoculate An. gambiae mosquitoes with spores of M. anisopliae. The inside vertical surface and the circular base of each cylinder were lined with white rough-surfaced velvex tissue papers that measured 28.6 × 14.3 cm (for vertical surface) and 9 cm in diameter (for circular base area). A piece of mosquito netting material was secured over the mouth of each cylinder using a rubber band. A hole was punched at the centre of the net to serve as an introduction point for experimental mosquitoes. Each cylinder was held in a slanting position and 0.1 g (approx. 1.0 × 1011 conidia/m2) of M. anisopliae spores were weighed and poured on the paper. Using both hands, the cylinders were rolled several times until the papers were uniformly covered by the spores. The inner and the base surfaces of the cylinder used for uninfected mosquitoes were lined with white rough paper without spores.\nFor survival experiments, a total of four cylinders with fungus and four cylinders without fungus were used. Of these, two cylinders with fungus and two cylinders without fungus were each used to infect male and female mosquitoes separately. Sixty 1-d-old female and male mosquitoes were introduced into each of their respective four cylinders. The insects were held for six hr being supplied with 6% glucose solution soaked in a cotton pad and placed on top of the netting material covering the cylinder. The mosquitoes were then transferred into four separate holding cages (30 × 30 × 30 cm) based on sex and treatment and were supplied with 6% glucose solution on filter paper wicks. The insects were maintained under ambient conditions inside a screenhouse at 28 ± 2°C and 70 ± 5% r.h. For studies to evaluate plant material this procedure was repeated to infect the mosquitoes but the insects were provided with floral parts of R. communis and P. hysterophorous separately as source of sugar instead of 6% glucose solution. The base of the floral parts was hooked on the netting material covering the mouth of each cylinder using a tooth pick to suspend the plant inside the cylinder.\nFor sugar quantity and digestion rate experiments, five cylinders with fungus and three cylinders without fungus were used to infect female mosquitoes. The same numbers of cylinders were used to infect male mosquitoes. The number of mosquitoes exposed to fungus was higher than for the uninfected group to adjust for mortality in the holding cages prior to the start of the experiments on days one and three post-exposure.", "Two plant species namely Ricinus communis (Castor oil plant) and Parthenium hysterophorus (Santa Maria feverfew weed) were used. Ricinus communis has been demonstrated to enhance survival of An. gambiae and P. hysterophorus as most frequently visited by this mosquito species [19]. Five, fresh stems cut from each plant with leaves and floral parts intact were used in the study. The cuttings were collected from the agricultural field at icipe, Mbita Point, western Kenya where the plants grow naturally.", "To study the effect of fungus on the survival of M. anisopliae-infected An. gambiae mosquitoes on plant sugars, one hundred male and 100 female An. gambiae mosquitoes exposed to M. anisopliae for six hours upon emergence were held in separate cages (30 × 30 × 30 cm). Each cage was supplied with 250-ml flat bottomed conical flask containing 200-ml filtered Lake Victoria water and five stems (with leaves and floral parts intact) of R. communis. The stems were replaced every two days. Mosquito mortality was recorded daily to determine the length of time over which the mosquitoes survived. Dead individuals were plated in a Petri dish lined with wet filter paper and incubated at 28 ± 2°C. Fungal growth on mosquito cadavers was observed after three days at 400× magnification under a compound microscope. The experiment was replicated four times. This procedure was repeated using P. hysterophorus as an alternative test plant and 6% glucose solution on filter paper wicks as a control. Glucose solution was changed every two days.", "In order to determine the quantity of sugar imbibed by M. anisopliae-infected An. gambiae mosquitoes, preliminary experiments were conducted to determine the length of time required for individual mosquitoes to feed fully. Three groups of female mosquitoes each composed of 50 individuals were fed on stems (with leaves and floral parts intact) of R. communis for separate periods of 6, 12 and 24 hours. The experiments were replicated four times over time. This procedure was repeated with male mosquitoes. Both male and female mosquitoes took 12 hours to satiate. Thus, the amount of sugar ingested from stems of R. communis by male and female mosquitoes, one and three days post-exposure to M. anisopliae, was evaluated after every 12 hours of feeding. One day after exposure to fungus, fifty male and female mosquitoes were aspirated, each from their respective uninfected and fungus-exposed cages and released into four separate cages. The insects were starved for 6 hrs prior to introduction of a 250-ml conical flask containing stems of R. communis in each cage. After 12 hours of feeding, the insects were removed from the cages and held in four separate collection cups. The insects were held inside a refrigerator at 4°C for 30 min and their sugar levels were quantified following the procedure earlier described on ‘detection of plant sugars in mosquitoes using anthrone test’. The experiment was replicated four times. This procedure was repeated with mosquitoes three days post-infection.", "The digestion rate of An. gambiae mosquitoes exposed to M. anisopliae was determined by feeding males and females on R. communis. One day after exposure to fungus, 50 mosquitoes were aspirated from each cage holding uninfected and fungus-exposed mosquitoes and released into four separate cages. The mosquitoes were starved for 6 hours prior to introduction of the plant in a 250-ml conical flask in each cage. Mosquitoes were allowed to feed on R. communis for 12 hours after which the flasks containing the plant were removed from the cages. Fifty mosquitoes that appeared fully fed were also removed and held in separate cages from where ten mosquitoes were removed at an interval of 8 hours starting from time zero through to 32 hours post-feeding. Removed mosquitoes were held inside a refrigerator at 4°C for 30 min and the quantity of sugar in them, and by extension digestion rate, determined following the procedure earlier described. The experiment was replicated four times. The same procedure was repeated with groups of mosquitoes three days post-exposure to M. anisopliae.", "Ethical approval for this study was given by the Kenya National Ethical Review Committee located at the Kenya Medical Research Institute (NON-SSC Protocol number 203).", "Survival of uninfected and M. anisopliae-infected mosquitoes on glucose (6%), R. communis and P. hysterophorous was calculated by expressing the number of mosquitoes that succumbed to mortality as a percentage of the total number tested. Differences in survival between uninfected and fungus-infected groups were estimated using Cox regression analysis. Mortality rates, expressed as Hazard Ratio (HR) were used to estimate the risk of dying when infected compared to when not infected with fungus. To evaluate effects of infection on the amount of sugar ingested by infected (one and three days post-exposure) and control mosquitoes, first the number of mosquitoes that had fed on R. communis was expressed as a percentage of the total number tested. Further, the number of mosquitoes that imbibed small, medium and large quantities of sugar, respectively, was expressed as the mean percentage of the total number of mosquitoes tested. The difference between control and fungus-infected mosquitoes was calculated with the Chi square (χ2) test [25]. The digestion rate of the sugar ingested by mosquitoes one and three days post-exposure was each calculated by logistic regression. Logistic relationships for uninfected and fungus-exposed mosquitoes were fitted to describe sugar detection success for each time elapsed since feeding. The difference between uninfected and fungus-exposed mosquitoes was estimated by the Chi square (χ2) test. All analyses were conducted using SPSS (version 17.0)", "Infection with M. anisopliae reduced the survival of both sexes of An. gambiae with 100% mortality occurring within seven days compared to ≥ seven days with uninfected mosquitoes irrespective of the nutritional source (Figure 1). Survival of infected male and female mosquitoes in each nutritional group was significantly different from their respective controls. For example, the daily risk of death for both sexes was eight-fold greater on 6% glucose; four-fold (males) and eight-fold (females) greater on R. communis and two-fold greater for both sexes on P. hysterophorus relative to their controls (Table 1). In uninfected mosquitoes, the daily risk of death was three-fold greater for both males (HR = 3.4 [95% CI = 2.91 - 4.21], P = 0.0001) and females (HR = 2.9 [95% CI = 2.45 - 3.55], P = 0.0001) fed on R. communis and 14-fold greater for males (HR = 14.1 [95% CI = 11.33 - 17.6], P = 0.0001) and 13-fold greater for females (HR = 13.4 [95% CI = 10.71 - 16.8], P = 0.0001) fed on P. hysterophorus relative to 6% glucose. Therefore, P. hysterophorus caused a drastic reduction in the survival of mosquitoes regardless of fungal infection. Between sexes, survival rate over time in each nutritional regime was not different. Mycosis test results indicated high infection rates (>77%) in fungus-exposed male and female mosquitoes. No fungal conidia were observed on the cadavers of the control mosquitoes.Figure 1\nSurvival of uninfected and\nM. anisopliae\n-infected\nAn. gambiae\nfemales (Panel A, C and E) and males (Panel B, D and F) when fed on: (i) 6%\nglucose (panel A and B); (ii)\nRicinus communis\n(panel C and D) and (iii)\nParthenium hysterophorus\n(Panel E and F). Uninfected and M. anisopliae-infected mosquitoes are depicted by closed squares and closed triangles, respectively.Table 1\nSurvival analysis of\nAn. gambiae\nmosquitoes infected with\nM. anisopliae\nand fed on different nutritional sources; data show Cox regression Hazard Ratio (HR) outcomes (95% CI), statistical p-values are relative to the relevant control (not exposed to fungus)\n\nNutritional sources\n\nHR (95%\nCI)\n\nMale mosquitoes\n\nP-value\n\nFemale mosquitoes\n\nP-value\nGlucose (6%)8.53 (6.68 - 10.89)0.00017.64 (5.99 - 9.75)0.0001\nRicinus communis\n4.33 (3.59 - 5.23)0.00018.21 (6.49 - 10.37)0.0001\nParthenium hysterophorus\n1.62 (1.40 - 1.89)0.00012.15 (1.85 - 2.50)0.0001\n\nSurvival of uninfected and\nM. anisopliae\n-infected\nAn. gambiae\nfemales (Panel A, C and E) and males (Panel B, D and F) when fed on: (i) 6%\nglucose (panel A and B); (ii)\nRicinus communis\n(panel C and D) and (iii)\nParthenium hysterophorus\n(Panel E and F). Uninfected and M. anisopliae-infected mosquitoes are depicted by closed squares and closed triangles, respectively.\n\nSurvival analysis of\nAn. gambiae\nmosquitoes infected with\nM. anisopliae\nand fed on different nutritional sources; data show Cox regression Hazard Ratio (HR) outcomes (95% CI), statistical p-values are relative to the relevant control (not exposed to fungus)\n", "The quantity of sugar detected ranged from 1–64 μg. For easy analysis of the data, the mosquitoes were categorised as consumers of small, medium or large meals [26] if they imbibed 1–4 μg, 8–16 μg or 32–64 μg of sugar, respectively. Significantly fewer male and female mosquitoes exposed to fungus imbibed sugar from R. communis compared to mosquitoes not exposed to fungus (Figure 2). More uninfected than fungus-exposed mosquitoes ingested plant sugar. Fewer mosquitoes imbibed plant sugars at three days post-exposure than at one day post-exposure. Although fungus-exposed mosquitoes ingested less sugar than uninfected counterparts the differences were generally insignificant except for medium-feeding females three days post-exposure (Table 2) and small-feeding males, one and three days post-exposure (Table 3). Results from the mycosis test demonstrated high infection rates (>75%) in fungus-exposed males and females. No fungal hyphae were observed on the cadavers of control mosquitoes.Figure 2\nMean (± S.E) percentage of uninfected and\nM. anisopliae\n- infected\nAn. gambiae\nmales (Panel A) and females (Panel B) that imbibed sugar on exposure to\nRicinus communis\nfor 12 hr. White and gray shaded bars represent uninfected and M. anisopliae-infected mosquitoes respectively. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.Table 2\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nfemale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hours\n\nSugar quantity\n\nDays post-exposure\n\nN\n\nMean% (± S.E) of males that imbibed sugar\n\nχ\n2\n\nP\n\nUninfected\n\nFungus-infected\nSmall1447.5 ± 8.5438.0 ± 7.963.690.055Medium429.0 ± 4.5126.0 ± 4.080.450.502Large418.5 ± 8.2219.5 ± 6.290.070.799Small3435.0 ± 4.1230.0 ± 5.61.140.286Medium422.0 ± 2.226.0 ± 2.1622.280.001Large412.0 ± 4.696.5 ± 6.53.600.058One and three d post-exposure females were tested.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.Table 3\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nmale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hr\n\nSugar quantity\n\nDays post-exposure\n\nN\n\nMean% (± S.E) of males that imbibed sugar\n\nχ\n2\n\nP\n\nUninfected\n\nFungus-infected\nSmall1456.0 ± 6.1641.0 ± 9.479.010.003Medium422.0 ± 3.5621.5 ± 4.190.020.903Large49.5 ± 1.716.5 ± 2.631.220.269Small3452.0 ± 8.4937.0 ± 6.149.110.003Medium416.5 ± 0.5010.5 ± 7.373.080.079Large44.0 ± 2.451.0 ± 1.03.690.055One and three d post-exposure males were tested.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and\nM. anisopliae\n- infected\nAn. gambiae\nmales (Panel A) and females (Panel B) that imbibed sugar on exposure to\nRicinus communis\nfor 12 hr. White and gray shaded bars represent uninfected and M. anisopliae-infected mosquitoes respectively. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nfemale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hours\n\nOne and three d post-exposure females were tested.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nmale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hr\n\nOne and three d post-exposure males were tested.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.", "The proportions of uninfected and M. anisopliae-infected mosquitoes testing positive for plant sugar consistently decreased over time (Figure 3). For each time period since feeding, more mosquitoes, one day post-exposure to fungus, tested positive for sugar than uninfected mosquitoes but the difference was not significant except in males at 32 hours (χ2 = 6.27; df = 1; P = 0.001) and in females at 24 hours (χ2 = 10.91; df = 1; P = 0.001) and 32 hours (χ2 = 11.25; df = 1; P = 0.001) of digestion. In addition, fewer mosquitoes, three days post-exposure, than controls tested positive for sugars until 16 hours in males and 24 hours in females after feeding with the differences significant at 32 hour of digestion (males: χ2 = 6.49; df = 1; P = 0.001; females χ2 = 7.67; df = 1; P = 0.006). Cumulative scores from time zero through to the 32 hour demonstrate that, more one day post-exposure males (52% versus 45%) and females (58% versus 39%) than controls tested positive for sugar. This was an overall indication that digestion rate was slower in fungus-exposed mosquitoes. The difference was only significant for females, one day post-exposure (Table 4). Further, the proportion of three day post-exposure males (43% versus 43%) and females (53% versus 53%) with sugar was equal to that of the controls. Hence, timing of fungal exposure only had an effect on sugar digestion in females. Results from mycosis tests indicated that, on average 73-81% of males and 78-85% of females were infected with fungus but no spores were observed on the cadaver of the control mosquitoes.Figure 3\nEffect of infection with\nM. anisopliae\non sugar detection success in\nAn. gambiae\nmosquitoes. Panels A and B represent sugar detection success in uninfected and in M. anisopliae-infected males and females respectively one day post-exposure with Panels C and D represent 3 d post-exposure when fed on Ricinus communis for 12 hr. Solid lines representing uninfected mosquitoes and dotted lines representing infected mosquitoes describe the fitted logistic relationships between sugar detection success for each time period since feeding: − Logit (sugar detection success) = ß0 + ß1 time. Circles denote observed values. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.Table 4\nProportion of uninfected and fungus-exposed\nAn. gambiae\nmosquitoes that tested positive for sugar within 32 hr after feeding on\nRicinus communis\nfor 12 hr\n\nSex\n\nDays post-exposure\n\nN\n\n% mosquitoes sugar positive\n\nχ\n2\n\nP\n\nPercent (± S.E) infection\n\nUninfected\n\nFungus-exposed\nMale1445521.960.16173.0 ± 3.87 (146)Female438.55815.290.00178.0 ± 4.16 (156)Male3443.5430.010.92081.0 ± 1.0 (162)Female452.5530.010.92085.0 ± 2.08 (170)Males and females were tested one and three d post-exposure.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nEffect of infection with\nM. anisopliae\non sugar detection success in\nAn. gambiae\nmosquitoes. Panels A and B represent sugar detection success in uninfected and in M. anisopliae-infected males and females respectively one day post-exposure with Panels C and D represent 3 d post-exposure when fed on Ricinus communis for 12 hr. Solid lines representing uninfected mosquitoes and dotted lines representing infected mosquitoes describe the fitted logistic relationships between sugar detection success for each time period since feeding: − Logit (sugar detection success) = ß0 + ß1 time. Circles denote observed values. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.\n\nProportion of uninfected and fungus-exposed\nAn. gambiae\nmosquitoes that tested positive for sugar within 32 hr after feeding on\nRicinus communis\nfor 12 hr\n\nMales and females were tested one and three d post-exposure.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes." ]

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[ "Background", "Methods", "Mosquitoes", "Detection of plant sugars in mosquitoes using the anthrone test", "Fungal isolate", "Fungal infection process", "Plant species", "Survival of M. anisopliae-infected An. gambiae mosquitoes fed on plant sugars", "Quantity of sugar imbibed by M. anisopliae-infected An. gambiae mosquitoes", "Sugar digestion rate of M. anisopliae-infected An. gambiae mosquitoes", "Ethical approval", "Statistics", "Results", "Survival of M. anisopliae-infected An. gambiae mosquitoes fed on plant sugars", "Quantity of sugar imbibed by M. anisopliae-infected An. gambiae mosquitoes", "Sugar digestion rate of M. anisopliae-infected An. gambiae mosquitoes", "Discussion", "Conclusions" ]

[ "Studies have shown that fungal pathogens reduce survival of Anopheles mosquitoes to a level that prevents transmission of malaria parasites [1-3]. The fungi achieve this by reducing mosquito blood feeding [4,5] and fecundity [5]. Plant sugar acquired from floral and extrafloral nectaries, honeydew, damaged fruits and leaves is essential for mosquito survival [6-8]. It is the only nutritional source of adult males and a dietary supplement to blood for females. Sugar feeding is an early priority for both sexes, which typically have limited energy reserves upon emergence [9-14]. Besides survival and building of energy reserves, sugar enhances maturation of ovarian follicles in females and reproductive fitness in males [15].\nSurvival of the malaria mosquito Anopheles gambiae is assured with frequent feeding and ingestion of sizeable amounts of sugar meals [16] or by ingestion of small amounts of sugar at a time [6,8]. Recent studies have shown that mosquitoes feed on a wide variety of plants common in their natural habitats [17-20] with males preferentially feeding on the most rewarding sugar sources [21]. Sugars from some of these plants promote longer survival of both sexes, which enhances the vectorial capacity of females [16,22] and fitness and reproductive capacity of the males [21]. Most studies, though, have targeted females due to their significant role in malaria transmission with the contribution of males in the whole process often overlooked. As sugar feeding is central in the biology of adult mosquitoes, it is imperative to assess whether infection of mosquitoes with entomopathogenic fungi impacts sugar feeding.\nThis study investigated three aspects of plant sugar feeding behaviour of adult male and female An. gambiae mosquitoes under natural climatic conditions. These included (a) determining the survival of fungus-exposed An. gambiae mosquitoes when fed on glucose or plant sugars, (b) establishing the feeding propensity and the quantity of sugar ingested from plants by the infected mosquitoes and (c) assessing the digestion rate of sugar imbibed by fungus-exposed mosquitoes.", " Mosquitoes Experiments were carried out using laboratory-reared Anopheles gambiae Giles sensu stricto (hereafter termed An. gambiae) mosquitoes obtained from a colony established from wild gravid females collected at Mbita Point (000 25’S, 340 13’E), western Kenya in 1999. All mosquito life stages were maintained under ambient conditions in a mosquito insectary present at the Thomas Odhiambo Campus (TOC) of the International Centre of Insect Physiology and Ecology (icipe) located near Mbita Point Township in western Kenya. Larval and adult stages of the mosquitoes were raised using procedures described previously [23]. Both sexes were separated at emergence and held under ambient conditions in 30 × 30 × 30 cm cages inside a screenhouse. Before experiments, the insects were maintained either on an aqueous 6% glucose solution presented on filter paper wicks or on stem cuttings of Ricinus communis (Castor oil plant) and Parthenium hysterophorus (Santa Maria feverfew weed).\nExperiments were carried out using laboratory-reared Anopheles gambiae Giles sensu stricto (hereafter termed An. gambiae) mosquitoes obtained from a colony established from wild gravid females collected at Mbita Point (000 25’S, 340 13’E), western Kenya in 1999. All mosquito life stages were maintained under ambient conditions in a mosquito insectary present at the Thomas Odhiambo Campus (TOC) of the International Centre of Insect Physiology and Ecology (icipe) located near Mbita Point Township in western Kenya. Larval and adult stages of the mosquitoes were raised using procedures described previously [23]. Both sexes were separated at emergence and held under ambient conditions in 30 × 30 × 30 cm cages inside a screenhouse. Before experiments, the insects were maintained either on an aqueous 6% glucose solution presented on filter paper wicks or on stem cuttings of Ricinus communis (Castor oil plant) and Parthenium hysterophorus (Santa Maria feverfew weed).\n Detection of plant sugars in mosquitoes using the anthrone test The anthrone test was used to determine the presence of sugars in the mosquitoes. To do so standard sucrose solutions of different strengths in the series 1, 2, 4, 8, 16, 32, 64, 128 and 256 μg/μl were prepared. Initially, 25.6 g of reagent grade sucrose was dissolved in 50 ml of distilled water. More water was added gradually while mixing to make a 100 ml solution from which the serial dilutions were prepared. Distilled water served as the neutral liquid. These solutions were stored at −4°C. Diluted sulphuric acid was then prepared by mixing 380 ml concentrated sulphuric acid with 150 ml distilled water in a fume hood. The whole solution was cooled for 5 hours at room temperature and further for 12 hours in the refrigerator at 5°C before use. Anthrone reagent was then prepared by mixing 0.15 g of anthrone powder per 100 ml of the diluted sulphuric acid.\nTwo test tube racks were used with each rack holding one hundred 5-ml test tubes. A third rack was used to hold 10 test tubes for the standard sucrose solutions. The standards were prepared by pipetting 1 μl from each of the nine standard sucrose solutions into the nine separate test tubes. The tenth tube contained 1 μl of distilled water. The other two racks were used to hold both sexes of uninfected and M. anisopliae-exposed An. gambiae mosquitoes. Each tube held one mosquito. One drop each of chloroform and methanol in the ratio 1:1 was added to each tube containing mosquitoes to dissolve the cuticle. The racks were held in a fume hood where 0.5 ml of anthrone reagent was added to the standards and the tubes containing mosquitoes. The racks were then transferred into a water bath at room temperature for one hr. In the presence of sugar, the colour of the solutions changed from green to green-blue and further dark-blue depending on the amount of sugar. In absence of sugar, the colour of the sample was transparent yellow. After one hour the results were read by comparing the colour change in tubes containing mosquitoes and those with standard solutions.\nThe anthrone test was used to determine the presence of sugars in the mosquitoes. To do so standard sucrose solutions of different strengths in the series 1, 2, 4, 8, 16, 32, 64, 128 and 256 μg/μl were prepared. Initially, 25.6 g of reagent grade sucrose was dissolved in 50 ml of distilled water. More water was added gradually while mixing to make a 100 ml solution from which the serial dilutions were prepared. Distilled water served as the neutral liquid. These solutions were stored at −4°C. Diluted sulphuric acid was then prepared by mixing 380 ml concentrated sulphuric acid with 150 ml distilled water in a fume hood. The whole solution was cooled for 5 hours at room temperature and further for 12 hours in the refrigerator at 5°C before use. Anthrone reagent was then prepared by mixing 0.15 g of anthrone powder per 100 ml of the diluted sulphuric acid.\nTwo test tube racks were used with each rack holding one hundred 5-ml test tubes. A third rack was used to hold 10 test tubes for the standard sucrose solutions. The standards were prepared by pipetting 1 μl from each of the nine standard sucrose solutions into the nine separate test tubes. The tenth tube contained 1 μl of distilled water. The other two racks were used to hold both sexes of uninfected and M. anisopliae-exposed An. gambiae mosquitoes. Each tube held one mosquito. One drop each of chloroform and methanol in the ratio 1:1 was added to each tube containing mosquitoes to dissolve the cuticle. The racks were held in a fume hood where 0.5 ml of anthrone reagent was added to the standards and the tubes containing mosquitoes. The racks were then transferred into a water bath at room temperature for one hr. In the presence of sugar, the colour of the solutions changed from green to green-blue and further dark-blue depending on the amount of sugar. In absence of sugar, the colour of the sample was transparent yellow. After one hour the results were read by comparing the colour change in tubes containing mosquitoes and those with standard solutions.\n Fungal isolate The entomopathogenic fungus Metarhizium anisopliae isolate ICIPE 30 was used (courtesy Dr. N.K. Maniania). This fungus was originally isolated from the stem borer Busseola fusca (Lepidoptera, Noctuidea) in Kendu Bay, western Kenya, in 1999 and has been maintained at the icipe’s Germplasm Centre. Conidia were produced on long rice as substrate [24]. Harvested spores were dried for 48 hours in a desiccator containing active silica gel and stored in a refrigerator (4-6°C) until required. The viability of spores was determined before being used in the experiments. Germination rates >85% after 24 hours on Sabouraud dextrose agar was considered adequate for use in the experiments.\nThe entomopathogenic fungus Metarhizium anisopliae isolate ICIPE 30 was used (courtesy Dr. N.K. Maniania). This fungus was originally isolated from the stem borer Busseola fusca (Lepidoptera, Noctuidea) in Kendu Bay, western Kenya, in 1999 and has been maintained at the icipe’s Germplasm Centre. Conidia were produced on long rice as substrate [24]. Harvested spores were dried for 48 hours in a desiccator containing active silica gel and stored in a refrigerator (4-6°C) until required. The viability of spores was determined before being used in the experiments. Germination rates >85% after 24 hours on Sabouraud dextrose agar was considered adequate for use in the experiments.\n Fungal infection process Transparent plastic cylinders (9 cm diameter; 15 cm height) were used to inoculate An. gambiae mosquitoes with spores of M. anisopliae. The inside vertical surface and the circular base of each cylinder were lined with white rough-surfaced velvex tissue papers that measured 28.6 × 14.3 cm (for vertical surface) and 9 cm in diameter (for circular base area). A piece of mosquito netting material was secured over the mouth of each cylinder using a rubber band. A hole was punched at the centre of the net to serve as an introduction point for experimental mosquitoes. Each cylinder was held in a slanting position and 0.1 g (approx. 1.0 × 1011 conidia/m2) of M. anisopliae spores were weighed and poured on the paper. Using both hands, the cylinders were rolled several times until the papers were uniformly covered by the spores. The inner and the base surfaces of the cylinder used for uninfected mosquitoes were lined with white rough paper without spores.\nFor survival experiments, a total of four cylinders with fungus and four cylinders without fungus were used. Of these, two cylinders with fungus and two cylinders without fungus were each used to infect male and female mosquitoes separately. Sixty 1-d-old female and male mosquitoes were introduced into each of their respective four cylinders. The insects were held for six hr being supplied with 6% glucose solution soaked in a cotton pad and placed on top of the netting material covering the cylinder. The mosquitoes were then transferred into four separate holding cages (30 × 30 × 30 cm) based on sex and treatment and were supplied with 6% glucose solution on filter paper wicks. The insects were maintained under ambient conditions inside a screenhouse at 28 ± 2°C and 70 ± 5% r.h. For studies to evaluate plant material this procedure was repeated to infect the mosquitoes but the insects were provided with floral parts of R. communis and P. hysterophorous separately as source of sugar instead of 6% glucose solution. The base of the floral parts was hooked on the netting material covering the mouth of each cylinder using a tooth pick to suspend the plant inside the cylinder.\nFor sugar quantity and digestion rate experiments, five cylinders with fungus and three cylinders without fungus were used to infect female mosquitoes. The same numbers of cylinders were used to infect male mosquitoes. The number of mosquitoes exposed to fungus was higher than for the uninfected group to adjust for mortality in the holding cages prior to the start of the experiments on days one and three post-exposure.\nTransparent plastic cylinders (9 cm diameter; 15 cm height) were used to inoculate An. gambiae mosquitoes with spores of M. anisopliae. The inside vertical surface and the circular base of each cylinder were lined with white rough-surfaced velvex tissue papers that measured 28.6 × 14.3 cm (for vertical surface) and 9 cm in diameter (for circular base area). A piece of mosquito netting material was secured over the mouth of each cylinder using a rubber band. A hole was punched at the centre of the net to serve as an introduction point for experimental mosquitoes. Each cylinder was held in a slanting position and 0.1 g (approx. 1.0 × 1011 conidia/m2) of M. anisopliae spores were weighed and poured on the paper. Using both hands, the cylinders were rolled several times until the papers were uniformly covered by the spores. The inner and the base surfaces of the cylinder used for uninfected mosquitoes were lined with white rough paper without spores.\nFor survival experiments, a total of four cylinders with fungus and four cylinders without fungus were used. Of these, two cylinders with fungus and two cylinders without fungus were each used to infect male and female mosquitoes separately. Sixty 1-d-old female and male mosquitoes were introduced into each of their respective four cylinders. The insects were held for six hr being supplied with 6% glucose solution soaked in a cotton pad and placed on top of the netting material covering the cylinder. The mosquitoes were then transferred into four separate holding cages (30 × 30 × 30 cm) based on sex and treatment and were supplied with 6% glucose solution on filter paper wicks. The insects were maintained under ambient conditions inside a screenhouse at 28 ± 2°C and 70 ± 5% r.h. For studies to evaluate plant material this procedure was repeated to infect the mosquitoes but the insects were provided with floral parts of R. communis and P. hysterophorous separately as source of sugar instead of 6% glucose solution. The base of the floral parts was hooked on the netting material covering the mouth of each cylinder using a tooth pick to suspend the plant inside the cylinder.\nFor sugar quantity and digestion rate experiments, five cylinders with fungus and three cylinders without fungus were used to infect female mosquitoes. The same numbers of cylinders were used to infect male mosquitoes. The number of mosquitoes exposed to fungus was higher than for the uninfected group to adjust for mortality in the holding cages prior to the start of the experiments on days one and three post-exposure.\n Plant species Two plant species namely Ricinus communis (Castor oil plant) and Parthenium hysterophorus (Santa Maria feverfew weed) were used. Ricinus communis has been demonstrated to enhance survival of An. gambiae and P. hysterophorus as most frequently visited by this mosquito species [19]. Five, fresh stems cut from each plant with leaves and floral parts intact were used in the study. The cuttings were collected from the agricultural field at icipe, Mbita Point, western Kenya where the plants grow naturally.\nTwo plant species namely Ricinus communis (Castor oil plant) and Parthenium hysterophorus (Santa Maria feverfew weed) were used. Ricinus communis has been demonstrated to enhance survival of An. gambiae and P. hysterophorus as most frequently visited by this mosquito species [19]. Five, fresh stems cut from each plant with leaves and floral parts intact were used in the study. The cuttings were collected from the agricultural field at icipe, Mbita Point, western Kenya where the plants grow naturally.\n Survival of M. anisopliae-infected An. gambiae mosquitoes fed on plant sugars To study the effect of fungus on the survival of M. anisopliae-infected An. gambiae mosquitoes on plant sugars, one hundred male and 100 female An. gambiae mosquitoes exposed to M. anisopliae for six hours upon emergence were held in separate cages (30 × 30 × 30 cm). Each cage was supplied with 250-ml flat bottomed conical flask containing 200-ml filtered Lake Victoria water and five stems (with leaves and floral parts intact) of R. communis. The stems were replaced every two days. Mosquito mortality was recorded daily to determine the length of time over which the mosquitoes survived. Dead individuals were plated in a Petri dish lined with wet filter paper and incubated at 28 ± 2°C. Fungal growth on mosquito cadavers was observed after three days at 400× magnification under a compound microscope. The experiment was replicated four times. This procedure was repeated using P. hysterophorus as an alternative test plant and 6% glucose solution on filter paper wicks as a control. Glucose solution was changed every two days.\nTo study the effect of fungus on the survival of M. anisopliae-infected An. gambiae mosquitoes on plant sugars, one hundred male and 100 female An. gambiae mosquitoes exposed to M. anisopliae for six hours upon emergence were held in separate cages (30 × 30 × 30 cm). Each cage was supplied with 250-ml flat bottomed conical flask containing 200-ml filtered Lake Victoria water and five stems (with leaves and floral parts intact) of R. communis. The stems were replaced every two days. Mosquito mortality was recorded daily to determine the length of time over which the mosquitoes survived. Dead individuals were plated in a Petri dish lined with wet filter paper and incubated at 28 ± 2°C. Fungal growth on mosquito cadavers was observed after three days at 400× magnification under a compound microscope. The experiment was replicated four times. This procedure was repeated using P. hysterophorus as an alternative test plant and 6% glucose solution on filter paper wicks as a control. Glucose solution was changed every two days.\n Quantity of sugar imbibed by M. anisopliae-infected An. gambiae mosquitoes In order to determine the quantity of sugar imbibed by M. anisopliae-infected An. gambiae mosquitoes, preliminary experiments were conducted to determine the length of time required for individual mosquitoes to feed fully. Three groups of female mosquitoes each composed of 50 individuals were fed on stems (with leaves and floral parts intact) of R. communis for separate periods of 6, 12 and 24 hours. The experiments were replicated four times over time. This procedure was repeated with male mosquitoes. Both male and female mosquitoes took 12 hours to satiate. Thus, the amount of sugar ingested from stems of R. communis by male and female mosquitoes, one and three days post-exposure to M. anisopliae, was evaluated after every 12 hours of feeding. One day after exposure to fungus, fifty male and female mosquitoes were aspirated, each from their respective uninfected and fungus-exposed cages and released into four separate cages. The insects were starved for 6 hrs prior to introduction of a 250-ml conical flask containing stems of R. communis in each cage. After 12 hours of feeding, the insects were removed from the cages and held in four separate collection cups. The insects were held inside a refrigerator at 4°C for 30 min and their sugar levels were quantified following the procedure earlier described on ‘detection of plant sugars in mosquitoes using anthrone test’. The experiment was replicated four times. This procedure was repeated with mosquitoes three days post-infection.\nIn order to determine the quantity of sugar imbibed by M. anisopliae-infected An. gambiae mosquitoes, preliminary experiments were conducted to determine the length of time required for individual mosquitoes to feed fully. Three groups of female mosquitoes each composed of 50 individuals were fed on stems (with leaves and floral parts intact) of R. communis for separate periods of 6, 12 and 24 hours. The experiments were replicated four times over time. This procedure was repeated with male mosquitoes. Both male and female mosquitoes took 12 hours to satiate. Thus, the amount of sugar ingested from stems of R. communis by male and female mosquitoes, one and three days post-exposure to M. anisopliae, was evaluated after every 12 hours of feeding. One day after exposure to fungus, fifty male and female mosquitoes were aspirated, each from their respective uninfected and fungus-exposed cages and released into four separate cages. The insects were starved for 6 hrs prior to introduction of a 250-ml conical flask containing stems of R. communis in each cage. After 12 hours of feeding, the insects were removed from the cages and held in four separate collection cups. The insects were held inside a refrigerator at 4°C for 30 min and their sugar levels were quantified following the procedure earlier described on ‘detection of plant sugars in mosquitoes using anthrone test’. The experiment was replicated four times. This procedure was repeated with mosquitoes three days post-infection.\n Sugar digestion rate of M. anisopliae-infected An. gambiae mosquitoes The digestion rate of An. gambiae mosquitoes exposed to M. anisopliae was determined by feeding males and females on R. communis. One day after exposure to fungus, 50 mosquitoes were aspirated from each cage holding uninfected and fungus-exposed mosquitoes and released into four separate cages. The mosquitoes were starved for 6 hours prior to introduction of the plant in a 250-ml conical flask in each cage. Mosquitoes were allowed to feed on R. communis for 12 hours after which the flasks containing the plant were removed from the cages. Fifty mosquitoes that appeared fully fed were also removed and held in separate cages from where ten mosquitoes were removed at an interval of 8 hours starting from time zero through to 32 hours post-feeding. Removed mosquitoes were held inside a refrigerator at 4°C for 30 min and the quantity of sugar in them, and by extension digestion rate, determined following the procedure earlier described. The experiment was replicated four times. The same procedure was repeated with groups of mosquitoes three days post-exposure to M. anisopliae.\nThe digestion rate of An. gambiae mosquitoes exposed to M. anisopliae was determined by feeding males and females on R. communis. One day after exposure to fungus, 50 mosquitoes were aspirated from each cage holding uninfected and fungus-exposed mosquitoes and released into four separate cages. The mosquitoes were starved for 6 hours prior to introduction of the plant in a 250-ml conical flask in each cage. Mosquitoes were allowed to feed on R. communis for 12 hours after which the flasks containing the plant were removed from the cages. Fifty mosquitoes that appeared fully fed were also removed and held in separate cages from where ten mosquitoes were removed at an interval of 8 hours starting from time zero through to 32 hours post-feeding. Removed mosquitoes were held inside a refrigerator at 4°C for 30 min and the quantity of sugar in them, and by extension digestion rate, determined following the procedure earlier described. The experiment was replicated four times. The same procedure was repeated with groups of mosquitoes three days post-exposure to M. anisopliae.\n Ethical approval Ethical approval for this study was given by the Kenya National Ethical Review Committee located at the Kenya Medical Research Institute (NON-SSC Protocol number 203).\nEthical approval for this study was given by the Kenya National Ethical Review Committee located at the Kenya Medical Research Institute (NON-SSC Protocol number 203).\n Statistics Survival of uninfected and M. anisopliae-infected mosquitoes on glucose (6%), R. communis and P. hysterophorous was calculated by expressing the number of mosquitoes that succumbed to mortality as a percentage of the total number tested. Differences in survival between uninfected and fungus-infected groups were estimated using Cox regression analysis. Mortality rates, expressed as Hazard Ratio (HR) were used to estimate the risk of dying when infected compared to when not infected with fungus. To evaluate effects of infection on the amount of sugar ingested by infected (one and three days post-exposure) and control mosquitoes, first the number of mosquitoes that had fed on R. communis was expressed as a percentage of the total number tested. Further, the number of mosquitoes that imbibed small, medium and large quantities of sugar, respectively, was expressed as the mean percentage of the total number of mosquitoes tested. The difference between control and fungus-infected mosquitoes was calculated with the Chi square (χ2) test [25]. The digestion rate of the sugar ingested by mosquitoes one and three days post-exposure was each calculated by logistic regression. Logistic relationships for uninfected and fungus-exposed mosquitoes were fitted to describe sugar detection success for each time elapsed since feeding. The difference between uninfected and fungus-exposed mosquitoes was estimated by the Chi square (χ2) test. All analyses were conducted using SPSS (version 17.0)\nSurvival of uninfected and M. anisopliae-infected mosquitoes on glucose (6%), R. communis and P. hysterophorous was calculated by expressing the number of mosquitoes that succumbed to mortality as a percentage of the total number tested. Differences in survival between uninfected and fungus-infected groups were estimated using Cox regression analysis. Mortality rates, expressed as Hazard Ratio (HR) were used to estimate the risk of dying when infected compared to when not infected with fungus. To evaluate effects of infection on the amount of sugar ingested by infected (one and three days post-exposure) and control mosquitoes, first the number of mosquitoes that had fed on R. communis was expressed as a percentage of the total number tested. Further, the number of mosquitoes that imbibed small, medium and large quantities of sugar, respectively, was expressed as the mean percentage of the total number of mosquitoes tested. The difference between control and fungus-infected mosquitoes was calculated with the Chi square (χ2) test [25]. The digestion rate of the sugar ingested by mosquitoes one and three days post-exposure was each calculated by logistic regression. Logistic relationships for uninfected and fungus-exposed mosquitoes were fitted to describe sugar detection success for each time elapsed since feeding. The difference between uninfected and fungus-exposed mosquitoes was estimated by the Chi square (χ2) test. All analyses were conducted using SPSS (version 17.0)", "Experiments were carried out using laboratory-reared Anopheles gambiae Giles sensu stricto (hereafter termed An. gambiae) mosquitoes obtained from a colony established from wild gravid females collected at Mbita Point (000 25’S, 340 13’E), western Kenya in 1999. All mosquito life stages were maintained under ambient conditions in a mosquito insectary present at the Thomas Odhiambo Campus (TOC) of the International Centre of Insect Physiology and Ecology (icipe) located near Mbita Point Township in western Kenya. Larval and adult stages of the mosquitoes were raised using procedures described previously [23]. Both sexes were separated at emergence and held under ambient conditions in 30 × 30 × 30 cm cages inside a screenhouse. Before experiments, the insects were maintained either on an aqueous 6% glucose solution presented on filter paper wicks or on stem cuttings of Ricinus communis (Castor oil plant) and Parthenium hysterophorus (Santa Maria feverfew weed).", "The anthrone test was used to determine the presence of sugars in the mosquitoes. To do so standard sucrose solutions of different strengths in the series 1, 2, 4, 8, 16, 32, 64, 128 and 256 μg/μl were prepared. Initially, 25.6 g of reagent grade sucrose was dissolved in 50 ml of distilled water. More water was added gradually while mixing to make a 100 ml solution from which the serial dilutions were prepared. Distilled water served as the neutral liquid. These solutions were stored at −4°C. Diluted sulphuric acid was then prepared by mixing 380 ml concentrated sulphuric acid with 150 ml distilled water in a fume hood. The whole solution was cooled for 5 hours at room temperature and further for 12 hours in the refrigerator at 5°C before use. Anthrone reagent was then prepared by mixing 0.15 g of anthrone powder per 100 ml of the diluted sulphuric acid.\nTwo test tube racks were used with each rack holding one hundred 5-ml test tubes. A third rack was used to hold 10 test tubes for the standard sucrose solutions. The standards were prepared by pipetting 1 μl from each of the nine standard sucrose solutions into the nine separate test tubes. The tenth tube contained 1 μl of distilled water. The other two racks were used to hold both sexes of uninfected and M. anisopliae-exposed An. gambiae mosquitoes. Each tube held one mosquito. One drop each of chloroform and methanol in the ratio 1:1 was added to each tube containing mosquitoes to dissolve the cuticle. The racks were held in a fume hood where 0.5 ml of anthrone reagent was added to the standards and the tubes containing mosquitoes. The racks were then transferred into a water bath at room temperature for one hr. In the presence of sugar, the colour of the solutions changed from green to green-blue and further dark-blue depending on the amount of sugar. In absence of sugar, the colour of the sample was transparent yellow. After one hour the results were read by comparing the colour change in tubes containing mosquitoes and those with standard solutions.", "The entomopathogenic fungus Metarhizium anisopliae isolate ICIPE 30 was used (courtesy Dr. N.K. Maniania). This fungus was originally isolated from the stem borer Busseola fusca (Lepidoptera, Noctuidea) in Kendu Bay, western Kenya, in 1999 and has been maintained at the icipe’s Germplasm Centre. Conidia were produced on long rice as substrate [24]. Harvested spores were dried for 48 hours in a desiccator containing active silica gel and stored in a refrigerator (4-6°C) until required. The viability of spores was determined before being used in the experiments. Germination rates >85% after 24 hours on Sabouraud dextrose agar was considered adequate for use in the experiments.", "Transparent plastic cylinders (9 cm diameter; 15 cm height) were used to inoculate An. gambiae mosquitoes with spores of M. anisopliae. The inside vertical surface and the circular base of each cylinder were lined with white rough-surfaced velvex tissue papers that measured 28.6 × 14.3 cm (for vertical surface) and 9 cm in diameter (for circular base area). A piece of mosquito netting material was secured over the mouth of each cylinder using a rubber band. A hole was punched at the centre of the net to serve as an introduction point for experimental mosquitoes. Each cylinder was held in a slanting position and 0.1 g (approx. 1.0 × 1011 conidia/m2) of M. anisopliae spores were weighed and poured on the paper. Using both hands, the cylinders were rolled several times until the papers were uniformly covered by the spores. The inner and the base surfaces of the cylinder used for uninfected mosquitoes were lined with white rough paper without spores.\nFor survival experiments, a total of four cylinders with fungus and four cylinders without fungus were used. Of these, two cylinders with fungus and two cylinders without fungus were each used to infect male and female mosquitoes separately. Sixty 1-d-old female and male mosquitoes were introduced into each of their respective four cylinders. The insects were held for six hr being supplied with 6% glucose solution soaked in a cotton pad and placed on top of the netting material covering the cylinder. The mosquitoes were then transferred into four separate holding cages (30 × 30 × 30 cm) based on sex and treatment and were supplied with 6% glucose solution on filter paper wicks. The insects were maintained under ambient conditions inside a screenhouse at 28 ± 2°C and 70 ± 5% r.h. For studies to evaluate plant material this procedure was repeated to infect the mosquitoes but the insects were provided with floral parts of R. communis and P. hysterophorous separately as source of sugar instead of 6% glucose solution. The base of the floral parts was hooked on the netting material covering the mouth of each cylinder using a tooth pick to suspend the plant inside the cylinder.\nFor sugar quantity and digestion rate experiments, five cylinders with fungus and three cylinders without fungus were used to infect female mosquitoes. The same numbers of cylinders were used to infect male mosquitoes. The number of mosquitoes exposed to fungus was higher than for the uninfected group to adjust for mortality in the holding cages prior to the start of the experiments on days one and three post-exposure.", "Two plant species namely Ricinus communis (Castor oil plant) and Parthenium hysterophorus (Santa Maria feverfew weed) were used. Ricinus communis has been demonstrated to enhance survival of An. gambiae and P. hysterophorus as most frequently visited by this mosquito species [19]. Five, fresh stems cut from each plant with leaves and floral parts intact were used in the study. The cuttings were collected from the agricultural field at icipe, Mbita Point, western Kenya where the plants grow naturally.", "To study the effect of fungus on the survival of M. anisopliae-infected An. gambiae mosquitoes on plant sugars, one hundred male and 100 female An. gambiae mosquitoes exposed to M. anisopliae for six hours upon emergence were held in separate cages (30 × 30 × 30 cm). Each cage was supplied with 250-ml flat bottomed conical flask containing 200-ml filtered Lake Victoria water and five stems (with leaves and floral parts intact) of R. communis. The stems were replaced every two days. Mosquito mortality was recorded daily to determine the length of time over which the mosquitoes survived. Dead individuals were plated in a Petri dish lined with wet filter paper and incubated at 28 ± 2°C. Fungal growth on mosquito cadavers was observed after three days at 400× magnification under a compound microscope. The experiment was replicated four times. This procedure was repeated using P. hysterophorus as an alternative test plant and 6% glucose solution on filter paper wicks as a control. Glucose solution was changed every two days.", "In order to determine the quantity of sugar imbibed by M. anisopliae-infected An. gambiae mosquitoes, preliminary experiments were conducted to determine the length of time required for individual mosquitoes to feed fully. Three groups of female mosquitoes each composed of 50 individuals were fed on stems (with leaves and floral parts intact) of R. communis for separate periods of 6, 12 and 24 hours. The experiments were replicated four times over time. This procedure was repeated with male mosquitoes. Both male and female mosquitoes took 12 hours to satiate. Thus, the amount of sugar ingested from stems of R. communis by male and female mosquitoes, one and three days post-exposure to M. anisopliae, was evaluated after every 12 hours of feeding. One day after exposure to fungus, fifty male and female mosquitoes were aspirated, each from their respective uninfected and fungus-exposed cages and released into four separate cages. The insects were starved for 6 hrs prior to introduction of a 250-ml conical flask containing stems of R. communis in each cage. After 12 hours of feeding, the insects were removed from the cages and held in four separate collection cups. The insects were held inside a refrigerator at 4°C for 30 min and their sugar levels were quantified following the procedure earlier described on ‘detection of plant sugars in mosquitoes using anthrone test’. The experiment was replicated four times. This procedure was repeated with mosquitoes three days post-infection.", "The digestion rate of An. gambiae mosquitoes exposed to M. anisopliae was determined by feeding males and females on R. communis. One day after exposure to fungus, 50 mosquitoes were aspirated from each cage holding uninfected and fungus-exposed mosquitoes and released into four separate cages. The mosquitoes were starved for 6 hours prior to introduction of the plant in a 250-ml conical flask in each cage. Mosquitoes were allowed to feed on R. communis for 12 hours after which the flasks containing the plant were removed from the cages. Fifty mosquitoes that appeared fully fed were also removed and held in separate cages from where ten mosquitoes were removed at an interval of 8 hours starting from time zero through to 32 hours post-feeding. Removed mosquitoes were held inside a refrigerator at 4°C for 30 min and the quantity of sugar in them, and by extension digestion rate, determined following the procedure earlier described. The experiment was replicated four times. The same procedure was repeated with groups of mosquitoes three days post-exposure to M. anisopliae.", "Ethical approval for this study was given by the Kenya National Ethical Review Committee located at the Kenya Medical Research Institute (NON-SSC Protocol number 203).", "Survival of uninfected and M. anisopliae-infected mosquitoes on glucose (6%), R. communis and P. hysterophorous was calculated by expressing the number of mosquitoes that succumbed to mortality as a percentage of the total number tested. Differences in survival between uninfected and fungus-infected groups were estimated using Cox regression analysis. Mortality rates, expressed as Hazard Ratio (HR) were used to estimate the risk of dying when infected compared to when not infected with fungus. To evaluate effects of infection on the amount of sugar ingested by infected (one and three days post-exposure) and control mosquitoes, first the number of mosquitoes that had fed on R. communis was expressed as a percentage of the total number tested. Further, the number of mosquitoes that imbibed small, medium and large quantities of sugar, respectively, was expressed as the mean percentage of the total number of mosquitoes tested. The difference between control and fungus-infected mosquitoes was calculated with the Chi square (χ2) test [25]. The digestion rate of the sugar ingested by mosquitoes one and three days post-exposure was each calculated by logistic regression. Logistic relationships for uninfected and fungus-exposed mosquitoes were fitted to describe sugar detection success for each time elapsed since feeding. The difference between uninfected and fungus-exposed mosquitoes was estimated by the Chi square (χ2) test. All analyses were conducted using SPSS (version 17.0)", " Survival of M. anisopliae-infected An. gambiae mosquitoes fed on plant sugars Infection with M. anisopliae reduced the survival of both sexes of An. gambiae with 100% mortality occurring within seven days compared to ≥ seven days with uninfected mosquitoes irrespective of the nutritional source (Figure 1). Survival of infected male and female mosquitoes in each nutritional group was significantly different from their respective controls. For example, the daily risk of death for both sexes was eight-fold greater on 6% glucose; four-fold (males) and eight-fold (females) greater on R. communis and two-fold greater for both sexes on P. hysterophorus relative to their controls (Table 1). In uninfected mosquitoes, the daily risk of death was three-fold greater for both males (HR = 3.4 [95% CI = 2.91 - 4.21], P = 0.0001) and females (HR = 2.9 [95% CI = 2.45 - 3.55], P = 0.0001) fed on R. communis and 14-fold greater for males (HR = 14.1 [95% CI = 11.33 - 17.6], P = 0.0001) and 13-fold greater for females (HR = 13.4 [95% CI = 10.71 - 16.8], P = 0.0001) fed on P. hysterophorus relative to 6% glucose. Therefore, P. hysterophorus caused a drastic reduction in the survival of mosquitoes regardless of fungal infection. Between sexes, survival rate over time in each nutritional regime was not different. Mycosis test results indicated high infection rates (>77%) in fungus-exposed male and female mosquitoes. No fungal conidia were observed on the cadavers of the control mosquitoes.Figure 1\nSurvival of uninfected and\nM. anisopliae\n-infected\nAn. gambiae\nfemales (Panel A, C and E) and males (Panel B, D and F) when fed on: (i) 6%\nglucose (panel A and B); (ii)\nRicinus communis\n(panel C and D) and (iii)\nParthenium hysterophorus\n(Panel E and F). Uninfected and M. anisopliae-infected mosquitoes are depicted by closed squares and closed triangles, respectively.Table 1\nSurvival analysis of\nAn. gambiae\nmosquitoes infected with\nM. anisopliae\nand fed on different nutritional sources; data show Cox regression Hazard Ratio (HR) outcomes (95% CI), statistical p-values are relative to the relevant control (not exposed to fungus)\n\nNutritional sources\n\nHR (95%\nCI)\n\nMale mosquitoes\n\nP-value\n\nFemale mosquitoes\n\nP-value\nGlucose (6%)8.53 (6.68 - 10.89)0.00017.64 (5.99 - 9.75)0.0001\nRicinus communis\n4.33 (3.59 - 5.23)0.00018.21 (6.49 - 10.37)0.0001\nParthenium hysterophorus\n1.62 (1.40 - 1.89)0.00012.15 (1.85 - 2.50)0.0001\n\nSurvival of uninfected and\nM. anisopliae\n-infected\nAn. gambiae\nfemales (Panel A, C and E) and males (Panel B, D and F) when fed on: (i) 6%\nglucose (panel A and B); (ii)\nRicinus communis\n(panel C and D) and (iii)\nParthenium hysterophorus\n(Panel E and F). Uninfected and M. anisopliae-infected mosquitoes are depicted by closed squares and closed triangles, respectively.\n\nSurvival analysis of\nAn. gambiae\nmosquitoes infected with\nM. anisopliae\nand fed on different nutritional sources; data show Cox regression Hazard Ratio (HR) outcomes (95% CI), statistical p-values are relative to the relevant control (not exposed to fungus)\n\nInfection with M. anisopliae reduced the survival of both sexes of An. gambiae with 100% mortality occurring within seven days compared to ≥ seven days with uninfected mosquitoes irrespective of the nutritional source (Figure 1). Survival of infected male and female mosquitoes in each nutritional group was significantly different from their respective controls. For example, the daily risk of death for both sexes was eight-fold greater on 6% glucose; four-fold (males) and eight-fold (females) greater on R. communis and two-fold greater for both sexes on P. hysterophorus relative to their controls (Table 1). In uninfected mosquitoes, the daily risk of death was three-fold greater for both males (HR = 3.4 [95% CI = 2.91 - 4.21], P = 0.0001) and females (HR = 2.9 [95% CI = 2.45 - 3.55], P = 0.0001) fed on R. communis and 14-fold greater for males (HR = 14.1 [95% CI = 11.33 - 17.6], P = 0.0001) and 13-fold greater for females (HR = 13.4 [95% CI = 10.71 - 16.8], P = 0.0001) fed on P. hysterophorus relative to 6% glucose. Therefore, P. hysterophorus caused a drastic reduction in the survival of mosquitoes regardless of fungal infection. Between sexes, survival rate over time in each nutritional regime was not different. Mycosis test results indicated high infection rates (>77%) in fungus-exposed male and female mosquitoes. No fungal conidia were observed on the cadavers of the control mosquitoes.Figure 1\nSurvival of uninfected and\nM. anisopliae\n-infected\nAn. gambiae\nfemales (Panel A, C and E) and males (Panel B, D and F) when fed on: (i) 6%\nglucose (panel A and B); (ii)\nRicinus communis\n(panel C and D) and (iii)\nParthenium hysterophorus\n(Panel E and F). Uninfected and M. anisopliae-infected mosquitoes are depicted by closed squares and closed triangles, respectively.Table 1\nSurvival analysis of\nAn. gambiae\nmosquitoes infected with\nM. anisopliae\nand fed on different nutritional sources; data show Cox regression Hazard Ratio (HR) outcomes (95% CI), statistical p-values are relative to the relevant control (not exposed to fungus)\n\nNutritional sources\n\nHR (95%\nCI)\n\nMale mosquitoes\n\nP-value\n\nFemale mosquitoes\n\nP-value\nGlucose (6%)8.53 (6.68 - 10.89)0.00017.64 (5.99 - 9.75)0.0001\nRicinus communis\n4.33 (3.59 - 5.23)0.00018.21 (6.49 - 10.37)0.0001\nParthenium hysterophorus\n1.62 (1.40 - 1.89)0.00012.15 (1.85 - 2.50)0.0001\n\nSurvival of uninfected and\nM. anisopliae\n-infected\nAn. gambiae\nfemales (Panel A, C and E) and males (Panel B, D and F) when fed on: (i) 6%\nglucose (panel A and B); (ii)\nRicinus communis\n(panel C and D) and (iii)\nParthenium hysterophorus\n(Panel E and F). Uninfected and M. anisopliae-infected mosquitoes are depicted by closed squares and closed triangles, respectively.\n\nSurvival analysis of\nAn. gambiae\nmosquitoes infected with\nM. anisopliae\nand fed on different nutritional sources; data show Cox regression Hazard Ratio (HR) outcomes (95% CI), statistical p-values are relative to the relevant control (not exposed to fungus)\n\n Quantity of sugar imbibed by M. anisopliae-infected An. gambiae mosquitoes The quantity of sugar detected ranged from 1–64 μg. For easy analysis of the data, the mosquitoes were categorised as consumers of small, medium or large meals [26] if they imbibed 1–4 μg, 8–16 μg or 32–64 μg of sugar, respectively. Significantly fewer male and female mosquitoes exposed to fungus imbibed sugar from R. communis compared to mosquitoes not exposed to fungus (Figure 2). More uninfected than fungus-exposed mosquitoes ingested plant sugar. Fewer mosquitoes imbibed plant sugars at three days post-exposure than at one day post-exposure. Although fungus-exposed mosquitoes ingested less sugar than uninfected counterparts the differences were generally insignificant except for medium-feeding females three days post-exposure (Table 2) and small-feeding males, one and three days post-exposure (Table 3). Results from the mycosis test demonstrated high infection rates (>75%) in fungus-exposed males and females. No fungal hyphae were observed on the cadavers of control mosquitoes.Figure 2\nMean (± S.E) percentage of uninfected and\nM. anisopliae\n- infected\nAn. gambiae\nmales (Panel A) and females (Panel B) that imbibed sugar on exposure to\nRicinus communis\nfor 12 hr. White and gray shaded bars represent uninfected and M. anisopliae-infected mosquitoes respectively. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.Table 2\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nfemale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hours\n\nSugar quantity\n\nDays post-exposure\n\nN\n\nMean% (± S.E) of males that imbibed sugar\n\nχ\n2\n\nP\n\nUninfected\n\nFungus-infected\nSmall1447.5 ± 8.5438.0 ± 7.963.690.055Medium429.0 ± 4.5126.0 ± 4.080.450.502Large418.5 ± 8.2219.5 ± 6.290.070.799Small3435.0 ± 4.1230.0 ± 5.61.140.286Medium422.0 ± 2.226.0 ± 2.1622.280.001Large412.0 ± 4.696.5 ± 6.53.600.058One and three d post-exposure females were tested.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.Table 3\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nmale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hr\n\nSugar quantity\n\nDays post-exposure\n\nN\n\nMean% (± S.E) of males that imbibed sugar\n\nχ\n2\n\nP\n\nUninfected\n\nFungus-infected\nSmall1456.0 ± 6.1641.0 ± 9.479.010.003Medium422.0 ± 3.5621.5 ± 4.190.020.903Large49.5 ± 1.716.5 ± 2.631.220.269Small3452.0 ± 8.4937.0 ± 6.149.110.003Medium416.5 ± 0.5010.5 ± 7.373.080.079Large44.0 ± 2.451.0 ± 1.03.690.055One and three d post-exposure males were tested.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and\nM. anisopliae\n- infected\nAn. gambiae\nmales (Panel A) and females (Panel B) that imbibed sugar on exposure to\nRicinus communis\nfor 12 hr. White and gray shaded bars represent uninfected and M. anisopliae-infected mosquitoes respectively. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nfemale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hours\n\nOne and three d post-exposure females were tested.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nmale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hr\n\nOne and three d post-exposure males were tested.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\nThe quantity of sugar detected ranged from 1–64 μg. For easy analysis of the data, the mosquitoes were categorised as consumers of small, medium or large meals [26] if they imbibed 1–4 μg, 8–16 μg or 32–64 μg of sugar, respectively. Significantly fewer male and female mosquitoes exposed to fungus imbibed sugar from R. communis compared to mosquitoes not exposed to fungus (Figure 2). More uninfected than fungus-exposed mosquitoes ingested plant sugar. Fewer mosquitoes imbibed plant sugars at three days post-exposure than at one day post-exposure. Although fungus-exposed mosquitoes ingested less sugar than uninfected counterparts the differences were generally insignificant except for medium-feeding females three days post-exposure (Table 2) and small-feeding males, one and three days post-exposure (Table 3). Results from the mycosis test demonstrated high infection rates (>75%) in fungus-exposed males and females. No fungal hyphae were observed on the cadavers of control mosquitoes.Figure 2\nMean (± S.E) percentage of uninfected and\nM. anisopliae\n- infected\nAn. gambiae\nmales (Panel A) and females (Panel B) that imbibed sugar on exposure to\nRicinus communis\nfor 12 hr. White and gray shaded bars represent uninfected and M. anisopliae-infected mosquitoes respectively. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.Table 2\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nfemale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hours\n\nSugar quantity\n\nDays post-exposure\n\nN\n\nMean% (± S.E) of males that imbibed sugar\n\nχ\n2\n\nP\n\nUninfected\n\nFungus-infected\nSmall1447.5 ± 8.5438.0 ± 7.963.690.055Medium429.0 ± 4.5126.0 ± 4.080.450.502Large418.5 ± 8.2219.5 ± 6.290.070.799Small3435.0 ± 4.1230.0 ± 5.61.140.286Medium422.0 ± 2.226.0 ± 2.1622.280.001Large412.0 ± 4.696.5 ± 6.53.600.058One and three d post-exposure females were tested.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.Table 3\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nmale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hr\n\nSugar quantity\n\nDays post-exposure\n\nN\n\nMean% (± S.E) of males that imbibed sugar\n\nχ\n2\n\nP\n\nUninfected\n\nFungus-infected\nSmall1456.0 ± 6.1641.0 ± 9.479.010.003Medium422.0 ± 3.5621.5 ± 4.190.020.903Large49.5 ± 1.716.5 ± 2.631.220.269Small3452.0 ± 8.4937.0 ± 6.149.110.003Medium416.5 ± 0.5010.5 ± 7.373.080.079Large44.0 ± 2.451.0 ± 1.03.690.055One and three d post-exposure males were tested.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and\nM. anisopliae\n- infected\nAn. gambiae\nmales (Panel A) and females (Panel B) that imbibed sugar on exposure to\nRicinus communis\nfor 12 hr. White and gray shaded bars represent uninfected and M. anisopliae-infected mosquitoes respectively. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nfemale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hours\n\nOne and three d post-exposure females were tested.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nmale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hr\n\nOne and three d post-exposure males were tested.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n Sugar digestion rate of M. anisopliae-infected An. gambiae mosquitoes The proportions of uninfected and M. anisopliae-infected mosquitoes testing positive for plant sugar consistently decreased over time (Figure 3). For each time period since feeding, more mosquitoes, one day post-exposure to fungus, tested positive for sugar than uninfected mosquitoes but the difference was not significant except in males at 32 hours (χ2 = 6.27; df = 1; P = 0.001) and in females at 24 hours (χ2 = 10.91; df = 1; P = 0.001) and 32 hours (χ2 = 11.25; df = 1; P = 0.001) of digestion. In addition, fewer mosquitoes, three days post-exposure, than controls tested positive for sugars until 16 hours in males and 24 hours in females after feeding with the differences significant at 32 hour of digestion (males: χ2 = 6.49; df = 1; P = 0.001; females χ2 = 7.67; df = 1; P = 0.006). Cumulative scores from time zero through to the 32 hour demonstrate that, more one day post-exposure males (52% versus 45%) and females (58% versus 39%) than controls tested positive for sugar. This was an overall indication that digestion rate was slower in fungus-exposed mosquitoes. The difference was only significant for females, one day post-exposure (Table 4). Further, the proportion of three day post-exposure males (43% versus 43%) and females (53% versus 53%) with sugar was equal to that of the controls. Hence, timing of fungal exposure only had an effect on sugar digestion in females. Results from mycosis tests indicated that, on average 73-81% of males and 78-85% of females were infected with fungus but no spores were observed on the cadaver of the control mosquitoes.Figure 3\nEffect of infection with\nM. anisopliae\non sugar detection success in\nAn. gambiae\nmosquitoes. Panels A and B represent sugar detection success in uninfected and in M. anisopliae-infected males and females respectively one day post-exposure with Panels C and D represent 3 d post-exposure when fed on Ricinus communis for 12 hr. Solid lines representing uninfected mosquitoes and dotted lines representing infected mosquitoes describe the fitted logistic relationships between sugar detection success for each time period since feeding: − Logit (sugar detection success) = ß0 + ß1 time. Circles denote observed values. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.Table 4\nProportion of uninfected and fungus-exposed\nAn. gambiae\nmosquitoes that tested positive for sugar within 32 hr after feeding on\nRicinus communis\nfor 12 hr\n\nSex\n\nDays post-exposure\n\nN\n\n% mosquitoes sugar positive\n\nχ\n2\n\nP\n\nPercent (± S.E) infection\n\nUninfected\n\nFungus-exposed\nMale1445521.960.16173.0 ± 3.87 (146)Female438.55815.290.00178.0 ± 4.16 (156)Male3443.5430.010.92081.0 ± 1.0 (162)Female452.5530.010.92085.0 ± 2.08 (170)Males and females were tested one and three d post-exposure.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nEffect of infection with\nM. anisopliae\non sugar detection success in\nAn. gambiae\nmosquitoes. Panels A and B represent sugar detection success in uninfected and in M. anisopliae-infected males and females respectively one day post-exposure with Panels C and D represent 3 d post-exposure when fed on Ricinus communis for 12 hr. Solid lines representing uninfected mosquitoes and dotted lines representing infected mosquitoes describe the fitted logistic relationships between sugar detection success for each time period since feeding: − Logit (sugar detection success) = ß0 + ß1 time. Circles denote observed values. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.\n\nProportion of uninfected and fungus-exposed\nAn. gambiae\nmosquitoes that tested positive for sugar within 32 hr after feeding on\nRicinus communis\nfor 12 hr\n\nMales and females were tested one and three d post-exposure.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\nThe proportions of uninfected and M. anisopliae-infected mosquitoes testing positive for plant sugar consistently decreased over time (Figure 3). For each time period since feeding, more mosquitoes, one day post-exposure to fungus, tested positive for sugar than uninfected mosquitoes but the difference was not significant except in males at 32 hours (χ2 = 6.27; df = 1; P = 0.001) and in females at 24 hours (χ2 = 10.91; df = 1; P = 0.001) and 32 hours (χ2 = 11.25; df = 1; P = 0.001) of digestion. In addition, fewer mosquitoes, three days post-exposure, than controls tested positive for sugars until 16 hours in males and 24 hours in females after feeding with the differences significant at 32 hour of digestion (males: χ2 = 6.49; df = 1; P = 0.001; females χ2 = 7.67; df = 1; P = 0.006). Cumulative scores from time zero through to the 32 hour demonstrate that, more one day post-exposure males (52% versus 45%) and females (58% versus 39%) than controls tested positive for sugar. This was an overall indication that digestion rate was slower in fungus-exposed mosquitoes. The difference was only significant for females, one day post-exposure (Table 4). Further, the proportion of three day post-exposure males (43% versus 43%) and females (53% versus 53%) with sugar was equal to that of the controls. Hence, timing of fungal exposure only had an effect on sugar digestion in females. Results from mycosis tests indicated that, on average 73-81% of males and 78-85% of females were infected with fungus but no spores were observed on the cadaver of the control mosquitoes.Figure 3\nEffect of infection with\nM. anisopliae\non sugar detection success in\nAn. gambiae\nmosquitoes. Panels A and B represent sugar detection success in uninfected and in M. anisopliae-infected males and females respectively one day post-exposure with Panels C and D represent 3 d post-exposure when fed on Ricinus communis for 12 hr. Solid lines representing uninfected mosquitoes and dotted lines representing infected mosquitoes describe the fitted logistic relationships between sugar detection success for each time period since feeding: − Logit (sugar detection success) = ß0 + ß1 time. Circles denote observed values. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.Table 4\nProportion of uninfected and fungus-exposed\nAn. gambiae\nmosquitoes that tested positive for sugar within 32 hr after feeding on\nRicinus communis\nfor 12 hr\n\nSex\n\nDays post-exposure\n\nN\n\n% mosquitoes sugar positive\n\nχ\n2\n\nP\n\nPercent (± S.E) infection\n\nUninfected\n\nFungus-exposed\nMale1445521.960.16173.0 ± 3.87 (146)Female438.55815.290.00178.0 ± 4.16 (156)Male3443.5430.010.92081.0 ± 1.0 (162)Female452.5530.010.92085.0 ± 2.08 (170)Males and females were tested one and three d post-exposure.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nEffect of infection with\nM. anisopliae\non sugar detection success in\nAn. gambiae\nmosquitoes. Panels A and B represent sugar detection success in uninfected and in M. anisopliae-infected males and females respectively one day post-exposure with Panels C and D represent 3 d post-exposure when fed on Ricinus communis for 12 hr. Solid lines representing uninfected mosquitoes and dotted lines representing infected mosquitoes describe the fitted logistic relationships between sugar detection success for each time period since feeding: − Logit (sugar detection success) = ß0 + ß1 time. Circles denote observed values. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.\n\nProportion of uninfected and fungus-exposed\nAn. gambiae\nmosquitoes that tested positive for sugar within 32 hr after feeding on\nRicinus communis\nfor 12 hr\n\nMales and females were tested one and three d post-exposure.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.", "Infection with M. anisopliae reduced the survival of both sexes of An. gambiae with 100% mortality occurring within seven days compared to ≥ seven days with uninfected mosquitoes irrespective of the nutritional source (Figure 1). Survival of infected male and female mosquitoes in each nutritional group was significantly different from their respective controls. For example, the daily risk of death for both sexes was eight-fold greater on 6% glucose; four-fold (males) and eight-fold (females) greater on R. communis and two-fold greater for both sexes on P. hysterophorus relative to their controls (Table 1). In uninfected mosquitoes, the daily risk of death was three-fold greater for both males (HR = 3.4 [95% CI = 2.91 - 4.21], P = 0.0001) and females (HR = 2.9 [95% CI = 2.45 - 3.55], P = 0.0001) fed on R. communis and 14-fold greater for males (HR = 14.1 [95% CI = 11.33 - 17.6], P = 0.0001) and 13-fold greater for females (HR = 13.4 [95% CI = 10.71 - 16.8], P = 0.0001) fed on P. hysterophorus relative to 6% glucose. Therefore, P. hysterophorus caused a drastic reduction in the survival of mosquitoes regardless of fungal infection. Between sexes, survival rate over time in each nutritional regime was not different. Mycosis test results indicated high infection rates (>77%) in fungus-exposed male and female mosquitoes. No fungal conidia were observed on the cadavers of the control mosquitoes.Figure 1\nSurvival of uninfected and\nM. anisopliae\n-infected\nAn. gambiae\nfemales (Panel A, C and E) and males (Panel B, D and F) when fed on: (i) 6%\nglucose (panel A and B); (ii)\nRicinus communis\n(panel C and D) and (iii)\nParthenium hysterophorus\n(Panel E and F). Uninfected and M. anisopliae-infected mosquitoes are depicted by closed squares and closed triangles, respectively.Table 1\nSurvival analysis of\nAn. gambiae\nmosquitoes infected with\nM. anisopliae\nand fed on different nutritional sources; data show Cox regression Hazard Ratio (HR) outcomes (95% CI), statistical p-values are relative to the relevant control (not exposed to fungus)\n\nNutritional sources\n\nHR (95%\nCI)\n\nMale mosquitoes\n\nP-value\n\nFemale mosquitoes\n\nP-value\nGlucose (6%)8.53 (6.68 - 10.89)0.00017.64 (5.99 - 9.75)0.0001\nRicinus communis\n4.33 (3.59 - 5.23)0.00018.21 (6.49 - 10.37)0.0001\nParthenium hysterophorus\n1.62 (1.40 - 1.89)0.00012.15 (1.85 - 2.50)0.0001\n\nSurvival of uninfected and\nM. anisopliae\n-infected\nAn. gambiae\nfemales (Panel A, C and E) and males (Panel B, D and F) when fed on: (i) 6%\nglucose (panel A and B); (ii)\nRicinus communis\n(panel C and D) and (iii)\nParthenium hysterophorus\n(Panel E and F). Uninfected and M. anisopliae-infected mosquitoes are depicted by closed squares and closed triangles, respectively.\n\nSurvival analysis of\nAn. gambiae\nmosquitoes infected with\nM. anisopliae\nand fed on different nutritional sources; data show Cox regression Hazard Ratio (HR) outcomes (95% CI), statistical p-values are relative to the relevant control (not exposed to fungus)\n", "The quantity of sugar detected ranged from 1–64 μg. For easy analysis of the data, the mosquitoes were categorised as consumers of small, medium or large meals [26] if they imbibed 1–4 μg, 8–16 μg or 32–64 μg of sugar, respectively. Significantly fewer male and female mosquitoes exposed to fungus imbibed sugar from R. communis compared to mosquitoes not exposed to fungus (Figure 2). More uninfected than fungus-exposed mosquitoes ingested plant sugar. Fewer mosquitoes imbibed plant sugars at three days post-exposure than at one day post-exposure. Although fungus-exposed mosquitoes ingested less sugar than uninfected counterparts the differences were generally insignificant except for medium-feeding females three days post-exposure (Table 2) and small-feeding males, one and three days post-exposure (Table 3). Results from the mycosis test demonstrated high infection rates (>75%) in fungus-exposed males and females. No fungal hyphae were observed on the cadavers of control mosquitoes.Figure 2\nMean (± S.E) percentage of uninfected and\nM. anisopliae\n- infected\nAn. gambiae\nmales (Panel A) and females (Panel B) that imbibed sugar on exposure to\nRicinus communis\nfor 12 hr. White and gray shaded bars represent uninfected and M. anisopliae-infected mosquitoes respectively. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.Table 2\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nfemale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hours\n\nSugar quantity\n\nDays post-exposure\n\nN\n\nMean% (± S.E) of males that imbibed sugar\n\nχ\n2\n\nP\n\nUninfected\n\nFungus-infected\nSmall1447.5 ± 8.5438.0 ± 7.963.690.055Medium429.0 ± 4.5126.0 ± 4.080.450.502Large418.5 ± 8.2219.5 ± 6.290.070.799Small3435.0 ± 4.1230.0 ± 5.61.140.286Medium422.0 ± 2.226.0 ± 2.1622.280.001Large412.0 ± 4.696.5 ± 6.53.600.058One and three d post-exposure females were tested.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.Table 3\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nmale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hr\n\nSugar quantity\n\nDays post-exposure\n\nN\n\nMean% (± S.E) of males that imbibed sugar\n\nχ\n2\n\nP\n\nUninfected\n\nFungus-infected\nSmall1456.0 ± 6.1641.0 ± 9.479.010.003Medium422.0 ± 3.5621.5 ± 4.190.020.903Large49.5 ± 1.716.5 ± 2.631.220.269Small3452.0 ± 8.4937.0 ± 6.149.110.003Medium416.5 ± 0.5010.5 ± 7.373.080.079Large44.0 ± 2.451.0 ± 1.03.690.055One and three d post-exposure males were tested.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and\nM. anisopliae\n- infected\nAn. gambiae\nmales (Panel A) and females (Panel B) that imbibed sugar on exposure to\nRicinus communis\nfor 12 hr. White and gray shaded bars represent uninfected and M. anisopliae-infected mosquitoes respectively. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nfemale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hours\n\nOne and three d post-exposure females were tested.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nMean (± S.E) percentage of uninfected and fungus-infected\nAn. gambiae\nmale mosquitoes (see Figure\n1\n) that imbibed different amounts of sugar when fed on\nRicinus communis\nfor 12 hr\n\nOne and three d post-exposure males were tested.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.", "The proportions of uninfected and M. anisopliae-infected mosquitoes testing positive for plant sugar consistently decreased over time (Figure 3). For each time period since feeding, more mosquitoes, one day post-exposure to fungus, tested positive for sugar than uninfected mosquitoes but the difference was not significant except in males at 32 hours (χ2 = 6.27; df = 1; P = 0.001) and in females at 24 hours (χ2 = 10.91; df = 1; P = 0.001) and 32 hours (χ2 = 11.25; df = 1; P = 0.001) of digestion. In addition, fewer mosquitoes, three days post-exposure, than controls tested positive for sugars until 16 hours in males and 24 hours in females after feeding with the differences significant at 32 hour of digestion (males: χ2 = 6.49; df = 1; P = 0.001; females χ2 = 7.67; df = 1; P = 0.006). Cumulative scores from time zero through to the 32 hour demonstrate that, more one day post-exposure males (52% versus 45%) and females (58% versus 39%) than controls tested positive for sugar. This was an overall indication that digestion rate was slower in fungus-exposed mosquitoes. The difference was only significant for females, one day post-exposure (Table 4). Further, the proportion of three day post-exposure males (43% versus 43%) and females (53% versus 53%) with sugar was equal to that of the controls. Hence, timing of fungal exposure only had an effect on sugar digestion in females. Results from mycosis tests indicated that, on average 73-81% of males and 78-85% of females were infected with fungus but no spores were observed on the cadaver of the control mosquitoes.Figure 3\nEffect of infection with\nM. anisopliae\non sugar detection success in\nAn. gambiae\nmosquitoes. Panels A and B represent sugar detection success in uninfected and in M. anisopliae-infected males and females respectively one day post-exposure with Panels C and D represent 3 d post-exposure when fed on Ricinus communis for 12 hr. Solid lines representing uninfected mosquitoes and dotted lines representing infected mosquitoes describe the fitted logistic relationships between sugar detection success for each time period since feeding: − Logit (sugar detection success) = ß0 + ß1 time. Circles denote observed values. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.Table 4\nProportion of uninfected and fungus-exposed\nAn. gambiae\nmosquitoes that tested positive for sugar within 32 hr after feeding on\nRicinus communis\nfor 12 hr\n\nSex\n\nDays post-exposure\n\nN\n\n% mosquitoes sugar positive\n\nχ\n2\n\nP\n\nPercent (± S.E) infection\n\nUninfected\n\nFungus-exposed\nMale1445521.960.16173.0 ± 3.87 (146)Female438.55815.290.00178.0 ± 4.16 (156)Male3443.5430.010.92081.0 ± 1.0 (162)Female452.5530.010.92085.0 ± 2.08 (170)Males and females were tested one and three d post-exposure.Statistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.\n\nEffect of infection with\nM. anisopliae\non sugar detection success in\nAn. gambiae\nmosquitoes. Panels A and B represent sugar detection success in uninfected and in M. anisopliae-infected males and females respectively one day post-exposure with Panels C and D represent 3 d post-exposure when fed on Ricinus communis for 12 hr. Solid lines representing uninfected mosquitoes and dotted lines representing infected mosquitoes describe the fitted logistic relationships between sugar detection success for each time period since feeding: − Logit (sugar detection success) = ß0 + ß1 time. Circles denote observed values. Level of statistical difference between treatments was calculated by Chi square (\nχ\n2\n) test. Each treatment tested 200 mosquitoes.\n\nProportion of uninfected and fungus-exposed\nAn. gambiae\nmosquitoes that tested positive for sugar within 32 hr after feeding on\nRicinus communis\nfor 12 hr\n\nMales and females were tested one and three d post-exposure.\nStatistical significance (P value) between the number of uninfected and fungus-infected mosquitoes in each category of sugar quantity imbibed was calculated by Chi square (χ\n2) test. Each treatment tested 200 mosquitoes.", "Results of this study demonstrate that under ambient conditions, infection with the entomopathogenic fungus M. anisopliae reduced the daily survival of An. gambiae mosquitoes irrespective of the sugar source. Such a significant reduction in the survival of An. gambiae on glucose within 10 d after exposure to M. anisopliae has been reported previously under laboratory conditions [27-30]. Moreover, both R. communis and P. hysterophorus had a strong negative effect on survival of healthy mosquitoes. These findings are in agreement with other studies that reported longer survivorship of healthy An. gambiae mosquitoes fed on glucose than on plant-derived sugars [16,18,31].\nRecent studies have shown that An. gambiae feed from a wide variety of plants and the quantity of sugar affects their survival [17,31]. Moreover, although sugar is present in the leaves, stems and floral parts of the plants, it is in the latter that different sugar types are highly concentrated [19]. Therefore, the lower survival on plant sugars relative to 6% glucose in this study may be due to the limited choice of nectar sources provided i.e. one plant choice instead of mixed plant choices and insufficient production of sugar by nectaries of the cut plants or accumulation of toxic substances due to interruption of nutrient circulation in the plant cuttings. The longer survival of uninfected mosquitoes on R. communis than P. hysterophorus may be attributed to the high amounts of digestible sugars produced [32] and therefore consumed in larger amounts from R. communis than P. hysterophorus [31]. The drastic reduction in the survival of mosquitoes on P. hysterophorus may be associated with the limited feeding resource points on the plant as reported in other studies where mosquitoes ingested sugars from the leaves only when compared to sugars imbibed from leaves, stems and floral parts in the case of R. communis plant [19]. Interestingly, the negative effects of plant sugars from R. communis and P. hysterophorus as reported may be entirely overcome when the insects are additionally offered a blood meal and these sugars then are highly beneficial by extending the survivorship [13,22,33,34].\nSurvivorship is a key feature that defines the vectorial capacity of malaria vectors [35,36]. Survival of An. gambiae mosquitoes on R. communis in this study was longer than the extrinsic incubation period of a pathogen that is as short as 10d for the malaria parasite Plasmodium falciparum [37-39]. This concurs with other studies on survival of An. gambiae on plant sugars [16,31]. As this occurred under semi-field conditions, it is likely that in field situations mosquitoes forage on a wide variety of plants to complete their dietary requirements, sustain longevity and with blood-supplement become efficient as malaria vectors. Therefore, reduction in the life-span of both sexes of An. gambiae by entomopathogenic fungi as demonstrated in this study could lead to a considerable reduction in malaria transmission.\nInfection with fungi strongly reduced the proportion of mosquitoes that ingested sugar from R. communis independent of the time since infection. Interestingly, the feeding potential and the quantity of sugar assimilated by the mosquitoes that did feed remained similar between the treatment and the control groups. This is the first study to report on the impact of entomopathogenic fungi M. anisopliae on plant sugar feeding since reports to date about mosquitoes have addressed reduction of blood-feeding in fungus-infected females [1,2,5]. These findings corroborate our previous work that showed reduction in blood-feeding propensity in fungus-infected mosquitoes [40]. Both findings suggest that entomopathogenic fungi impose a similar effect on the feeding behaviour of mosquitoes irrespective of the food source. In other insect species, a significant reduction in feeding in the maize stem borer Chilo partellus (Swinhoe) larvae [41], adult thrips Megalurothrips sjostedti Trybom [42] and the variegated grasshopper, Zonocerus variegatus (Linnaeus) [43] occurred as early as one to four days after infection with the entomopathogenic fungus M. anisopliae. The normal feeding that we observed has also been reported in corn earworm, Heliothis zea (Boddie) larvae [44] infected with B. bassiana. The insects, however, die at a later stage, which may indicate that infection causes starvation due to physiological changes in infected hosts.\nThe reduction in sugar-feeding propensity may be attributed to three factors. First, infected mosquitoes may have fed as often as the uninfected ones but the sugar content was too low to be detected. Secondly, the sugar in infected mosquitoes may have already been digested and converted into a metabolic product which the anthrone test could not detect. Lastly, some of the ‘sick insects’ may have lost appetite [43], thus affecting their feeding ability [45-47]. The normal feeding in fungus-infected mosquitoes could be associated with dose [48] and the insect defense mechanism to fight the infection [49]. This is because the immune system of insects responds in defense of fungal attack as early as 12 h after exposure to the pathogen [50].\nThe study has further shown that infection by M. anisopliae has no effect on the digestion rate of sugar except in females, one day post-exposure. However, as the fungal infection progressed, fewer infected than uninfected mosquitoes (both sexes) tested sugar positive. Digestion of sugar in insects takes place in the crop and midgut and its rate is influenced by the meal size consumed, sugar concentration [51], metabolic rate [52] and the extent of energy reserves, among other factors. The mechanism that affects feeding rate due to pathogen attack may also affect the digestion process. Therefore, the slow digestion rate in early days of fungal infection is likely to be associated with the dose and the mechanical disruption of the midgut tissues by fungal toxins [46]. Furthermore, the increased breakdown of sugar as the infection advances could be associated with the need to replenish the teneral energy reserves depleted by invasive fungal pathogens in the insect haemolymph. These teneral reserves are critical for the survival of insects [13,53]. In the case where digestion rate between treatments was equal, it is likely that infected mosquitoes imbibed more sugar than controls for two purposes. First, to nourish the storage reserve this is the primary source of nourishment to the fungal pathogen [54]. Second, to replenish and store sugar in the crop for future use. This is because the accumulation of energy reserves retards digestion [6]. Between sexes, the proportion of individuals that tested positive to sugars did not differ in spite of their different synthesis of reserves. This concurs with what has been reported by van Handel [51].\nThe inability of fungus-exposed mosquitoes to sugar feed may pose some advantages. The life-span of both sexes could be reduced to less than five days. During this period, the mating ability of males may be compromised leading to fewer females getting inseminated. Although females can build their energy reserves from human blood, they may not survive long enough to become efficient malaria vectors. Therefore, if both sexes become infected early in life, this could lead to population suppression, incomplete development of the malaria parasite in females and reduction in malaria transmission [5,12]. Moreover, the ability of mosquitoes to feed on and digest sugars may negatively impact on the survival of both sexes and minimize human-mosquito contact. Thus, maintaining the normal rate of food consumption and digestion in fungus-infected insects for as long as possible benefits the fungal pathogen because this maximizes the amount of food available for the entomopathogen [33,55]. Further research however is needed to determine if a similar impact of fungus can occur in field situations.\nThe life of male mosquitoes is exclusively tied to the plant community. By focusing on fungal inoculation during plant feeding, therefore, both males and females are likely to become infected. Control strategies that target both sexes may lead to significant reduction in the prevalence and transmission of malaria and other mosquito borne diseases. In recent studies, the efficiency of plant attractants in attractive toxic sugar baits (ATSB) for the control of mosquitoes has been demonstrated [20,56-61]. The approach uses odour stationary traps baited with fermented ripe fruits and flower scent as attractants, a sugar solution as feeding stimulant and an oral pesticide [20]. The strategy can be adopted to infect and kill mosquitoes with entomopathogenic fungi during plant sugar feeding in two ways. Firstly, by spraying flowering plants with fungal conidia formulated in a suitable carrier that can withstand ultra-violent effects and retain spore virulence. This strategy however, requires assessment on the impact of fungal pathogens on non-target organisms especially pollinators as reported in a separate study with the use of ATSB [62]. Secondly, by spraying fungal conidia in traps baited with fruits and flowers and sugar solution or plant-derived synthetic odours to which mosquitoes respond to [63]. The use of plant odours in the traps will also increase the chance of infecting female mosquitoes harbouring malaria parasites with fungus since the ‘sick mosquitoes’ are reported to be highly attracted to sugar sources [64]. The first approach may be cost effective since preparation of attractants for the traps may be problematic. Also, more mosquitoes are likely to be targeted and killed by spraying the plants than by being attracted to the baited traps, as these are in competition with flowering plants. Nevertheless, research is needed to demonstrate the possibility of these proposed pathways and other unexplored approaches for infecting wild mosquitoes, particularly males, by entomopathogenic fungi.", "This study has demonstrated that infection with entomopathogenic fungi reduces survival and plant sugar-feeding ability of male and female An. gambiae mosquitoes, but not their potential to ingest and digest sugars, except in the late stages of fungal infection. By reducing survival, a fraction of the mosquito population is eliminated thus lowering the level of malaria transmission. The fact that infected mosquitoes continue to feed is an indication that they have a chance to sustain their physiological requirements including reproduction. This may delay mosquitoes from succumbing to infection quickly but may facilitate the occurrence of sub-lethal effects that can lead to reduction in fecundity and a further decline of mosquito population, hence disease transmission. The possibility of targeting male mosquitoes for population reduction by an entomopathogenic fungus opens a new strategy for mosquito vector control." ]

[ "introduction", "materials|methods", null, null, null, null, null, null, null, null, null, null, "results", null, null, null, "discussion", "conclusion" ]

[ "\nMetarhizium anisopliae\n", "\nAnopheles gambiae s.s\n", "Host plants", "Sugar feeding", "Malaria vector" ]

[ 178, 414, 130, 500, 92, 202, 281, 202, 30, 267, 704, 929, 912 ]

[ "mosquitoes", "sugar", "fungus", "infected", "uninfected", "exposure", "anisopliae", "gambiae", "communis", "post" ]

[ "mosquitoes irrespective nutritional", "sugar fewer mosquitoes", "plant sugars mosquitoes", "female mosquitoes nutritional", "sugars mosquitoes anthrone" ]

[ "Animals", "Biofilms", "Cattle", "Dental Enamel", "Dental Pellicle", "Hardness", "Microradiography", "Pasteurization", "Reference Values", "Saliva", "Sucrose", "Surface Properties", "Tooth Demineralization" ]

[ "Specimen preparation", "Baseline measurement and experimental groups", "Salivary bacterial model", "Biofilm model", "Saliva collection", "Saliva pasteurization", "Surface conditioning", "Biofilm growth", "Post-treatment analyses", "Surface microhardness change (VHNchange)", "Transverse microradiography", "Statistical analysis" ]

[ "Extracted bovine incisors were sectioned to obtain 5×5 mm enamel specimens using a Buehler IsometTM low-speed saw (Buehler, Ltd., Lake Bluff, IL, USA). Approximately 54 teeth were used to obtain 54 specimens. During preparation, the teeth were stored in deionized water with thymol. Using a Struers Rotopol 31/RotoForce 4 polishing unit (Struers Inc., Cleveland, PA, USA), all specimens were ground and polished to ensure flat parallel dentin/enamel surfaces. For the finishing process, the dentin side was ground using 500–grit silicon carbide grinding paper. Then, the enamel side was serially ground using 1,200, 2,400 and 4,000 grit papers. After that, specimens were polished using a 1–µm diamond polishing suspension on a polishing cloth to obtain a 5×5 mm polished enamel surface. All specimens were examined for cracks, white spots, or any other flaws that could exclude the specimen from the study, using Nikon SMZ 1500 stereomicroscope at ×20 magnification.", "All specimens were subjected to enamel surface microhardness test (VHNsound) to ensure standardization. A Vickers diamond identifier (Tukon 2100; Wilson-Instron, Norwood, MA, USA) was used with a load of 200 g for 15 s. Three indentations, approximately 100 µm apart, were placed on each specimen and averaged; the inclusion range was VHNsound between 300-380. Specimens were divided into two groups, based on the sucrose concentration to which the biofilm/enamel surface was subjected (0.5% and 1% sucrose concentrations). Each group was divided further into three subgroups (n=9/subgroup), according to the nature of the salivary conditioning to the enamel surface before biofilm formation. The three conditions tested were: filtered/pasteurized saliva; filtered saliva; and deionized water (DIW; negative control).", " Biofilm model After completing specimen preparation, specimens were mounted on the inside of a lid of a 6–well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol.1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation.21\nAfter completing specimen preparation, specimens were mounted on the inside of a lid of a 6–well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol.1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation.21\n Saliva collection Ethical approval was obtained from the Indiana University (IUPUI) institutional review board (IRB #1406440799) for saliva collection. Wax–stimulated saliva samples from three adult donors were collected and pooled (approx. 50 mL/donor). The inclusion criterion considered healthy participants (no systemic diseases) with normal salivary flow and absence of active caries or periodontal disease. To ensure standardization, participants refrained from oral hygiene measures overnight. Before bacterial inoculation or freezing, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars (MSSB and Rogosa agars, respectively). The results confirmed the presence of both species. A total of 5 mL of the pooled saliva and growth media mix (1:10 ratio) were incubated anaerobically overnight, then mixed with 10% glycerol and frozen immediately at −80ºC. This microcosm bacterial mix was used as the source for bacterial inoculum. The remaining pooled saliva was pasteurized as described below.\nEthical approval was obtained from the Indiana University (IUPUI) institutional review board (IRB #1406440799) for saliva collection. Wax–stimulated saliva samples from three adult donors were collected and pooled (approx. 50 mL/donor). The inclusion criterion considered healthy participants (no systemic diseases) with normal salivary flow and absence of active caries or periodontal disease. To ensure standardization, participants refrained from oral hygiene measures overnight. Before bacterial inoculation or freezing, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars (MSSB and Rogosa agars, respectively). The results confirmed the presence of both species. A total of 5 mL of the pooled saliva and growth media mix (1:10 ratio) were incubated anaerobically overnight, then mixed with 10% glycerol and frozen immediately at −80ºC. This microcosm bacterial mix was used as the source for bacterial inoculum. The remaining pooled saliva was pasteurized as described below.\n Saliva pasteurization The collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva. For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol.22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.\nThe collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva. For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol.22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.", "After completing specimen preparation, specimens were mounted on the inside of a lid of a 6–well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol.1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation.21", "Ethical approval was obtained from the Indiana University (IUPUI) institutional review board (IRB #1406440799) for saliva collection. Wax–stimulated saliva samples from three adult donors were collected and pooled (approx. 50 mL/donor). The inclusion criterion considered healthy participants (no systemic diseases) with normal salivary flow and absence of active caries or periodontal disease. To ensure standardization, participants refrained from oral hygiene measures overnight. Before bacterial inoculation or freezing, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars (MSSB and Rogosa agars, respectively). The results confirmed the presence of both species. A total of 5 mL of the pooled saliva and growth media mix (1:10 ratio) were incubated anaerobically overnight, then mixed with 10% glycerol and frozen immediately at −80ºC. This microcosm bacterial mix was used as the source for bacterial inoculum. The remaining pooled saliva was pasteurized as described below.", "The collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva. For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol.22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.", "All specimens were immersed in their corresponding solutions: filtered/pasteurized saliva, filtered saliva, or DIW as negative control. Specimens were incubated in their respective solution at 5% CO2 and 37ºC for 5 minutes to allow surface conditioning.", "Immediately after surface conditioning, specimens were transferred to a new, sterile 6–well plate containing growth culture media that was inoculated with the overnight bacterial culture (without washing the samples between the two steps). Microcosm biofilm was grown under anaerobic conditions at 37°C for 48 h. The growth media used to grow the biofilm was Brain Heart Infusion (BHI) broth, supplemented with 5 g/L yeast extract, 5% vitamin K and hemin (v/v) and supplemented with either 0.5% sucrose or 1% sucrose. After 48 h, the biofilm was collected by placing each specimen in an Eppendorf tube (containing 1 mL sterile saline), sonicated at 30 W for 10 seconds, and vortexed immediately for 10 seconds to completely detach biofilm from the enamel surface.", " Surface microhardness change (VHNchange) Post-treatment surface microhardness was measured following the same protocol used for the VHNsound. Three indentations were made at approximately 100 µm next to the baseline VHNsound indentations. VHNchange values were calculated using the formula VHNchange=100×(VHNsound−VHNpost)/VHNsound.\nPost-treatment surface microhardness was measured following the same protocol used for the VHNsound. Three indentations were made at approximately 100 µm next to the baseline VHNsound indentations. VHNchange values were calculated using the formula VHNchange=100×(VHNsound−VHNpost)/VHNsound.\n Transverse microradiography One section, approximately 100 µm thick, was cut from the center of each specimen and across the specimen using a Silverstone-Taylor Hard Tissue Microtome (Scientific Fabrications Laboratories, USA). All sections were placed in the TMR-D1 v.5.0.0.1 system and X-rayed at 45 kV and 45 mA at a fixed distance of 12 s. An aluminum step wedge was X-rayed under identical conditions. Digital images were analyzed using the TMR software v.3.0.0.18. Sound enamel was assumed to be 87% v/v mineral. The data obtained from this analysis were integrated mineral loss (∆Z; %vol.μm) and lesion depth (L; μm).\nOne section, approximately 100 µm thick, was cut from the center of each specimen and across the specimen using a Silverstone-Taylor Hard Tissue Microtome (Scientific Fabrications Laboratories, USA). All sections were placed in the TMR-D1 v.5.0.0.1 system and X-rayed at 45 kV and 45 mA at a fixed distance of 12 s. An aluminum step wedge was X-rayed under identical conditions. Digital images were analyzed using the TMR software v.3.0.0.18. Sound enamel was assumed to be 87% v/v mineral. The data obtained from this analysis were integrated mineral loss (∆Z; %vol.μm) and lesion depth (L; μm).", "Post-treatment surface microhardness was measured following the same protocol used for the VHNsound. Three indentations were made at approximately 100 µm next to the baseline VHNsound indentations. VHNchange values were calculated using the formula VHNchange=100×(VHNsound−VHNpost)/VHNsound.", "One section, approximately 100 µm thick, was cut from the center of each specimen and across the specimen using a Silverstone-Taylor Hard Tissue Microtome (Scientific Fabrications Laboratories, USA). All sections were placed in the TMR-D1 v.5.0.0.1 system and X-rayed at 45 kV and 45 mA at a fixed distance of 12 s. An aluminum step wedge was X-rayed under identical conditions. Digital images were analyzed using the TMR software v.3.0.0.18. Sound enamel was assumed to be 87% v/v mineral. The data obtained from this analysis were integrated mineral loss (∆Z; %vol.μm) and lesion depth (L; μm).", "All three variables (VHNchange, ∆Z, L) were analyzed using two-way ANOVA, with factors for sucrose concentration and surface conditioning as well as the interaction between them. All pair-wise comparisons from ANOVA analysis were made using Fisher’s Protected Least Significant Differences to control the overall significance level at 5%. Statistical analysis was performed using SAS version 9.4 (SAS Institute, Inc., Cary, NC)." ]

[ null, null, null, null, null, null, null, null, null, null, null, null ]

[ "Introduction", "Methodology", "Specimen preparation", "Baseline measurement and experimental groups", "Salivary bacterial model", "Biofilm model", "Saliva collection", "Saliva pasteurization", "Surface conditioning", "Biofilm growth", "Post-treatment analyses", "Surface microhardness change (VHNchange)", "Transverse microradiography", "Statistical analysis", "Results", "Discussion", "Conclusion" ]

[ "Dental caries is a multifactorial disease, in which acid–producing bacteria, dietary carbohydrates, time, and a susceptible host contribute to the disease initiation and progression.1 The process starts when oral bacteria, present in an equilibrium state, ferment carbohydrates; this equilibrium shifts to increased populations of acidogenic (acid–producing) and aciduric (acid–tolerant) bacteria.1 The consistent presence of acid in the environment disrupts the mineral equilibrium of the exposed dental structures (i.e. enamel and/or dentin), and, therefore, leads to carious lesions.1,2\nDental biofilm has been defined as “matrix–enclosed microbial communities in which cells adhere to each other and/or to surfaces or interfaces.”3 Over 700 bacterial species are present in the oral cavity.4 They are in all oral hard and soft tissue structures. These bacterial aggregations usually produce and become enclosed in extracellular polymeric substance (EPS). The formation of dental biofilm (or dental plaque) consists of several steps, which start with the formation of the acquired pellicle, followed by the initial adhesion of planktonic bacteria to the pellicle layer by binding sites, subsequent maturation of the bacterial biofilm, and, finally, the dispersion of biofilm with detachment of cells/clusters of cells.5\nThe formation of the acquired pellicle is the first step in dental biofilm formation, and it is a unique step distinguishing it from other biofilm types.5 It consists of several interactions between various salivary glycoproteins, and their interaction with the tooth surface. These biochemical interactions are based on Gibbs law of free enthalpy5,6; they lead to the attachment of salivary glycoproteins to a surface (i.e. the enamel). The resulting formed layer is a protein–rich layer with binding sites; these sites are ready for early colonizers to attach.6\nBased on this unique process, some studies suggested a new intervention to prevent biofilm formation: this intervention is in the form of preventing pellicle formation.7 Many microbial studies have explored and studied dental biofilm from many aspects using different cariogenic models.8-11 However, they omitted the step of surface conditioning by the formation of acquired pellicle. This leads to less clinical relevance, especially for this area of study (the significance of including the pellicle) has not been researched previously.\nAcquired enamel pellicle (AEP) has been explored previously for its composition and function.12-16 Studies have explored pellicles and found differences between AEP formed in vitro, in vivo, and in situ. These studies have reported ultrastructural variations, intrinsic and extrinsic maturation variations, as well as variation in the AEP morphology. Studies have found that in vitro AEP were superior to in vivo, which contain higher amounts of proteins. They are also superior in the overall amounts produced (due to the difficulty in collecting in vivo AEP).12-16\nIn in vitro studies, the salivary pellicle can typically form before exposure to bacteria–containing media, resulting in biofilm formation. Several methods have been used to form a salivary pellicle.17-19 In general, the dental surface is exposed to saliva (sterilized, free from bacteria) for a specific amount of time (ranges from minutes to several hours) before being exposed to oral bacteria for biofilm formation.17-19 The significance of surface conditioning before biofilm growth (to allow the formation of acquired enamel pellicle) in studying biofilm models was not evaluated previously and, therefore, needs to be explored. Hence, this study aims to explore the influence of salivary conditioning before biofilm formation on enamel demineralization.\nThe hypothesis was: 1) a significant difference between filtered/pasteurized saliva, filtered saliva, and deionized water (DIW; negative control) as conditioning agents on biofilm–mediated enamel demineralization; and 2) a significant difference between 0.5% and 1% sucrose–supplemented growth media on enamel demineralization.", " Specimen preparation Extracted bovine incisors were sectioned to obtain 5×5 mm enamel specimens using a Buehler IsometTM low-speed saw (Buehler, Ltd., Lake Bluff, IL, USA). Approximately 54 teeth were used to obtain 54 specimens. During preparation, the teeth were stored in deionized water with thymol. Using a Struers Rotopol 31/RotoForce 4 polishing unit (Struers Inc., Cleveland, PA, USA), all specimens were ground and polished to ensure flat parallel dentin/enamel surfaces. For the finishing process, the dentin side was ground using 500–grit silicon carbide grinding paper. Then, the enamel side was serially ground using 1,200, 2,400 and 4,000 grit papers. After that, specimens were polished using a 1–µm diamond polishing suspension on a polishing cloth to obtain a 5×5 mm polished enamel surface. All specimens were examined for cracks, white spots, or any other flaws that could exclude the specimen from the study, using Nikon SMZ 1500 stereomicroscope at ×20 magnification.\nExtracted bovine incisors were sectioned to obtain 5×5 mm enamel specimens using a Buehler IsometTM low-speed saw (Buehler, Ltd., Lake Bluff, IL, USA). Approximately 54 teeth were used to obtain 54 specimens. During preparation, the teeth were stored in deionized water with thymol. Using a Struers Rotopol 31/RotoForce 4 polishing unit (Struers Inc., Cleveland, PA, USA), all specimens were ground and polished to ensure flat parallel dentin/enamel surfaces. For the finishing process, the dentin side was ground using 500–grit silicon carbide grinding paper. Then, the enamel side was serially ground using 1,200, 2,400 and 4,000 grit papers. After that, specimens were polished using a 1–µm diamond polishing suspension on a polishing cloth to obtain a 5×5 mm polished enamel surface. All specimens were examined for cracks, white spots, or any other flaws that could exclude the specimen from the study, using Nikon SMZ 1500 stereomicroscope at ×20 magnification.\n Baseline measurement and experimental groups All specimens were subjected to enamel surface microhardness test (VHNsound) to ensure standardization. A Vickers diamond identifier (Tukon 2100; Wilson-Instron, Norwood, MA, USA) was used with a load of 200 g for 15 s. Three indentations, approximately 100 µm apart, were placed on each specimen and averaged; the inclusion range was VHNsound between 300-380. Specimens were divided into two groups, based on the sucrose concentration to which the biofilm/enamel surface was subjected (0.5% and 1% sucrose concentrations). Each group was divided further into three subgroups (n=9/subgroup), according to the nature of the salivary conditioning to the enamel surface before biofilm formation. The three conditions tested were: filtered/pasteurized saliva; filtered saliva; and deionized water (DIW; negative control).\nAll specimens were subjected to enamel surface microhardness test (VHNsound) to ensure standardization. A Vickers diamond identifier (Tukon 2100; Wilson-Instron, Norwood, MA, USA) was used with a load of 200 g for 15 s. Three indentations, approximately 100 µm apart, were placed on each specimen and averaged; the inclusion range was VHNsound between 300-380. Specimens were divided into two groups, based on the sucrose concentration to which the biofilm/enamel surface was subjected (0.5% and 1% sucrose concentrations). Each group was divided further into three subgroups (n=9/subgroup), according to the nature of the salivary conditioning to the enamel surface before biofilm formation. The three conditions tested were: filtered/pasteurized saliva; filtered saliva; and deionized water (DIW; negative control).\n Salivary bacterial model Biofilm model After completing specimen preparation, specimens were mounted on the inside of a lid of a 6–well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol.1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation.21\nAfter completing specimen preparation, specimens were mounted on the inside of a lid of a 6–well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol.1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation.21\n Saliva collection Ethical approval was obtained from the Indiana University (IUPUI) institutional review board (IRB #1406440799) for saliva collection. Wax–stimulated saliva samples from three adult donors were collected and pooled (approx. 50 mL/donor). The inclusion criterion considered healthy participants (no systemic diseases) with normal salivary flow and absence of active caries or periodontal disease. To ensure standardization, participants refrained from oral hygiene measures overnight. Before bacterial inoculation or freezing, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars (MSSB and Rogosa agars, respectively). The results confirmed the presence of both species. A total of 5 mL of the pooled saliva and growth media mix (1:10 ratio) were incubated anaerobically overnight, then mixed with 10% glycerol and frozen immediately at −80ºC. This microcosm bacterial mix was used as the source for bacterial inoculum. The remaining pooled saliva was pasteurized as described below.\nEthical approval was obtained from the Indiana University (IUPUI) institutional review board (IRB #1406440799) for saliva collection. Wax–stimulated saliva samples from three adult donors were collected and pooled (approx. 50 mL/donor). The inclusion criterion considered healthy participants (no systemic diseases) with normal salivary flow and absence of active caries or periodontal disease. To ensure standardization, participants refrained from oral hygiene measures overnight. Before bacterial inoculation or freezing, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars (MSSB and Rogosa agars, respectively). The results confirmed the presence of both species. A total of 5 mL of the pooled saliva and growth media mix (1:10 ratio) were incubated anaerobically overnight, then mixed with 10% glycerol and frozen immediately at −80ºC. This microcosm bacterial mix was used as the source for bacterial inoculum. The remaining pooled saliva was pasteurized as described below.\n Saliva pasteurization The collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva. For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol.22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.\nThe collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva. For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol.22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.\n Biofilm model After completing specimen preparation, specimens were mounted on the inside of a lid of a 6–well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol.1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation.21\nAfter completing specimen preparation, specimens were mounted on the inside of a lid of a 6–well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol.1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation.21\n Saliva collection Ethical approval was obtained from the Indiana University (IUPUI) institutional review board (IRB #1406440799) for saliva collection. Wax–stimulated saliva samples from three adult donors were collected and pooled (approx. 50 mL/donor). The inclusion criterion considered healthy participants (no systemic diseases) with normal salivary flow and absence of active caries or periodontal disease. To ensure standardization, participants refrained from oral hygiene measures overnight. Before bacterial inoculation or freezing, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars (MSSB and Rogosa agars, respectively). The results confirmed the presence of both species. A total of 5 mL of the pooled saliva and growth media mix (1:10 ratio) were incubated anaerobically overnight, then mixed with 10% glycerol and frozen immediately at −80ºC. This microcosm bacterial mix was used as the source for bacterial inoculum. The remaining pooled saliva was pasteurized as described below.\nEthical approval was obtained from the Indiana University (IUPUI) institutional review board (IRB #1406440799) for saliva collection. Wax–stimulated saliva samples from three adult donors were collected and pooled (approx. 50 mL/donor). The inclusion criterion considered healthy participants (no systemic diseases) with normal salivary flow and absence of active caries or periodontal disease. To ensure standardization, participants refrained from oral hygiene measures overnight. Before bacterial inoculation or freezing, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars (MSSB and Rogosa agars, respectively). The results confirmed the presence of both species. A total of 5 mL of the pooled saliva and growth media mix (1:10 ratio) were incubated anaerobically overnight, then mixed with 10% glycerol and frozen immediately at −80ºC. This microcosm bacterial mix was used as the source for bacterial inoculum. The remaining pooled saliva was pasteurized as described below.\n Saliva pasteurization The collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva. For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol.22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.\nThe collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva. For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol.22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.", "Extracted bovine incisors were sectioned to obtain 5×5 mm enamel specimens using a Buehler IsometTM low-speed saw (Buehler, Ltd., Lake Bluff, IL, USA). Approximately 54 teeth were used to obtain 54 specimens. During preparation, the teeth were stored in deionized water with thymol. Using a Struers Rotopol 31/RotoForce 4 polishing unit (Struers Inc., Cleveland, PA, USA), all specimens were ground and polished to ensure flat parallel dentin/enamel surfaces. For the finishing process, the dentin side was ground using 500–grit silicon carbide grinding paper. Then, the enamel side was serially ground using 1,200, 2,400 and 4,000 grit papers. After that, specimens were polished using a 1–µm diamond polishing suspension on a polishing cloth to obtain a 5×5 mm polished enamel surface. All specimens were examined for cracks, white spots, or any other flaws that could exclude the specimen from the study, using Nikon SMZ 1500 stereomicroscope at ×20 magnification.", "All specimens were subjected to enamel surface microhardness test (VHNsound) to ensure standardization. A Vickers diamond identifier (Tukon 2100; Wilson-Instron, Norwood, MA, USA) was used with a load of 200 g for 15 s. Three indentations, approximately 100 µm apart, were placed on each specimen and averaged; the inclusion range was VHNsound between 300-380. Specimens were divided into two groups, based on the sucrose concentration to which the biofilm/enamel surface was subjected (0.5% and 1% sucrose concentrations). Each group was divided further into three subgroups (n=9/subgroup), according to the nature of the salivary conditioning to the enamel surface before biofilm formation. The three conditions tested were: filtered/pasteurized saliva; filtered saliva; and deionized water (DIW; negative control).", " Biofilm model After completing specimen preparation, specimens were mounted on the inside of a lid of a 6–well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol.1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation.21\nAfter completing specimen preparation, specimens were mounted on the inside of a lid of a 6–well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol.1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation.21\n Saliva collection Ethical approval was obtained from the Indiana University (IUPUI) institutional review board (IRB #1406440799) for saliva collection. Wax–stimulated saliva samples from three adult donors were collected and pooled (approx. 50 mL/donor). The inclusion criterion considered healthy participants (no systemic diseases) with normal salivary flow and absence of active caries or periodontal disease. To ensure standardization, participants refrained from oral hygiene measures overnight. Before bacterial inoculation or freezing, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars (MSSB and Rogosa agars, respectively). The results confirmed the presence of both species. A total of 5 mL of the pooled saliva and growth media mix (1:10 ratio) were incubated anaerobically overnight, then mixed with 10% glycerol and frozen immediately at −80ºC. This microcosm bacterial mix was used as the source for bacterial inoculum. The remaining pooled saliva was pasteurized as described below.\nEthical approval was obtained from the Indiana University (IUPUI) institutional review board (IRB #1406440799) for saliva collection. Wax–stimulated saliva samples from three adult donors were collected and pooled (approx. 50 mL/donor). The inclusion criterion considered healthy participants (no systemic diseases) with normal salivary flow and absence of active caries or periodontal disease. To ensure standardization, participants refrained from oral hygiene measures overnight. Before bacterial inoculation or freezing, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars (MSSB and Rogosa agars, respectively). The results confirmed the presence of both species. A total of 5 mL of the pooled saliva and growth media mix (1:10 ratio) were incubated anaerobically overnight, then mixed with 10% glycerol and frozen immediately at −80ºC. This microcosm bacterial mix was used as the source for bacterial inoculum. The remaining pooled saliva was pasteurized as described below.\n Saliva pasteurization The collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva. For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol.22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.\nThe collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva. For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol.22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.", "After completing specimen preparation, specimens were mounted on the inside of a lid of a 6–well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol.1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation.21", "Ethical approval was obtained from the Indiana University (IUPUI) institutional review board (IRB #1406440799) for saliva collection. Wax–stimulated saliva samples from three adult donors were collected and pooled (approx. 50 mL/donor). The inclusion criterion considered healthy participants (no systemic diseases) with normal salivary flow and absence of active caries or periodontal disease. To ensure standardization, participants refrained from oral hygiene measures overnight. Before bacterial inoculation or freezing, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars (MSSB and Rogosa agars, respectively). The results confirmed the presence of both species. A total of 5 mL of the pooled saliva and growth media mix (1:10 ratio) were incubated anaerobically overnight, then mixed with 10% glycerol and frozen immediately at −80ºC. This microcosm bacterial mix was used as the source for bacterial inoculum. The remaining pooled saliva was pasteurized as described below.", "The collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva. For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol.22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.", "All specimens were immersed in their corresponding solutions: filtered/pasteurized saliva, filtered saliva, or DIW as negative control. Specimens were incubated in their respective solution at 5% CO2 and 37ºC for 5 minutes to allow surface conditioning.", "Immediately after surface conditioning, specimens were transferred to a new, sterile 6–well plate containing growth culture media that was inoculated with the overnight bacterial culture (without washing the samples between the two steps). Microcosm biofilm was grown under anaerobic conditions at 37°C for 48 h. The growth media used to grow the biofilm was Brain Heart Infusion (BHI) broth, supplemented with 5 g/L yeast extract, 5% vitamin K and hemin (v/v) and supplemented with either 0.5% sucrose or 1% sucrose. After 48 h, the biofilm was collected by placing each specimen in an Eppendorf tube (containing 1 mL sterile saline), sonicated at 30 W for 10 seconds, and vortexed immediately for 10 seconds to completely detach biofilm from the enamel surface.", " Surface microhardness change (VHNchange) Post-treatment surface microhardness was measured following the same protocol used for the VHNsound. Three indentations were made at approximately 100 µm next to the baseline VHNsound indentations. VHNchange values were calculated using the formula VHNchange=100×(VHNsound−VHNpost)/VHNsound.\nPost-treatment surface microhardness was measured following the same protocol used for the VHNsound. Three indentations were made at approximately 100 µm next to the baseline VHNsound indentations. VHNchange values were calculated using the formula VHNchange=100×(VHNsound−VHNpost)/VHNsound.\n Transverse microradiography One section, approximately 100 µm thick, was cut from the center of each specimen and across the specimen using a Silverstone-Taylor Hard Tissue Microtome (Scientific Fabrications Laboratories, USA). All sections were placed in the TMR-D1 v.5.0.0.1 system and X-rayed at 45 kV and 45 mA at a fixed distance of 12 s. An aluminum step wedge was X-rayed under identical conditions. Digital images were analyzed using the TMR software v.3.0.0.18. Sound enamel was assumed to be 87% v/v mineral. The data obtained from this analysis were integrated mineral loss (∆Z; %vol.μm) and lesion depth (L; μm).\nOne section, approximately 100 µm thick, was cut from the center of each specimen and across the specimen using a Silverstone-Taylor Hard Tissue Microtome (Scientific Fabrications Laboratories, USA). All sections were placed in the TMR-D1 v.5.0.0.1 system and X-rayed at 45 kV and 45 mA at a fixed distance of 12 s. An aluminum step wedge was X-rayed under identical conditions. Digital images were analyzed using the TMR software v.3.0.0.18. Sound enamel was assumed to be 87% v/v mineral. The data obtained from this analysis were integrated mineral loss (∆Z; %vol.μm) and lesion depth (L; μm).", "Post-treatment surface microhardness was measured following the same protocol used for the VHNsound. Three indentations were made at approximately 100 µm next to the baseline VHNsound indentations. VHNchange values were calculated using the formula VHNchange=100×(VHNsound−VHNpost)/VHNsound.", "One section, approximately 100 µm thick, was cut from the center of each specimen and across the specimen using a Silverstone-Taylor Hard Tissue Microtome (Scientific Fabrications Laboratories, USA). All sections were placed in the TMR-D1 v.5.0.0.1 system and X-rayed at 45 kV and 45 mA at a fixed distance of 12 s. An aluminum step wedge was X-rayed under identical conditions. Digital images were analyzed using the TMR software v.3.0.0.18. Sound enamel was assumed to be 87% v/v mineral. The data obtained from this analysis were integrated mineral loss (∆Z; %vol.μm) and lesion depth (L; μm).", "All three variables (VHNchange, ∆Z, L) were analyzed using two-way ANOVA, with factors for sucrose concentration and surface conditioning as well as the interaction between them. All pair-wise comparisons from ANOVA analysis were made using Fisher’s Protected Least Significant Differences to control the overall significance level at 5%. Statistical analysis was performed using SAS version 9.4 (SAS Institute, Inc., Cary, NC).", "The two-way interaction sucrose concentration × surface conditioning was not significant for VHNchange (p=0.872), ∆Z (p=0.662) or L (p=0.436). Surface conditioning affected VHNchange significantly (p=0.0079); however, it did not affect ∆Z (p=0.7383) or L (p=0.7323). Sucrose concentration impacted ∆Z (p<0.0001) and L (p<0.0001); however, it did not affect VHNchange (p=0.2877). Table 1 shows the data for all measured variables for each subgroup.\nThe VHNchange pairwise multiple comparison analyses indicated that the pellicle type created a significant difference between groups. In both sucrose concentrations, surface conditioning with filtered/pasteurized saliva resulted in the lowest VHNchange values, when compared with other surface conditioning groups. The difference between filtered/pasteurized subgroups and the two other surface conditionings was significant (filtered/pasteurized and filtered saliva subgroups p=0.006; filtered/pasteurized and DIW subgroups p=0.0075), while difference was insignificant between filtered saliva and DIW subgroups (p= 0.9312) (Table 1).\nFor the ∆Z values, the pairwise comparisons indicated a statistically significant difference only between 0.5% and 1% sucrose concentration (p<0.0001), and not based on the surface conditioning status. Growing the biofilm in 1% sucrose always resulted in lesions with higher ∆Z values, indicating more severe lesions.\nSimilarly, the pairwise comparisons for L values indicated a statistically significant difference between 0.5% and 1% sucrose (p<0.0001). Also, the L values were always higher in 1% incubation conditions, which means more severe carious lesions (Table 1).", "This study aimed to evaluate the influence of surface conditioning using human saliva before biofilm formation in vitro on enamel demineralization. The statistical analysis results showed the hardness data were only affected by pellicle type, whereas the TMR data were only affected by sucrose concentration. To fully understand this contradiction, one should consider the differences between the variables studied.\nSurface microhardness is a measurement of how a material responds to deformation. It is mainly influenced by surface integrity and rather than by structural characteristics or mineral content of the bulk substrate. One of the pellicle functions in the oral cavity is its masking effect: it coats dental surfaces and other structures, which may lead to different patterns of bacterial biofilm formation according to the presence/absence or the quality of the pellicle.23-25 The presence or absence of a pellicle layer, therefore, will affect surface characteristics, and this may explain the significant differences between pellicle subgroups in our study. On the other hand, TMR measures are based on mineral content rather than structure. Therefore, the expectation is to observe differences only when carious lesions with different mineral contents and/or distributions form during demineralization.26\nSurface microhardness testing is straightforward and nondestructive. In some studies, it is coupled with transverse microradiography based on their objective. The minerals loss within the outer enamel was found to be proportional with the degree of the indenter penetration. However, deeper lesions cannot be quantitatively measured using surface microhardness.27 Moreover, surface microhardness is most effective in analyzing homogenous materials and shallow lesions only (e.g. enamel outer surface).28 White28 (1987) reported, in a study in which they evaluated the differences between surface microhardness and microradiography, that surface microhardness could detect remineralization in early lesions (or at least hardening of the surface without remineralization).28 Evaluating mineral content within the outermost layers of the enamel using microradiography is difficult. Therefore, the two analyses are usually considered complementary to each other in demineralization/remineralization studies.28\nIn this study, an active attachment model adopted from a previously published model was used.1,20 Despite still lacking more complex features that lead to more clinical relevance (e.g. pulsation of nutrients into the environment),29 an active attachment has the advantage of ensuring that the bacterial layers formed over the surface are not just sedimented cells, but rather attached to the enamel surface and to each other.20\nA salivary bacterial mix was used to create a largely undefined microcosm biofilm. Before bacterial inoculation, the pooled saliva was tested for the presence of Streptococcus mutans and Lactobacilli using selective agars. The results confirmed the presence of both species. In vitro studies use various approaches with biofilms formed from monospecies (such as Streptococcus mutans or Lactobacilli),20,30 two or multiple species (3–10 species),1,31 or a microcosm biofilm.32,33 While single or multiple, defined species allow for greater control, employing a microcosm biofilm can result in greater clinical relevance. The acquired pellicle can be formed from saliva or plaque samples collected and pooled from single and/or multiple donors.21 This study used an approach based on conclusions drawn from previous studies.21,32 Some studies limited their salivary (or plaque) mix to be collected from a single donor,33 other studies collected samples from two or more donors.34 In this study, wax–stimulated saliva samples from three donors were pooled, thereby increasing the translational value of the findings.\nOne could suggest that using a microcosm biofilm source may result in large variability. However, the variability of biofilm characteristics in in vitro studies was explored previously,21 and most studies33-35 concluded that collection of saliva samples from the same donor at different times does not affect biofilm diversity. Moreover, the involvement of sucrose over time can lead to a dominance of certain bacterial strains (mainly cariogenic bacteria), thus overcoming initial differences between different samples (either from different donors or collected at different times from the same donor).21\nThe formation of acquired pellicle in in vitro studies can be conducted by exposing the surface of interest to sterile saliva solution for a certain period. Although including salivary pellicle in the model seems to be more clinically relevant, this step requires an expensive and time–consuming saliva sterilization (to ensure a bacterium–free solution that still contains salivary glycoproteins). Pasteurization was the method chosen to sterilize the diluted, pooled saliva samples.22 Although the samples were exposed to 60ºC for 30 minutes, this method still preserved salivary glycoproteins, as the heat needed for irreversible protein denaturation is at least 80ºC.\nIt was documented previously that the time required to form the pellicle in vitro ranges from 3 minutes to 7 days. The same studies reported minor relevance of pellicle maturation (i.e., aging).36-38 Based on that, the choice was to incubate the enamel samples in three surface conditioning media types for 5 minutes in this study. Exposing enamel surface in vitro to 1:10 diluted saliva for 5 minutes is still expected to maintain clinical relevance; the longer exposure to glycoproteins overcomes the dilution factor.\nThe second variable explored was sucrose concentration. Carbohydrate concentration within the growth media has been reported to impact the biofilm composition.39 Consequently, the biofilm cariogenicity may also be affected. As mentioned before, acquired pellicle formation is an integral step that precedes bacterial attachment to dental and oral surfaces. The formation of acquired pellicle generally consists of two stages.40 The first stage is very rapid and includes adsorption of salivary glycoproteins to the substrate. However, the second stage occurs immediately after the first stage in vivo.40 It is characterized by more adsorption of biomolecules, being the oral fluids the source of these biomolecules.40 Therefore, two different conditions of sources for the salivary pellicle (filtered/pasteurized and filtered saliva) were included in this study to represent these two stages and explore their influence in the pattern of demineralization. Although salivary pellicle formed from filtered/pasteurized saliva (which becomes free of viable bacteria) makes the in vitro study more controllable and the model more applicable if used in studies involving single/multiple species biofilm, using filtered saliva ensures more clinical relevance as the only eliminated element is food debris.\nThis study focused mainly on pellicle involvement in in vitro microbial studies, and on the influence of this factor on the hard tissue substrate characteristics. It did not test the influence of the presence of acquired pellicle on the cariogenicity of a microcosm biofilm. This can be tested in a similar study by collecting 48–hour biofilm and analyzing cariogenicity (e.g., lactic acid production). The bacterial source (i.e., saliva vs. plaque samples) may also be tested, since it was already reported that biofilms formed from saliva vs. plaque have different characteristics.21 Furthermore, different incubation times may affect pellicle formation and maturation. Lastly, pellicle formation can also be achieved by exposing specimens to the oral cavity for different periods of time, which provides material for future research. Lastly, all the variables tested may be evaluated in a prolonged study (more than 48 h) to observe the lesion characteristics, especially TMR data.", "Considering the limitations of this study, the presence or absence of an artificially induced acquired pellicle layer does not influence biofilm–mediated enamel caries lesion formation as measured by TMR. Some differences were observed using surface microhardness, indicating a complex interaction between pellicle proteins and biofilm–mediated demineralization of the enamel surface.\n\nTable 1Percentage surface microhardness (VHNchange), mineral loss (∆Z; %volmin.μm), and lesion depth (L; μm) data (mean ± standard deviation) for all treatment groupsSurface ConditioningSucrose Concen- trationVHNchange∆ZL Filtered/ Pasteurized SalivaFiltered SalivaControl (DIW)Filtered/ Pasteurized SalivaFiltered SalivaControl (DIW)Filtered/ Pasteurized SalivaFiltered SalivaControl (DIW)0.5%32.7a, A51.5b, A48b, A820a, A1257a, A867a, A38.8a, A47.8a, A37.3a, A ±20.7 ±18.4 ±17.1 ±266 ±406 ±338 ±9 ±9.6 ±14.2 1%24.8a, A43.9b, A46.2b, A2623a, B2505a, B2428a, B78.4a, B73.3a, B74.6a, B ±22.4 ±21 ±18.8 ±1014 ±1480 ±1208 ±17.5 ±26.7 ±20.6 Upper case letters indicate statistically significant differences between surface conditioning methods within sucrose concentrationsLower case letters indicate statistically significant differences between sucrose concentrations within surface conditioning methods\n\nUpper case letters indicate statistically significant differences between surface conditioning methods within sucrose concentrations\nLower case letters indicate statistically significant differences between sucrose concentrations within surface conditioning methods" ]

[ "intro", "methods", null, null, null, null, null, null, null, null, null, null, null, null, "results", "discussion", "conclusions" ]

[ "Dental caries", "Biofilms", "Salivary pellicle", "Saliva" ]

[ 184, 156, 759, 62, 177, 133, 44, 150, 342, 40, 124, 80 ]

[ "saliva", "surface", "filtered", "pellicle", "biofilm", "bacterial", "enamel", "10", "specimens", "pooled" ]

[ "biofilm formation enamel", "oral bacteria biofilm", "dental biofilm defined", "studied dental biofilm", "lesions dental biofilm" ]

[ "Allografts", "Anterior Cruciate Ligament Reconstruction", "Autografts", "Female", "Hamstring Tendons", "Humans", "Lysholm Knee Score", "Male", "Retrospective Studies" ]

[]

[]

[]

[ "METHODS", "RESULTS", "DISCUSSION" ]

[ "This study is a retrospective, non-drug, observational clinical trial. Local Ethics Committee approval was received from the Gaziosmanpasa Training and Research Hospital (2017), and consent form was obtained from the patients included in the study.\nA total of 168 patients who underwent ACL reconstruction with autografts or allografts between 2013 and 2015 were retrospectively reviewed. Exclusion criteria were graft size above 8 mm for autografts and below 10 mm for allografts, the presence of additional fractures around the knee joint, multiligamentous knee injury, chondral lesions, and medial or lateral meniscus injuries. Fifty-one patients who underwent ACL reconstruction with hamstring tendon autografts (size ≤ 8 mm) and 21 patients who underwent ACL reconstruction with ATT allografts (size ≥ 10 mm) were included in our study. Patient's preoperative age, sex, time from injury to surgery, and average follow-up time were evaluated and there were no significant differences between autograft and allograft groups (p < 0.05) (Table 1).\nAll surgical procedures were performed by the senior author (HB) at the same center. All knees were reconstructed with single-bundle ACL grafts. All grafts were fixed with EndoButton. Patients in both groups followed the same aggressive early postoperative rehabilitation program at the same center.1718) The patients did not wear a postoperative knee brace or an immobilizer. Passive motion exercises without restrictions on hyperextension were started immediately after surgery. At the same time, range of motion exercise was started with a continuous passive-motion machine from 0° to 50°. Patients began weight bearing as tolerated immediately after surgery and used bilateral axillary crutches. Phase 1 exercises were started to facilitate early motion and muscle activation in the first postoperative week. Mobilization began with full weight-bearing without an assistive device. The phase 1 exercises were continued in the second postoperative week. Phase 2 exercises were started in the fourth postoperative week, phase 3 exercises in the eighth postoperative week, and phase 4 exercises in the twelfth postoperative week.\nPreoperative and final follow-up clinical evaluations were performed with the Lysholm knee scoring scale19) and International Knee Documentation Committee (IKDC) questionnaire.20) In addition, special tests for ACL (anterior drawer, Lachman, and pivot shift tests) were conducted preoperatively and at the final follow-up. The preoperative and final follow-up values were compared between groups and within each group. All patients were assessed by the same evaluator at different centers.\nFor statistical analysis, patients' mean age, mean time from injury to surgery, and mean follow-up period were compared with a 2-tailed independent sample t-test, whereas all other parameters were compared with a chi-square test. Statistical significance was set at p < 0.05.", "The patients were divided into 2 groups according to the graft type. There were no significant differences between the 2 groups regarding the men-to-women ratio, mean age, mean follow-up period, and mean time from injury to surgery (p > 0.05) (Table 1). The mean diameter of the autografts was 7.48 ± 0.33 mm and the mean diameter of the ATT allografts was 10.76 ± 0.67 mm. The diameter of the allografts was significantly larger than that of the autografts (p < 0.001). The final follow-up results of the anterior drawer, Lachman, and pivot shift tests were significantly better than the preoperative test results in both autograft and allograft groups. Therefore, there were no significant differences between groups regarding the preoperative and final follow-up ACL test results (p > 0.05) (Table 2).\nAccording to the Lysholm knee scoring scale and IKDC questionnaire, the final follow-up results were significantly better than the preoperative values in both autograft and allograft groups. When the groups were compared within them according to their preoperative and final follow-up results, statistically better results were obtained at the final follow-up in both groups (p < 0.001). Therefore, there were no significant differences between groups preoperatively and at the final follow-up (p > 0.05) (Table 3). Infection developed in a patient in the allograft group at the third postoperative month. Allograft and all implants were extracted and she received antibiotic therapy for 3 weeks. In the autograft group, 2 patients were diagnosed with rerupture and revision surgery was performed.", "There are many graft options for ACL reconstruction. Although the ideal graft for ACL reconstruction is still controversial, the autograft remains a popular graft choice.21) Graft tissue type, bone containing or all soft tissue, is also controversial among surgeons reconstructing the ACL.5) Bone-containing autografts (bone-patellar tendon-bone graft) were most preferred previously. However, recent studies have shown that all soft-tissue autografts are as effective as bone-containing autografts in terms of stiffness, strength, and clinical outcomes.67) Nowadays, hamstring tendon grafts are the most preferred all soft-tissue autografts.678) In addition, the problems of bone-patellar tendon-bone autografts, such as donor site morbidity, postoperative knee motion restriction, and anterior knee pain, are minimized with the use of hamstring tendon autografts.910)\nThe use of allografts has become more common in ACL reconstruction than in the past. Although studies with allografts showed satisfactory results, complications such as longer vascularization time and potential risks including immune rejection and disease transmission are still unavoidable.22) The incorporation of allografts is slower than that of autografts. In a study by Jackson et al.,16) at a 6-month follow-up, the cross-sectional area of allografts was smaller than that of autografts and the graft strength was weaker for allografts. Therefore, the study suggested that rehabilitation should be implemented slowly in a more controlled setting in allograft reconstruction patients.\nIn our study, we used all soft-tissue grafts for autografts and allografts. We preferred the smaller size hamstring tendon graft as autografts and the larger size ATT as allografts. The same rehabilitation program was used for all patients who received autografts or allografts. Progressive rehabilitation was started in the early postoperative period in both groups. However, there was no significant difference between autograft and allograft groups in terms of final follow-up results and graft insufficiency.\nThe size of the graft is important in ACL reconstruction. One study showed that the larger graft diameter was associated with lower meniscal stress, decreased joint laxity, and less articular cartilage contact stress.12) In the study, 5-mm-diameter grafts exhibited 30% more anteroposterior translation than 9-mm grafts. The data of the study suggest that an increased graft size confers a biomechanical advantage in the ACL-reconstructed knees. In addition, the smaller the graft size, the higher the rate of revision.1323) Revision rate decreases 0.82 times per 0.5-mm graft diameter increase between 7-mm and 9-mm graft diameter.13) Small graft diameter also adversely affects clinical outcomes.11) Therefore, current studies suggest that increased graft size provides both biomechanical advantages and better functional and clinical outcomes in the ACL reconstructed knees.1124)\nACL reconstruction with 8-mm and larger diameter grafts reduces the risk of failure. In a study by Conte et al.,11) the failure rate was higher in reconstructions performed with grafts below 8 mm in diameter, especially in patients under 20 years of age.\nBesides the higher revision rate, the smaller graft size was a predictor of poorer functional score at 2 years after primary ACL reconstruction in a study by Magnussen et al.23) There are not enough studies evaluating the effect of graft size on the results of ACL reconstruction in the literature. Some studies compared autografts with different diameters,11121324) but there is no study on the allograft size in the literature. Several studies have reported the average diameter of the 4-strand hamstring tendon grafts to be between 7.7 mm and 8.5 mm.142526) The 4-strand hamstring tendon graft can be small in some patients, which increases the risk of failure and worse results after reconstruction. Thus, surgeons apply various methods to increase the graft size either by using the hamstring tendon of the other healthy extremity or by augmenting with the tendon graft from another part of the body. Some surgeons increase the size of these grafts by augmentation with allografts. However, it has been demonstrated in the literature that augmentation with allografts increases the risk of graft failure and retear rates.15)\nIn studies on autograft augmentation with allografts, it was found that the purity of the autograft was more important than the size. According to the KT-1000 test scores, autografts were significantly superior to hybrid grafts. It has been reported that increasing the size of autografts with allografts is meaningless.2728)\nThe graft diameter can be determined preoperatively in allograft ACL reconstruction. Surgeons prefer large diameter grafts to small diameter grafts in ACL reconstruction. We included patients who underwent ACL reconstruction with 10 mm and 11 mm diameter ATT allografts. In the autograft group, we included patients who underwent ACL reconstruction with a 4-stranded hamstring tendon graft with a diameter of 8 mm or less. However, we could not find any significant difference between the 2 groups in terms of inadequacy, rerupture, and final follow-up functional results. Although allografts were significantly larger than autografts, we did not see the positive effects of larger size grafts as described in the literature.\nStudies have shown that allografts and autografts undergo a similar remodeling sequence, which consists of graft necrosis, cellular repopulation, revascularization, and collagen remodeling. Allografts and autografts have similar phases during their biologic incorporation, but these processes may proceed at a slower rate in allografts than in autografts.1629) We think that this slow biological incorporation in allografts causes negative effects on the results. Therefore, smaller size autografts will give as good results as larger allografts.\nWe evaluated the early results of ACL reconstruction with a small sample size in this retrospective study; late results were not evaluated. If ACL reconstruction with larger size autografts and smaller size allografts had been included, the importance of this study would have been increased." ]

[ "methods", "results", "discussion" ]

[ "Anterior cruciate ligament injury", "Graft size", "Allograft", "Autograft" ]

[]

[ "graft", "autografts", "allografts", "acl", "size", "reconstruction", "mm", "patients", "results", "diameter" ]

[ "acl grafts grafts", "allografts common acl", "acl reconstruction hamstring", "preoperatively allograft acl", "acl reconstructed knees" ]

[ "Aged", "Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis", "Blood Sedimentation", "Body Mass Index", "C-Reactive Protein", "Cardiovascular Diseases", "Female", "Humans", "Kidney Failure, Chronic", "Male", "Middle Aged", "Sex Factors", "Stroke", "Survival Rate" ]

[ "Patients", "Clinical and laboratory data at diagnosis and during follow-up", "Statistical analyses", "Ethics statement", "Comparison of variables at diagnosis", "Comparison of variables during follow-up", "Comparison of cumulative survival rates", "Cox hazard model analyses" ]

[ "The medical records of 223 immunosuppressive drug-naïve patients with AAV were reviewed. They had been initially diagnosed or reclassified as AAV at the Division of Rheumatology, the Department of Internal Medicine, Yonsei University College of Medicine, Severance Hospital, from October 2000 to March 2020. All patients met the 2007 European medicines Agency algorithm for polyarteritis nodosa and AAV as well as the 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides.12 All patients had the medical records well-documented enough to either collect or assess AAV-specific indices including Birmingham vasculitis activity score (BVAS) version 3 and five-factor score (FFS).67 They had the initial results of ANCA by both an indirect immunofluorescence assay (IFA) for perinuclear (P)-ANCA and cytoplasmic (C)-ANCA and an antigen-specific assay for myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA. Patients negative by antigen-specific assay but positive for ANCA by IFA were considered to have MPO-ANCA or PR3-ANCA when AAV was strongly suspected by the clinical and laboratory features.8 Patients, who had serious medical conditions mimicking the clinical features of AAV at diagnosis such as malignancies, infectious diseases and autoimmune diseases other than AAV, were excluded from this study. In addition, patients, who had been received immunosuppressive drugs prior to diagnosis, were also excluded. All patients had been followed up for at least more than 3 months from the time of diagnosis.", "In terms of variables at diagnosis, sex, age, body mass index (BMI) and smoking history were collected as demographic data. AAV subtypes, ANCA positivity, clinical features based on BVAS items,6 and AAV-specific indices were obtained. As acute-phase reactants, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were also obtained. In terms of variables during follow-up, all-cause mortality, ESRD, cerebrovascular accident (CVA) and cardiovascular disease (CVD) were assessed as the poor outcomes of AAV. ESRD was defined as the status requiring for renal replacement therapy due to estimated glomerular filtration rate of less than 15 mL/min/1.73m2.5 On the basis of sub-items of cardiovascular and nervous systemic items of BVAS,6 CVA was defined as both ischaemic and haemorrhagic strokes, whereas CVD was defined as loss of pulses, vascular heart disease, pericarditis, ischaemic cardiac pain, cardiomyopathy and congestive cardiac failure. Since all patients were classified as AAV in our institute and most of them have been followed up, we could easily access the medical record in our institute to obtain data on the date of death and the first date of diagnosis of ESRD, CVA and CVD. In addition, information regarding all-cause mortality, ESRD, CVA and CVD in patients, who were not followed up in our institute, could be obtained by the Korean National Health Insurance Service system. The follow-up period based on all-cause mortality was defined as the periods from the time of diagnosis of AAV to the death for deceased patients, and those to the last visit for survived patients. On the other hand, the follow-up periods based on ESRD, CVA and CVD were also defined as the periods from diagnosis to either the first renal-replacement, the first diagnoses of CVA or that of CVD, respectively. We counted the number of patients who received each drug among glucocorticoid and immunosuppressive drugs.", "All statistical analyses were conducted using SPSS software (version 23 for Windows; IBM Corp., Armonk, NY, USA). Continuous variables were expressed as a mean ± standard deviation, and categorical variables were expressed as number and the percentage. Significant differences in categorical variables between the two groups were analysed using the χ2 and Fisher's exact tests. Significant differences in continuous variables between the two groups were compared using the Mann-Whitney test. Comparison of the cumulative survivals rates between the two groups was analysed by the Kaplan-Meier survival analysis with the log-rank test. The multivariable Cox hazard model using variables with statistical significance in the univariable Cox hazard model was conducted to appropriately obtain the hazard ratios (HRs) during the considerable follow-up period. P values less than 0.05 were considered statistically significant.", "This study was approved by the Institutional Review Board (IRB) of Severance Hospital (4-2017-0673), and the patient's written informed consent was waived by the approving IRB, as this was a retrospective study.", "The median age was 59.0 years and 74 of 223 AAV patients (64.8%) were men. AAV patients were divided into two groups based on sex and variables at diagnosis were compared between the two groups. Male patients exhibited a higher median BMI than female patients (23.2 vs. 22.0 kg/m2, P = 0.004). Age, smoking history, AAV subtypes, ANCA positivity and the clinical features based on BVAS items did not significantly differ between male and female patients. Also, there were no significant differences in AAV-specific indices and acute-phase reactants between the two groups (Table 1).\nValues are expressed as a median (interquartile range) or number (%).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, BMI = body mass index, MPA = microscopic polyangiitis, GPA = granulomatosis with polyangiitis, EGPA = eosinophilic granulomatosis with polyangiitis, MPO = myeloperoxidase, P = perinuclear, PR3 = proteinase 3, C = cytoplasmic, BVAS = Birmingham vasculitis activity score, FFS = five-factor score, ESR = erythrocyte sedimentation rate, CRP = C-reactive protein.", "With regard to the poor outcomes of AAV, there were no significant differences in the frequencies of the poor outcomes of AAV between the two groups. Among glucocorticoid and immunosuppressive drugs administered during follow-up, male patients received cyclophosphamide more frequently compared to female patients (62.2% vs. 44.3%, P = 0.012) (Table 2).\nValues are expressed as amedian (interquartile range) or number (%).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, ESRD = end-stage renal disease, CVA = cerebrovascular accident, CVD = cardiovascular disease.", "Among the four poor outcomes of AAV, male patients exhibited a significantly lower cumulative patients' survival rate than female patients during the follow-up period based on all-cause mortality (P = 0.037). Meanwhile, male patients tended to have a lower CVD-free survival rate compared to female patients but it did not reach statistical significance (P = 0.057) (Fig. 1).\nAmong all-cause mortality, ESRD, CVA and CVD, only a cumulative patients' survival rate diffed between male and female AAV patients. Male patients exhibited a significantly lower cumulative patients' survival rate than female patients.\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, ESRD = end-stage renal disease, CVA = cerebrovascular accident, CVD = cardiovascular disease.", "In the univariable analysis, age (HR, 1.055), male sex (HR, 2.264), smoking history (HR, 6.052), BVAS (HR, 1.096) and FFS (HR, 2.142) at diagnosis were significantly associated with all-cause mortality during follow-up. In the multivariable analysis, both male sex (HR, 2.378; 95% confidence interval [CI], 1.050–5.384) and FFS (HR, 1.693; 95% CI, 1.071–2.676) at diagnosis were significantly and independently associated with all-cause mortality during follow-up (Table 3).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, HR = hazard ratio, CI = confidence interval, BMI = body mass index, MPA = microscopic polyangiitis, GPA = granulomatosis with polyangiitis, EGPA = eosinophilic GPA, MPO = myeloperoxidase, P = perinuclear, PR3 = proteinase 3, C = cytoplasmic, BVAS = Birmingham vasculitis activity score, FFS = five-factor score, ESR = erythrocyte sedimentation rate, CRP = C-reactive protein." ]

[ null, null, null, null, null, null, null, null ]

[ "INTRODUCTION", "METHODS", "Patients", "Clinical and laboratory data at diagnosis and during follow-up", "Statistical analyses", "Ethics statement", "RESULTS", "Comparison of variables at diagnosis", "Comparison of variables during follow-up", "Comparison of cumulative survival rates", "Cox hazard model analyses", "DISCUSSION" ]

[ "Based on the 2012 revised international Chapel Hill Consensus Conference nomenclature of vasculitides, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) has currently been defined as the necrotising vasculitis without definite immune-complex deposition. AAV primarily affects small vessels, including small intraparenchymal arteries, arterioles, capillaries and venules and occasionally medium-sized arteries and veins.1 In addition, AAV can be further classified three subtypes based on pathogenesis, histological findings, clinical symptoms and signs and laboratory results such as microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA) and eosinophilic GPA (EGPA).12\nEach systemic vasculitis has its own typical sex difference in the incidence: for instance, among large vessel vasculitis, giant cell arteritis has the male to female ratio of 1:3, whereas Takayasu arteritis has the male to female ratio of 1:9.3 Moreover, each systemic vasculitis has a sex differences in the clinical features: for instance, with regard to Korean patients with Behcet's disease, female patients exhibited more frequently genital ulcers, peripheral arthritis, and inflammatory low back pain, whereas male patients showed a higher frequency of skin lesions.4 There was a previous study pertaining to the sex difference in AAV patients, which reported that male patients were vulnerable to the progression to end-stage renal disease (ESRD) compared to female patients. However, this study included only ANCA-positive AAV patients with histologically proven pauci-immune necrotising glomerulonephritis. For this reason, the results could not be generalised to all AAV patients.5 Also, given the ethnic and geographical differences affecting both the clinical manifestation and the poor outcomes of AAV, a need for a study investigating the sex difference in Korean patients with AAV is still raised but there has been no study on it to date. Hence, in this study, we investigated and compared the initial clinical features at diagnosis and the poor outcomes during follow-up in Korean patients with AAV based on sex.", "Patients The medical records of 223 immunosuppressive drug-naïve patients with AAV were reviewed. They had been initially diagnosed or reclassified as AAV at the Division of Rheumatology, the Department of Internal Medicine, Yonsei University College of Medicine, Severance Hospital, from October 2000 to March 2020. All patients met the 2007 European medicines Agency algorithm for polyarteritis nodosa and AAV as well as the 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides.12 All patients had the medical records well-documented enough to either collect or assess AAV-specific indices including Birmingham vasculitis activity score (BVAS) version 3 and five-factor score (FFS).67 They had the initial results of ANCA by both an indirect immunofluorescence assay (IFA) for perinuclear (P)-ANCA and cytoplasmic (C)-ANCA and an antigen-specific assay for myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA. Patients negative by antigen-specific assay but positive for ANCA by IFA were considered to have MPO-ANCA or PR3-ANCA when AAV was strongly suspected by the clinical and laboratory features.8 Patients, who had serious medical conditions mimicking the clinical features of AAV at diagnosis such as malignancies, infectious diseases and autoimmune diseases other than AAV, were excluded from this study. In addition, patients, who had been received immunosuppressive drugs prior to diagnosis, were also excluded. All patients had been followed up for at least more than 3 months from the time of diagnosis.\nThe medical records of 223 immunosuppressive drug-naïve patients with AAV were reviewed. They had been initially diagnosed or reclassified as AAV at the Division of Rheumatology, the Department of Internal Medicine, Yonsei University College of Medicine, Severance Hospital, from October 2000 to March 2020. All patients met the 2007 European medicines Agency algorithm for polyarteritis nodosa and AAV as well as the 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides.12 All patients had the medical records well-documented enough to either collect or assess AAV-specific indices including Birmingham vasculitis activity score (BVAS) version 3 and five-factor score (FFS).67 They had the initial results of ANCA by both an indirect immunofluorescence assay (IFA) for perinuclear (P)-ANCA and cytoplasmic (C)-ANCA and an antigen-specific assay for myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA. Patients negative by antigen-specific assay but positive for ANCA by IFA were considered to have MPO-ANCA or PR3-ANCA when AAV was strongly suspected by the clinical and laboratory features.8 Patients, who had serious medical conditions mimicking the clinical features of AAV at diagnosis such as malignancies, infectious diseases and autoimmune diseases other than AAV, were excluded from this study. In addition, patients, who had been received immunosuppressive drugs prior to diagnosis, were also excluded. All patients had been followed up for at least more than 3 months from the time of diagnosis.\nClinical and laboratory data at diagnosis and during follow-up In terms of variables at diagnosis, sex, age, body mass index (BMI) and smoking history were collected as demographic data. AAV subtypes, ANCA positivity, clinical features based on BVAS items,6 and AAV-specific indices were obtained. As acute-phase reactants, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were also obtained. In terms of variables during follow-up, all-cause mortality, ESRD, cerebrovascular accident (CVA) and cardiovascular disease (CVD) were assessed as the poor outcomes of AAV. ESRD was defined as the status requiring for renal replacement therapy due to estimated glomerular filtration rate of less than 15 mL/min/1.73m2.5 On the basis of sub-items of cardiovascular and nervous systemic items of BVAS,6 CVA was defined as both ischaemic and haemorrhagic strokes, whereas CVD was defined as loss of pulses, vascular heart disease, pericarditis, ischaemic cardiac pain, cardiomyopathy and congestive cardiac failure. Since all patients were classified as AAV in our institute and most of them have been followed up, we could easily access the medical record in our institute to obtain data on the date of death and the first date of diagnosis of ESRD, CVA and CVD. In addition, information regarding all-cause mortality, ESRD, CVA and CVD in patients, who were not followed up in our institute, could be obtained by the Korean National Health Insurance Service system. The follow-up period based on all-cause mortality was defined as the periods from the time of diagnosis of AAV to the death for deceased patients, and those to the last visit for survived patients. On the other hand, the follow-up periods based on ESRD, CVA and CVD were also defined as the periods from diagnosis to either the first renal-replacement, the first diagnoses of CVA or that of CVD, respectively. We counted the number of patients who received each drug among glucocorticoid and immunosuppressive drugs.\nIn terms of variables at diagnosis, sex, age, body mass index (BMI) and smoking history were collected as demographic data. AAV subtypes, ANCA positivity, clinical features based on BVAS items,6 and AAV-specific indices were obtained. As acute-phase reactants, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were also obtained. In terms of variables during follow-up, all-cause mortality, ESRD, cerebrovascular accident (CVA) and cardiovascular disease (CVD) were assessed as the poor outcomes of AAV. ESRD was defined as the status requiring for renal replacement therapy due to estimated glomerular filtration rate of less than 15 mL/min/1.73m2.5 On the basis of sub-items of cardiovascular and nervous systemic items of BVAS,6 CVA was defined as both ischaemic and haemorrhagic strokes, whereas CVD was defined as loss of pulses, vascular heart disease, pericarditis, ischaemic cardiac pain, cardiomyopathy and congestive cardiac failure. Since all patients were classified as AAV in our institute and most of them have been followed up, we could easily access the medical record in our institute to obtain data on the date of death and the first date of diagnosis of ESRD, CVA and CVD. In addition, information regarding all-cause mortality, ESRD, CVA and CVD in patients, who were not followed up in our institute, could be obtained by the Korean National Health Insurance Service system. The follow-up period based on all-cause mortality was defined as the periods from the time of diagnosis of AAV to the death for deceased patients, and those to the last visit for survived patients. On the other hand, the follow-up periods based on ESRD, CVA and CVD were also defined as the periods from diagnosis to either the first renal-replacement, the first diagnoses of CVA or that of CVD, respectively. We counted the number of patients who received each drug among glucocorticoid and immunosuppressive drugs.\nStatistical analyses All statistical analyses were conducted using SPSS software (version 23 for Windows; IBM Corp., Armonk, NY, USA). Continuous variables were expressed as a mean ± standard deviation, and categorical variables were expressed as number and the percentage. Significant differences in categorical variables between the two groups were analysed using the χ2 and Fisher's exact tests. Significant differences in continuous variables between the two groups were compared using the Mann-Whitney test. Comparison of the cumulative survivals rates between the two groups was analysed by the Kaplan-Meier survival analysis with the log-rank test. The multivariable Cox hazard model using variables with statistical significance in the univariable Cox hazard model was conducted to appropriately obtain the hazard ratios (HRs) during the considerable follow-up period. P values less than 0.05 were considered statistically significant.\nAll statistical analyses were conducted using SPSS software (version 23 for Windows; IBM Corp., Armonk, NY, USA). Continuous variables were expressed as a mean ± standard deviation, and categorical variables were expressed as number and the percentage. Significant differences in categorical variables between the two groups were analysed using the χ2 and Fisher's exact tests. Significant differences in continuous variables between the two groups were compared using the Mann-Whitney test. Comparison of the cumulative survivals rates between the two groups was analysed by the Kaplan-Meier survival analysis with the log-rank test. The multivariable Cox hazard model using variables with statistical significance in the univariable Cox hazard model was conducted to appropriately obtain the hazard ratios (HRs) during the considerable follow-up period. P values less than 0.05 were considered statistically significant.\nEthics statement This study was approved by the Institutional Review Board (IRB) of Severance Hospital (4-2017-0673), and the patient's written informed consent was waived by the approving IRB, as this was a retrospective study.\nThis study was approved by the Institutional Review Board (IRB) of Severance Hospital (4-2017-0673), and the patient's written informed consent was waived by the approving IRB, as this was a retrospective study.", "The medical records of 223 immunosuppressive drug-naïve patients with AAV were reviewed. They had been initially diagnosed or reclassified as AAV at the Division of Rheumatology, the Department of Internal Medicine, Yonsei University College of Medicine, Severance Hospital, from October 2000 to March 2020. All patients met the 2007 European medicines Agency algorithm for polyarteritis nodosa and AAV as well as the 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides.12 All patients had the medical records well-documented enough to either collect or assess AAV-specific indices including Birmingham vasculitis activity score (BVAS) version 3 and five-factor score (FFS).67 They had the initial results of ANCA by both an indirect immunofluorescence assay (IFA) for perinuclear (P)-ANCA and cytoplasmic (C)-ANCA and an antigen-specific assay for myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA. Patients negative by antigen-specific assay but positive for ANCA by IFA were considered to have MPO-ANCA or PR3-ANCA when AAV was strongly suspected by the clinical and laboratory features.8 Patients, who had serious medical conditions mimicking the clinical features of AAV at diagnosis such as malignancies, infectious diseases and autoimmune diseases other than AAV, were excluded from this study. In addition, patients, who had been received immunosuppressive drugs prior to diagnosis, were also excluded. All patients had been followed up for at least more than 3 months from the time of diagnosis.", "In terms of variables at diagnosis, sex, age, body mass index (BMI) and smoking history were collected as demographic data. AAV subtypes, ANCA positivity, clinical features based on BVAS items,6 and AAV-specific indices were obtained. As acute-phase reactants, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were also obtained. In terms of variables during follow-up, all-cause mortality, ESRD, cerebrovascular accident (CVA) and cardiovascular disease (CVD) were assessed as the poor outcomes of AAV. ESRD was defined as the status requiring for renal replacement therapy due to estimated glomerular filtration rate of less than 15 mL/min/1.73m2.5 On the basis of sub-items of cardiovascular and nervous systemic items of BVAS,6 CVA was defined as both ischaemic and haemorrhagic strokes, whereas CVD was defined as loss of pulses, vascular heart disease, pericarditis, ischaemic cardiac pain, cardiomyopathy and congestive cardiac failure. Since all patients were classified as AAV in our institute and most of them have been followed up, we could easily access the medical record in our institute to obtain data on the date of death and the first date of diagnosis of ESRD, CVA and CVD. In addition, information regarding all-cause mortality, ESRD, CVA and CVD in patients, who were not followed up in our institute, could be obtained by the Korean National Health Insurance Service system. The follow-up period based on all-cause mortality was defined as the periods from the time of diagnosis of AAV to the death for deceased patients, and those to the last visit for survived patients. On the other hand, the follow-up periods based on ESRD, CVA and CVD were also defined as the periods from diagnosis to either the first renal-replacement, the first diagnoses of CVA or that of CVD, respectively. We counted the number of patients who received each drug among glucocorticoid and immunosuppressive drugs.", "All statistical analyses were conducted using SPSS software (version 23 for Windows; IBM Corp., Armonk, NY, USA). Continuous variables were expressed as a mean ± standard deviation, and categorical variables were expressed as number and the percentage. Significant differences in categorical variables between the two groups were analysed using the χ2 and Fisher's exact tests. Significant differences in continuous variables between the two groups were compared using the Mann-Whitney test. Comparison of the cumulative survivals rates between the two groups was analysed by the Kaplan-Meier survival analysis with the log-rank test. The multivariable Cox hazard model using variables with statistical significance in the univariable Cox hazard model was conducted to appropriately obtain the hazard ratios (HRs) during the considerable follow-up period. P values less than 0.05 were considered statistically significant.", "This study was approved by the Institutional Review Board (IRB) of Severance Hospital (4-2017-0673), and the patient's written informed consent was waived by the approving IRB, as this was a retrospective study.", "Comparison of variables at diagnosis The median age was 59.0 years and 74 of 223 AAV patients (64.8%) were men. AAV patients were divided into two groups based on sex and variables at diagnosis were compared between the two groups. Male patients exhibited a higher median BMI than female patients (23.2 vs. 22.0 kg/m2, P = 0.004). Age, smoking history, AAV subtypes, ANCA positivity and the clinical features based on BVAS items did not significantly differ between male and female patients. Also, there were no significant differences in AAV-specific indices and acute-phase reactants between the two groups (Table 1).\nValues are expressed as a median (interquartile range) or number (%).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, BMI = body mass index, MPA = microscopic polyangiitis, GPA = granulomatosis with polyangiitis, EGPA = eosinophilic granulomatosis with polyangiitis, MPO = myeloperoxidase, P = perinuclear, PR3 = proteinase 3, C = cytoplasmic, BVAS = Birmingham vasculitis activity score, FFS = five-factor score, ESR = erythrocyte sedimentation rate, CRP = C-reactive protein.\nThe median age was 59.0 years and 74 of 223 AAV patients (64.8%) were men. AAV patients were divided into two groups based on sex and variables at diagnosis were compared between the two groups. Male patients exhibited a higher median BMI than female patients (23.2 vs. 22.0 kg/m2, P = 0.004). Age, smoking history, AAV subtypes, ANCA positivity and the clinical features based on BVAS items did not significantly differ between male and female patients. Also, there were no significant differences in AAV-specific indices and acute-phase reactants between the two groups (Table 1).\nValues are expressed as a median (interquartile range) or number (%).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, BMI = body mass index, MPA = microscopic polyangiitis, GPA = granulomatosis with polyangiitis, EGPA = eosinophilic granulomatosis with polyangiitis, MPO = myeloperoxidase, P = perinuclear, PR3 = proteinase 3, C = cytoplasmic, BVAS = Birmingham vasculitis activity score, FFS = five-factor score, ESR = erythrocyte sedimentation rate, CRP = C-reactive protein.\nComparison of variables during follow-up With regard to the poor outcomes of AAV, there were no significant differences in the frequencies of the poor outcomes of AAV between the two groups. Among glucocorticoid and immunosuppressive drugs administered during follow-up, male patients received cyclophosphamide more frequently compared to female patients (62.2% vs. 44.3%, P = 0.012) (Table 2).\nValues are expressed as amedian (interquartile range) or number (%).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, ESRD = end-stage renal disease, CVA = cerebrovascular accident, CVD = cardiovascular disease.\nWith regard to the poor outcomes of AAV, there were no significant differences in the frequencies of the poor outcomes of AAV between the two groups. Among glucocorticoid and immunosuppressive drugs administered during follow-up, male patients received cyclophosphamide more frequently compared to female patients (62.2% vs. 44.3%, P = 0.012) (Table 2).\nValues are expressed as amedian (interquartile range) or number (%).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, ESRD = end-stage renal disease, CVA = cerebrovascular accident, CVD = cardiovascular disease.\nComparison of cumulative survival rates Among the four poor outcomes of AAV, male patients exhibited a significantly lower cumulative patients' survival rate than female patients during the follow-up period based on all-cause mortality (P = 0.037). Meanwhile, male patients tended to have a lower CVD-free survival rate compared to female patients but it did not reach statistical significance (P = 0.057) (Fig. 1).\nAmong all-cause mortality, ESRD, CVA and CVD, only a cumulative patients' survival rate diffed between male and female AAV patients. Male patients exhibited a significantly lower cumulative patients' survival rate than female patients.\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, ESRD = end-stage renal disease, CVA = cerebrovascular accident, CVD = cardiovascular disease.\nAmong the four poor outcomes of AAV, male patients exhibited a significantly lower cumulative patients' survival rate than female patients during the follow-up period based on all-cause mortality (P = 0.037). Meanwhile, male patients tended to have a lower CVD-free survival rate compared to female patients but it did not reach statistical significance (P = 0.057) (Fig. 1).\nAmong all-cause mortality, ESRD, CVA and CVD, only a cumulative patients' survival rate diffed between male and female AAV patients. Male patients exhibited a significantly lower cumulative patients' survival rate than female patients.\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, ESRD = end-stage renal disease, CVA = cerebrovascular accident, CVD = cardiovascular disease.\nCox hazard model analyses In the univariable analysis, age (HR, 1.055), male sex (HR, 2.264), smoking history (HR, 6.052), BVAS (HR, 1.096) and FFS (HR, 2.142) at diagnosis were significantly associated with all-cause mortality during follow-up. In the multivariable analysis, both male sex (HR, 2.378; 95% confidence interval [CI], 1.050–5.384) and FFS (HR, 1.693; 95% CI, 1.071–2.676) at diagnosis were significantly and independently associated with all-cause mortality during follow-up (Table 3).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, HR = hazard ratio, CI = confidence interval, BMI = body mass index, MPA = microscopic polyangiitis, GPA = granulomatosis with polyangiitis, EGPA = eosinophilic GPA, MPO = myeloperoxidase, P = perinuclear, PR3 = proteinase 3, C = cytoplasmic, BVAS = Birmingham vasculitis activity score, FFS = five-factor score, ESR = erythrocyte sedimentation rate, CRP = C-reactive protein.\nIn the univariable analysis, age (HR, 1.055), male sex (HR, 2.264), smoking history (HR, 6.052), BVAS (HR, 1.096) and FFS (HR, 2.142) at diagnosis were significantly associated with all-cause mortality during follow-up. In the multivariable analysis, both male sex (HR, 2.378; 95% confidence interval [CI], 1.050–5.384) and FFS (HR, 1.693; 95% CI, 1.071–2.676) at diagnosis were significantly and independently associated with all-cause mortality during follow-up (Table 3).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, HR = hazard ratio, CI = confidence interval, BMI = body mass index, MPA = microscopic polyangiitis, GPA = granulomatosis with polyangiitis, EGPA = eosinophilic GPA, MPO = myeloperoxidase, P = perinuclear, PR3 = proteinase 3, C = cytoplasmic, BVAS = Birmingham vasculitis activity score, FFS = five-factor score, ESR = erythrocyte sedimentation rate, CRP = C-reactive protein.", "The median age was 59.0 years and 74 of 223 AAV patients (64.8%) were men. AAV patients were divided into two groups based on sex and variables at diagnosis were compared between the two groups. Male patients exhibited a higher median BMI than female patients (23.2 vs. 22.0 kg/m2, P = 0.004). Age, smoking history, AAV subtypes, ANCA positivity and the clinical features based on BVAS items did not significantly differ between male and female patients. Also, there were no significant differences in AAV-specific indices and acute-phase reactants between the two groups (Table 1).\nValues are expressed as a median (interquartile range) or number (%).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, BMI = body mass index, MPA = microscopic polyangiitis, GPA = granulomatosis with polyangiitis, EGPA = eosinophilic granulomatosis with polyangiitis, MPO = myeloperoxidase, P = perinuclear, PR3 = proteinase 3, C = cytoplasmic, BVAS = Birmingham vasculitis activity score, FFS = five-factor score, ESR = erythrocyte sedimentation rate, CRP = C-reactive protein.", "With regard to the poor outcomes of AAV, there were no significant differences in the frequencies of the poor outcomes of AAV between the two groups. Among glucocorticoid and immunosuppressive drugs administered during follow-up, male patients received cyclophosphamide more frequently compared to female patients (62.2% vs. 44.3%, P = 0.012) (Table 2).\nValues are expressed as amedian (interquartile range) or number (%).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, ESRD = end-stage renal disease, CVA = cerebrovascular accident, CVD = cardiovascular disease.", "Among the four poor outcomes of AAV, male patients exhibited a significantly lower cumulative patients' survival rate than female patients during the follow-up period based on all-cause mortality (P = 0.037). Meanwhile, male patients tended to have a lower CVD-free survival rate compared to female patients but it did not reach statistical significance (P = 0.057) (Fig. 1).\nAmong all-cause mortality, ESRD, CVA and CVD, only a cumulative patients' survival rate diffed between male and female AAV patients. Male patients exhibited a significantly lower cumulative patients' survival rate than female patients.\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, ESRD = end-stage renal disease, CVA = cerebrovascular accident, CVD = cardiovascular disease.", "In the univariable analysis, age (HR, 1.055), male sex (HR, 2.264), smoking history (HR, 6.052), BVAS (HR, 1.096) and FFS (HR, 2.142) at diagnosis were significantly associated with all-cause mortality during follow-up. In the multivariable analysis, both male sex (HR, 2.378; 95% confidence interval [CI], 1.050–5.384) and FFS (HR, 1.693; 95% CI, 1.071–2.676) at diagnosis were significantly and independently associated with all-cause mortality during follow-up (Table 3).\nANCA = antineutrophil cytoplasmic antibody, AAV = ANCA-associated vasculitis, HR = hazard ratio, CI = confidence interval, BMI = body mass index, MPA = microscopic polyangiitis, GPA = granulomatosis with polyangiitis, EGPA = eosinophilic GPA, MPO = myeloperoxidase, P = perinuclear, PR3 = proteinase 3, C = cytoplasmic, BVAS = Birmingham vasculitis activity score, FFS = five-factor score, ESR = erythrocyte sedimentation rate, CRP = C-reactive protein.", "In this study comparing the clinical features based on the sex difference in AAV patients, we discovered the following three new findings. First, at the time of diagnosis, the clinical features and laboratory related to AAV, such as AAV subtypes, ANCA positivity, clinical manifestations and AAV-specific indices, did not significantly differ between male and female patients. Second, during the follow-up period, male patients exhibited a significantly lower cumulative patients' survival rate compared to female patients. Third, male sex together with FFS was proved to be an independent predictor of all-cause mortality during the follow-up period in AAV patients.\nOnly male sex itself does not seem to simply increase the rate of all-cause mortality in this study. A previous study reported introduced dietary risks, tobacco smoking, high BMI, high blood pressure and high fasting plasma glucose as the most common risk factors for mortality. However, male sex itself was not clearly defined as a primary risk factor for mortality.9 Another previous study denied the fact that the life expectancy of men was meaningfully lower than that of women. Instead, it suggested the different clinical features based on the sex difference.10 Meanwhile, although there were no differences in clinical manifestations at diagnosis between male and female AAV patients, in the multivariable Cox analysis, male sex was an independent predictor of all-cause mortality during follow-up. It is difficult to suggest the exact mechanism, but it can be assumed that in a condition with persistent AAV, male patients are more frequently exposed to situations that may increase the rate of all-cause mortality compared to female patients. The result that cyclophosphamide had been administered to male patients more frequently than female patients during the follow-up period may support our assumption.\nAt diagnosis, male patients showed a higher mean BMI than female patients.\nWe wondered whether a high BMI in male patients has influenced an increase in all-cause mortality compared to female patients. To get the clue to prove this, we compared BMI between survived and dead patients with AAV and found that there was no significant difference between the two groups (22.1 vs. 23.0 kg/m2, P = 0.292). In addition, in the multivariable Cox analysis, BMI was not significantly associated with all-cause mortality (Table 3). Why did not the high calculated BMI in male patients contribute to an increased all-cause mortality rate in male patients? According to the previous studies, the rate of all-cause mortality showed a U-shape with BMI between 22.5 and 25 kg/m2 as a reference range: the rate of all-cause mortality tended to increase not only in the BMI range of below 22.5 (or 25) kg/m2 but also in BMI range of above 25 kg/m2.1112 However, unlike the previous studies, in this study, the BMI range, where the largest number of AAV patients died (44.0%), was between 22.1 and 25.0 kg/m2. It could be assumed that this discrepancy was derived from the different study-subjects between general people and AAV patients and furthermore, it might offset the high calculated BMI in male patients from contributing to an increased all-cause mortality rate.\nA previous study, male sex was significantly associated with ESRD occurrence compared to female sex in AAV patients with histologically proven pauci-immune necrotising glomerulonephritis.5 However, unlike the previous study, no significant difference in the cumulative ESRD-free survival rate between male and female patients in this study. Although not all patients with renal involvement underwent renal biopsy, to reproduce the result of the previous study, we included only AAV patients with renal involvement (50 men and 86 women) and analysed it again. However, we could find no significant difference in the cumulative ESRD-free survival rates between male and female patients during the follow-up period based on ESRD (P = 0.994) (Supplementary Fig. 1). Therefore, although focusing on AAV patients with renal involvement, we conclude that the male sex was turned out to be not a good predictor of ESRD during follow-up.\nWe wondered whether male sex may differently affect the cumulative patients' survival rates among AAV-subtypes. Therefore, we conducted the Kaplan-Meier survival analysis for comparing the cumulative patients' survival rates between male and female patients in each AAV subtypes. Among MPA patients, male patients exhibited a significantly lower cumulative survival rate than female patients. However, no significant difference in the cumulative survival rates was observed between male and female patients among GPA patients (Supplementary Fig. 2). On the other hand, since all EGPA patients survived during the follow-up period, the analysis regarding the clinical significance of sex for all-cause mortality was not performed in EGPA patients. With these results, we conclude that the male sex could reduce significantly the cumulative patients' survival rates in MPA patients compared to GPA patients. However, in real clinical settings, since there are not a few cases where the differential diagnosis between MPA and GPA may be difficult, we intend to maintain the current title including AAV patients rather than MPA patients.\nIn this study, variables with P value less than 0.005 in the univariable Cox hazard model analysis were restrictively included in the multivariable analysis. However, since we cannot ignore the variables that were found to be associated with mortality in previous studies, we considered including these variables in multivariate analysis. Since BMI showed a ‘U shape’ contribution to the rate of all-cause mortality, BMI cannot be included in the multivariable analysis. Whereas, ANCA type was reported to be associated with the mortality rate or the infectious cause of death in AAV patients.13 Therefore, we consider including MPO-ANCA (or P-ANCA) and PR3-ANCA (or C-ANCA) in the multivariable Cox hazards model analysis, however, both of them exhibited too low statistical significance in the univariable analysis to be included in the multivariable analysis. On the other hand, CRP tended to be significantly associated with all-cause mortality during the follow-up period (P = 0.073) and furthermore, an index consisting of CRP and other variables was reported to be an independent predictor of all-cause mortality in AAV patients.14 Therefore, we conducted the multivariable Cox hazards model analysis by including CRP. However, similar to the results from Table 3, only male sex and FFS at diagnosis exhibited statically significant HR in the multivariable analysis, despite the addition of CRP. Therefore, we decided to maintain the results of Table 3.\nThe retrospective design might weaken the power of the clinical implication of the sex difference that our study provided. Also, the number of patients was not large enough to represent all Korean patients with AAV. However, this study might be valuable in that this is the first study which provided information regarding the differences in the clinical features and prognosis in the course of AAV between male and female AAV patients in Korea. Also, the limitation of the monocentric study may paradoxically minimise the inter-centric variation or bias, which could enhance the reliability of our study. Most deaths from septic shock or cancer were more frequent than death from persistent haemoptysis or damage to major organs such as the brain and heart. However, since there were many cases in which it was impossible to accurately classify the cause of their death, this study used the item of all-cause mortality without classifying death by cause, which was an additional limitation of this study. We believe that a prospective future study with a larger number of patients by recruiting the institutes, where the same inclusion criteria can be applied and the electronic medical records can be shared, will provide more reliable and valuable information.\nIn conclusion, male sex did not affect the clinical features at diagnosis, however, it increased the rate of all-cause mortality during the follow-up of AAV and was proved to be an independent predictor of all-cause mortality in AAV patients." ]

[ "intro", "methods", null, null, null, null, "results", null, null, null, null, "discussion" ]

[ "Sex", "Difference", "Antineutrophil Cytoplasmic Antibody Vasculitis", "Clinical Features", "Prognosis" ]

[ 263, 370, 155, 44, 216, 113, 151, 203 ]

[ "patients", "aav", "anca", "male", "mortality", "diagnosis", "cause", "female", "follow", "cause mortality" ]

[ "vasculitis definite", "ratio systemic vasculitis", "associated vasculitis aav", "systemic vasculitis sex", "vasculitis sex differences" ]

[ "Aged", "Aged, 80 and over", "Caregivers", "Diabetes Mellitus, Type 2", "Female", "Humans", "Hypoglycemic Agents", "Male", "Middle Aged", "Personal Satisfaction", "Psychometrics", "Quality of Life", "Reproducibility of Results", "Spain", "Surveys and Questionnaires" ]

[ "Patients and methods", "Preparation of the questionnaire", "Validation of the questionnaire", "Conducting the surveys", "Implementation of the questionnaire", "Conducting the surveys", "Statistical analysis", "Ethical considerations", "Questionnaire", "Description of the sample", "Validation of the current-status version of the questionnaire", "Validation of the change version of the questionnaire", "Administration of the current-status version of the questionnaire", "Administration of the change version of the questionnaire" ]

[ "This was an observational, descriptive, epidemiological study conducted in the Los Montalvos Internal Medicine Department at Complejo Asistencial Universitario de Salamanca (Spain). Caregivers of dependent type 2 diabetic patients were invited to take part when the patients were on drug treatment to control their blood glucose levels, had been going to the department for 12 consecutive months, and had been admitted for reasons other than metabolic decompensation of their diabetes mellitus. Cases were included successively in order of admission according to these selection criteria.\nFor the purposes of this study, patients were considered to be dependent if they scored 0 in the “responsibility for own medication” items on the Lawton and Brody scale.12 Caregivers were considered to be those involved in the care, support, and day-to-day looking after of the patient13 and responsible for administering medication to the patients (responsibility sometimes shared with other caregivers).\nThe patients were treated in line with the routine clinical criteria of the medical team whose care they were under. A change in therapy was considered to be any modification to the type of drug or the number of times the blood glucose-lowering medication was administered. A simple dose modification was not considered a change.\n Preparation of the questionnaire The research team, experienced in assessing satisfaction in a hospital setting, selected the dimensions they considered most relevant, and established the content for both versions of the questionnaire for assessing treatment satisfaction. The questions were then written with the characteristics of the target population (cultural level, degree of involvement, age, etc) in mind and the fact that the questionnaire would be administered in person-to-person interviews and interviews conducted over the telephone. Twelve questions were initially set, from which seven were selected by consensus. A pilot study was then carried out with ten participants to check whether or not the items were clear and easy to complete. Results of the pilot study indicated that questions 2, 4, and 5 of both versions needed to be rewritten (Tables 1 and 2). Additionally, a question regarding the caregiver degree of satisfaction with the patient’s blood glucose levels was included on the form.\nThe research team, experienced in assessing satisfaction in a hospital setting, selected the dimensions they considered most relevant, and established the content for both versions of the questionnaire for assessing treatment satisfaction. The questions were then written with the characteristics of the target population (cultural level, degree of involvement, age, etc) in mind and the fact that the questionnaire would be administered in person-to-person interviews and interviews conducted over the telephone. Twelve questions were initially set, from which seven were selected by consensus. A pilot study was then carried out with ten participants to check whether or not the items were clear and easy to complete. Results of the pilot study indicated that questions 2, 4, and 5 of both versions needed to be rewritten (Tables 1 and 2). Additionally, a question regarding the caregiver degree of satisfaction with the patient’s blood glucose levels was included on the form.\n Validation of the questionnaire Conducting the surveys The data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\n Statistical analysis The minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.\nThe minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.\n Conducting the surveys The data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\n Statistical analysis The minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.\nThe minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.\n Implementation of the questionnaire Conducting the surveys As stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\nAs stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\n Statistical analysis A descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.\nA descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.\n Conducting the surveys As stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\nAs stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\n Statistical analysis A descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.\nA descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.\n Ethical considerations Informed consent in writing was requested for participation in the study. The study was assessed and approved by the Complejo Asistencial Universitario de Salamanca Independent Ethics Committee.\nInformed consent in writing was requested for participation in the study. The study was assessed and approved by the Complejo Asistencial Universitario de Salamanca Independent Ethics Committee.", "The research team, experienced in assessing satisfaction in a hospital setting, selected the dimensions they considered most relevant, and established the content for both versions of the questionnaire for assessing treatment satisfaction. The questions were then written with the characteristics of the target population (cultural level, degree of involvement, age, etc) in mind and the fact that the questionnaire would be administered in person-to-person interviews and interviews conducted over the telephone. Twelve questions were initially set, from which seven were selected by consensus. A pilot study was then carried out with ten participants to check whether or not the items were clear and easy to complete. Results of the pilot study indicated that questions 2, 4, and 5 of both versions needed to be rewritten (Tables 1 and 2). Additionally, a question regarding the caregiver degree of satisfaction with the patient’s blood glucose levels was included on the form.", " Conducting the surveys The data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\n Statistical analysis The minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.\nThe minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.", "The data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.", " Conducting the surveys As stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\nAs stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\n Statistical analysis A descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.\nA descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.", "As stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.", "A descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.", "Informed consent in writing was requested for participation in the study. The study was assessed and approved by the Complejo Asistencial Universitario de Salamanca Independent Ethics Committee.", "Two versions of the questionnaire were created (Tables 1 and 2): the first (STCD2-a) to assess satisfaction with current treatment (“over the last few weeks”), and the second (STCD2-c) to assess satisfaction after a change in treatment (“over the last few months”). Both consist of seven questions with a five-response scale, from “Very satisfied” to “Very dissatisfied” in the first six items and from “I would definitely recommend it” to “I wouldn’t recommend it at all” in the last item. The first item refers to overall treatment satisfaction, the next five assess satisfaction in specific areas, and the final item concerns whether or not they would recommend the patient’s current treatment.", "A summary of the clinical and demographic data for the 219 patients included in the study and their caregivers is shown in Tables 3 and 4.", "The internal consistency of the STCD2-a was assessed in the entire sample (219 cases), with Cronbach’s α-coefficient being 0.93 (values above 0.7 are considered adequate).15 All the items correlated with each other (r=0.36–0.91, P<0.001). Test–retest reliability was analyzed in 88 cases interviewed by telephone after the hospital stay. The intraclass correlation coefficient was 0.96 (95% confidence interval [CI] 0.94–0.97, P<0.001).\nConstruct validity was demonstrated in the correlation matrix: all the items, as well as the overall satisfaction score, were correlated with the degree of satisfaction and with the blood glucose levels (r=0.40–0.86, P<0.001). All the items were correlated with the HbA1c figures (r=−0.24 to −0.35, P<0.001), except for satisfaction with their knowledge about diabetes (r=−0.07, P<0.05).\nIn the analysis of longitudinal validity, a significant correlation was demonstrated in the increase in the overall satisfaction index and the increased satisfaction with the blood glucose levels between the initial survey and the one carried out to assess the change (r=0.77, P<0.001). In four cases (1.8%) and 0 cases, respectively, extreme overall satisfaction scores were achieved (floor/ceiling effect).", "The validation of the change survey was carried out in 127 cases. Cronbach’s α-coefficient was 0.92. Correlation was demonstrated between all the items (r=0.33–0.79, P<0.001). In the test–retest reliability analysis, the intraclass correlation coefficient was 0.96 (95% CI 0.95–0.97, P<0.001).\nThe scores for all the questionnaire items and the overall satisfaction score were correlated with the satisfaction scores for the blood glucose levels (r=0.44–0.78, P<0.001). In eight cases (6.2%) and 0 cases, respectively, extreme scores were achieved (floor/ceiling effect).", "The STCD2-a questionnaire was administered to 219 caregivers. The average HbA1c levels of the patients they looked after were 7.4% (95% CI 7.2–7.5). A total of 101 (46.1%) cases were on insulin treatment, of which 14 (6.4%) were receiving treatment with rapid-acting insulin or analog, eleven (5.0%) with intermediate-acting insulin or analog, 24 (11.0%) with preloaded mixtures of rapid-acting insulin or analog plus intermediate-acting insulin or analog, and 69 (31.5%) with long-acting analog. A total of 141 (64.4%) were receiving an oral antidiabetic agent, and 23 (10.5%) cases insulin plus an oral antidiabetic agent.\nThe median number of daily administrations for the antidiabetic medication (insulin and/or oral antidiabetics) was two (one to six). The median caregivers’ satisfaction with the blood glucose levels was three (one to five), with a mean overall satisfaction of 24.8 points (95% CI 24.0–25.6). Caregivers were “very” or “somewhat” satisfied with what they knew about the treatment of diabetes in 32 (39.4%) cases. The caregivers with the highest scores for overall satisfaction were those who looked after the patients in a residential care setting (26.0, 95% CI 24.6–27.5), as opposed to those who did so at home (24.2, 95% CI 23.2–25.2; P<0.05). Among those caring at home, there was a correlation between the satisfaction score and the number of hours dedicated to caring for the patient (r=0.29, P<0.001). The caregivers who had no help at home (n=38) were more satisfied than those who received help (n=109) (26.4, 95% CI 22.3–24.5 versus 23.4, 95% CI 2.3–24.5; P<0.01), as did those who received payment for their work (n=8) than the informal caregivers (n=139) (28.3, 95% CI 25.6–30.9 versus 23.9, 95% CI 22.9–25.0; P<0.05).\nIn terms of the treatment administered, correlation was observed between the degree of satisfaction and the number of daily administrations of the blood glucose-lowering medication (r=−0.21, P<0.05).\nOverall satisfaction in each of the groups of global treatment is shown in Figure 1. Caregivers of patients treated with insulin plus an oral antidiabetic agent (n=23) had lower average satisfaction levels than the rest of the caregivers (n=196) (20.0, 95% CI 17.0–23.0 versus 25.3, 95% CI 24.5–26.1; P<0.001). This was also the case with caregivers of patients treated with rapid-acting insulin or analog (n=14) (20.6, 95% CI 16.5–24.8 versus 25.1, 95% CI 24.2–25.9; P<0.01) or intermediate-acting insulin or analog (n=11) (21.1, 95% CI 17.2–25.0 versus 25.0, 95% CI 24.1–25.8; P<0.05). On the other hand, caregivers of patients treated only with a long-acting insulin analog (n=52) had higher average satisfaction levels than the rest of the caregivers (n=167) (26.4, 95% CI 25.0–27.8 versus 24.3, 95% CI 23.3–25.2; P<0.01).", "The STCD2-c questionnaire was given to 127 caregivers of patients in whom the blood glucose-lowering treatment had been modified as described earlier. These caregivers had a lower average satisfaction score in the STCD2-c than the caregivers of patients in whom no treatment changes had been made (22.9, 95% CI 21.8–24.0 versus 27.3, 95% CI 26.3–28.3; P<0.001) and lower satisfaction scores for blood glucose levels (3.0, 95% CI 2.8–3.2 versus 3.7, 95% CI 3.5–3.9; P<0.001). There were no statistically significant differences between the two groups in HbA1c levels during the hospital stay.\nThe changes made to treatment were as follows: change in dosage, 39 cases (30.7%); switch from oral antidiabetic drug to insulin therapy, 25 cases (19.7%); discontinuation of all blood glucose-lowering medication, 19 cases (15%); change of oral antidiabetic drug, 16 cases (12.6%); change of type of insulin, 14 cases (11.0%); discontinuation of oral antidiabetic drug in an insulin-plus-antidiabetic regimen, 12 cases (9.4%); addition of a new insulin to the previous insulin regimen, one case (0.8%); and discontinuation of insulin therapy in an insulin-plus-antidiabetic regimen, one case (0.8%). Of the 40 cases in which insulin was added to the treatment, this was a long-acting insulin analog in 31 cases.\nThe median number of daily administrations for the anti-diabetic medication (insulin and/or oral antidiabetic) was one (zero to four). The degree of satisfaction with the change (the sum of scores for each of the items in the change questionnaire) was 27.3 (95% CI 26.4–28.2). Statistically significant differences in the questionnaire score were found based on the change in treatment between those with a change in the dosage and those with no dosage change (25.1, 95% CI 23.8–26.4 versus 28.3, 95% CI 27.1–29.5; P<0.01) and between those in whom all blood glucose-lowering medication was discontinued and all other changes (30.8, 95% CI 27.8–33.8 versus 26.6, 95% CI 25.7–27.5; P<0.01). Additionally, the cases in which a long-acting insulin analog was included in the new treatment had higher scores for the item referring to greater satisfaction with the patient’s blood glucose levels (4.6, 95% CI 4.3–4.9 versus 3.9, 95% CI 3.7–4.1; P<0.001) and for the item asking about greater satisfaction in terms of continuing with the treatment after the change (4.5, 95% CI 4.2–4.9 versus 4.0, 95% CI 3.8–4.2; P<0.01).\nCaregivers of patients receiving more frequent administration of their antidiabetic medication prior to the change were more satisfied with the change (r=0.24, P<0.001). Similarly, correlation was found between the number of daily administrations for blood glucose-lowering medication after the change and the degree of satisfaction (r=−0.43, P<0.001)." ]

[ "methods", null, null, "methods", null, "methods", "methods", null, null, null, null, null, null, null ]

[ "Introduction", "Patients and methods", "Preparation of the questionnaire", "Validation of the questionnaire", "Conducting the surveys", "Statistical analysis", "Implementation of the questionnaire", "Conducting the surveys", "Statistical analysis", "Ethical considerations", "Results", "Questionnaire", "Description of the sample", "Validation of the current-status version of the questionnaire", "Validation of the change version of the questionnaire", "Administration of the current-status version of the questionnaire", "Administration of the change version of the questionnaire", "Discussion" ]

[ "Satisfaction with treatment, knowledge about the disease, and assessment of the impact on the patient’s quality of life are all considered measurements for assessing results in clinical practice.1\nIn the case of diabetes mellitus, a number of different questionnaires have been designed to assess patient satisfaction with treatment. Some, like the Diabetes Treatment Satisfaction Questionnaire,2 are specific, while others, such as the Diabetes-Specific Quality-of-Life Scale3 and the Diabetes Quality of Life Clinical Trial Questionnaire,4 come under the category of quality-of-life assessments.\nHowever, when assessing satisfaction in dependent patients, the opinion of their carers becomes more important, since they are the ones administering the medication and it is they who will detect any side effects. Complex therapy regimens, insufficient or excessive treatments, poorly controlled disease, and other incidents can make the disease more difficult for caregivers to manage, requiring greater vigilance and increasing the caregivers’ workload. From this perspective, it is important to know how satisfied caregivers are with the treatments they administer.5\nSeveral studies have assessed caregiver/parent satisfaction with treatment in cases of children with type 1 diabetes.6,7 However, there is no such information available specifically for dependent type 2 diabetic patients. Nor have the characteristics which make up the caregiver profile in these cases been identified. Some studies point to the reduction in quality of life resulting from their workload,8,9 others report an increase in mental stress,10 while others emphasize the lack of specific training in relation to the burden of responsibility they take on.11\nIn this context, our objective was to develop, validate, and then administer two versions of a specific questionnaire to assess satisfaction with blood glucose-lowering treatment in caregivers of dependent type 2 diabetic patients. The first version (current-status version) was designed to assess current satisfaction with the treatment received, while the second (change version) was intended to assess satisfaction after introducing changes in blood glucose-lowering treatment.", "This was an observational, descriptive, epidemiological study conducted in the Los Montalvos Internal Medicine Department at Complejo Asistencial Universitario de Salamanca (Spain). Caregivers of dependent type 2 diabetic patients were invited to take part when the patients were on drug treatment to control their blood glucose levels, had been going to the department for 12 consecutive months, and had been admitted for reasons other than metabolic decompensation of their diabetes mellitus. Cases were included successively in order of admission according to these selection criteria.\nFor the purposes of this study, patients were considered to be dependent if they scored 0 in the “responsibility for own medication” items on the Lawton and Brody scale.12 Caregivers were considered to be those involved in the care, support, and day-to-day looking after of the patient13 and responsible for administering medication to the patients (responsibility sometimes shared with other caregivers).\nThe patients were treated in line with the routine clinical criteria of the medical team whose care they were under. A change in therapy was considered to be any modification to the type of drug or the number of times the blood glucose-lowering medication was administered. A simple dose modification was not considered a change.\n Preparation of the questionnaire The research team, experienced in assessing satisfaction in a hospital setting, selected the dimensions they considered most relevant, and established the content for both versions of the questionnaire for assessing treatment satisfaction. The questions were then written with the characteristics of the target population (cultural level, degree of involvement, age, etc) in mind and the fact that the questionnaire would be administered in person-to-person interviews and interviews conducted over the telephone. Twelve questions were initially set, from which seven were selected by consensus. A pilot study was then carried out with ten participants to check whether or not the items were clear and easy to complete. Results of the pilot study indicated that questions 2, 4, and 5 of both versions needed to be rewritten (Tables 1 and 2). Additionally, a question regarding the caregiver degree of satisfaction with the patient’s blood glucose levels was included on the form.\nThe research team, experienced in assessing satisfaction in a hospital setting, selected the dimensions they considered most relevant, and established the content for both versions of the questionnaire for assessing treatment satisfaction. The questions were then written with the characteristics of the target population (cultural level, degree of involvement, age, etc) in mind and the fact that the questionnaire would be administered in person-to-person interviews and interviews conducted over the telephone. Twelve questions were initially set, from which seven were selected by consensus. A pilot study was then carried out with ten participants to check whether or not the items were clear and easy to complete. Results of the pilot study indicated that questions 2, 4, and 5 of both versions needed to be rewritten (Tables 1 and 2). Additionally, a question regarding the caregiver degree of satisfaction with the patient’s blood glucose levels was included on the form.\n Validation of the questionnaire Conducting the surveys The data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\n Statistical analysis The minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.\nThe minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.\n Conducting the surveys The data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\n Statistical analysis The minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.\nThe minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.\n Implementation of the questionnaire Conducting the surveys As stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\nAs stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\n Statistical analysis A descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.\nA descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.\n Conducting the surveys As stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\nAs stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\n Statistical analysis A descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.\nA descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.\n Ethical considerations Informed consent in writing was requested for participation in the study. The study was assessed and approved by the Complejo Asistencial Universitario de Salamanca Independent Ethics Committee.\nInformed consent in writing was requested for participation in the study. The study was assessed and approved by the Complejo Asistencial Universitario de Salamanca Independent Ethics Committee.", "The research team, experienced in assessing satisfaction in a hospital setting, selected the dimensions they considered most relevant, and established the content for both versions of the questionnaire for assessing treatment satisfaction. The questions were then written with the characteristics of the target population (cultural level, degree of involvement, age, etc) in mind and the fact that the questionnaire would be administered in person-to-person interviews and interviews conducted over the telephone. Twelve questions were initially set, from which seven were selected by consensus. A pilot study was then carried out with ten participants to check whether or not the items were clear and easy to complete. Results of the pilot study indicated that questions 2, 4, and 5 of both versions needed to be rewritten (Tables 1 and 2). Additionally, a question regarding the caregiver degree of satisfaction with the patient’s blood glucose levels was included on the form.", " Conducting the surveys The data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.\n Statistical analysis The minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.\nThe minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.", "The data used to validate the current-status version of the questionnaire (Satisfacción con el Tratamiento, de los Cuidadores de pacientes Diabéticos Dependientes tipo 2 [versión actual] [STCD2-a]; [Treatment Satisfaction among Caregivers of Dependent Type 2 Diabetic Patients]) were obtained in person-to-person interviews with caregivers and in telephone interviews conducted 4 weeks later in which the questionnaire was repeated.\nThe data used to validate the change version of the questionnaire (STCD2-c) were obtained in telephone interviews carried out 8 weeks after discharge from hospital (in those cases where the blood glucose-lowering medication had been changed during hospital admission) and further telephone interviews 4 weeks later in which the questionnaire was repeated.", "The minimum number of participants necessary to validate each of the versions was determined using the Streiner and Norman methodology.14 A scoring system was established ranging from 5 points to 1 point for each of the questionnaire items (5= “Very satisfied”, 4= “Somewhat satisfied”, 3= “Neither satisfied nor dissatisfied”, 2= “Somewhat dissatisfied”, and 1= “Very dissatisfied”). An overall satisfaction index was defined as the sum of the scores for the seven items (minimum 7 points, maximum 35 points).\nThe internal consistency of the survey was calculated using Cronbach’s α-coefficient. Test–retest reliability was analyzed by calculating the intraclass correlation coefficient between the two administration times for each version of the questionnaire. Construct validity was investigated by estimating the Spearman correlation between the individual and overall scores for the questionnaires and the HbA1c figures and/or levels of satisfaction with the patient’s blood glucose levels. The longitudinal validity of the STCD2-a was analyzed by studying the Spearman correlation between the variations in overall satisfaction scores and those for satisfaction with the patient’s blood glucose levels at the time the STCD2-c was administered.", " Conducting the surveys As stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\nAs stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.\n Statistical analysis A descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.\nA descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.", "As stated earlier, the STCD2-a was administered to caregivers in a person-to-person interview conducted at the hospital during the time the patients were admitted. The STCD2-c was administered over the telephone 8 weeks after discharge from hospital in those cases in which the treatment had been modified in some way.\nDuring the hospital stay, clinical and epidemiological data were collected for the patient, along with demographic information about the caregiver. Both the initial questionnaires and those carried out after the change in medication (when applicable) were administered by members of the investigating medical team.", "A descriptive analysis was done of the epidemiological and clinical variables collected for the patients and their caregivers during the hospital stay. The Spearman test was used to study correlations. Quantitative variables (including the overall score for the questionnaires) were compared using Student’s t-statistic, while ordinal variables (scores for the individual items) were compared using the Mann–Whitney U-test in the case of independent samples or the Wilcoxon rank test for paired data. SPSS 15.0 for Windows statistics software was used. In all cases, a significance level of α=0.05 was established as the limit for statistical significance.", "Informed consent in writing was requested for participation in the study. The study was assessed and approved by the Complejo Asistencial Universitario de Salamanca Independent Ethics Committee.", " Questionnaire Two versions of the questionnaire were created (Tables 1 and 2): the first (STCD2-a) to assess satisfaction with current treatment (“over the last few weeks”), and the second (STCD2-c) to assess satisfaction after a change in treatment (“over the last few months”). Both consist of seven questions with a five-response scale, from “Very satisfied” to “Very dissatisfied” in the first six items and from “I would definitely recommend it” to “I wouldn’t recommend it at all” in the last item. The first item refers to overall treatment satisfaction, the next five assess satisfaction in specific areas, and the final item concerns whether or not they would recommend the patient’s current treatment.\nTwo versions of the questionnaire were created (Tables 1 and 2): the first (STCD2-a) to assess satisfaction with current treatment (“over the last few weeks”), and the second (STCD2-c) to assess satisfaction after a change in treatment (“over the last few months”). Both consist of seven questions with a five-response scale, from “Very satisfied” to “Very dissatisfied” in the first six items and from “I would definitely recommend it” to “I wouldn’t recommend it at all” in the last item. The first item refers to overall treatment satisfaction, the next five assess satisfaction in specific areas, and the final item concerns whether or not they would recommend the patient’s current treatment.\n Description of the sample A summary of the clinical and demographic data for the 219 patients included in the study and their caregivers is shown in Tables 3 and 4.\nA summary of the clinical and demographic data for the 219 patients included in the study and their caregivers is shown in Tables 3 and 4.\n Validation of the current-status version of the questionnaire The internal consistency of the STCD2-a was assessed in the entire sample (219 cases), with Cronbach’s α-coefficient being 0.93 (values above 0.7 are considered adequate).15 All the items correlated with each other (r=0.36–0.91, P<0.001). Test–retest reliability was analyzed in 88 cases interviewed by telephone after the hospital stay. The intraclass correlation coefficient was 0.96 (95% confidence interval [CI] 0.94–0.97, P<0.001).\nConstruct validity was demonstrated in the correlation matrix: all the items, as well as the overall satisfaction score, were correlated with the degree of satisfaction and with the blood glucose levels (r=0.40–0.86, P<0.001). All the items were correlated with the HbA1c figures (r=−0.24 to −0.35, P<0.001), except for satisfaction with their knowledge about diabetes (r=−0.07, P<0.05).\nIn the analysis of longitudinal validity, a significant correlation was demonstrated in the increase in the overall satisfaction index and the increased satisfaction with the blood glucose levels between the initial survey and the one carried out to assess the change (r=0.77, P<0.001). In four cases (1.8%) and 0 cases, respectively, extreme overall satisfaction scores were achieved (floor/ceiling effect).\nThe internal consistency of the STCD2-a was assessed in the entire sample (219 cases), with Cronbach’s α-coefficient being 0.93 (values above 0.7 are considered adequate).15 All the items correlated with each other (r=0.36–0.91, P<0.001). Test–retest reliability was analyzed in 88 cases interviewed by telephone after the hospital stay. The intraclass correlation coefficient was 0.96 (95% confidence interval [CI] 0.94–0.97, P<0.001).\nConstruct validity was demonstrated in the correlation matrix: all the items, as well as the overall satisfaction score, were correlated with the degree of satisfaction and with the blood glucose levels (r=0.40–0.86, P<0.001). All the items were correlated with the HbA1c figures (r=−0.24 to −0.35, P<0.001), except for satisfaction with their knowledge about diabetes (r=−0.07, P<0.05).\nIn the analysis of longitudinal validity, a significant correlation was demonstrated in the increase in the overall satisfaction index and the increased satisfaction with the blood glucose levels between the initial survey and the one carried out to assess the change (r=0.77, P<0.001). In four cases (1.8%) and 0 cases, respectively, extreme overall satisfaction scores were achieved (floor/ceiling effect).\n Validation of the change version of the questionnaire The validation of the change survey was carried out in 127 cases. Cronbach’s α-coefficient was 0.92. Correlation was demonstrated between all the items (r=0.33–0.79, P<0.001). In the test–retest reliability analysis, the intraclass correlation coefficient was 0.96 (95% CI 0.95–0.97, P<0.001).\nThe scores for all the questionnaire items and the overall satisfaction score were correlated with the satisfaction scores for the blood glucose levels (r=0.44–0.78, P<0.001). In eight cases (6.2%) and 0 cases, respectively, extreme scores were achieved (floor/ceiling effect).\nThe validation of the change survey was carried out in 127 cases. Cronbach’s α-coefficient was 0.92. Correlation was demonstrated between all the items (r=0.33–0.79, P<0.001). In the test–retest reliability analysis, the intraclass correlation coefficient was 0.96 (95% CI 0.95–0.97, P<0.001).\nThe scores for all the questionnaire items and the overall satisfaction score were correlated with the satisfaction scores for the blood glucose levels (r=0.44–0.78, P<0.001). In eight cases (6.2%) and 0 cases, respectively, extreme scores were achieved (floor/ceiling effect).\n Administration of the current-status version of the questionnaire The STCD2-a questionnaire was administered to 219 caregivers. The average HbA1c levels of the patients they looked after were 7.4% (95% CI 7.2–7.5). A total of 101 (46.1%) cases were on insulin treatment, of which 14 (6.4%) were receiving treatment with rapid-acting insulin or analog, eleven (5.0%) with intermediate-acting insulin or analog, 24 (11.0%) with preloaded mixtures of rapid-acting insulin or analog plus intermediate-acting insulin or analog, and 69 (31.5%) with long-acting analog. A total of 141 (64.4%) were receiving an oral antidiabetic agent, and 23 (10.5%) cases insulin plus an oral antidiabetic agent.\nThe median number of daily administrations for the antidiabetic medication (insulin and/or oral antidiabetics) was two (one to six). The median caregivers’ satisfaction with the blood glucose levels was three (one to five), with a mean overall satisfaction of 24.8 points (95% CI 24.0–25.6). Caregivers were “very” or “somewhat” satisfied with what they knew about the treatment of diabetes in 32 (39.4%) cases. The caregivers with the highest scores for overall satisfaction were those who looked after the patients in a residential care setting (26.0, 95% CI 24.6–27.5), as opposed to those who did so at home (24.2, 95% CI 23.2–25.2; P<0.05). Among those caring at home, there was a correlation between the satisfaction score and the number of hours dedicated to caring for the patient (r=0.29, P<0.001). The caregivers who had no help at home (n=38) were more satisfied than those who received help (n=109) (26.4, 95% CI 22.3–24.5 versus 23.4, 95% CI 2.3–24.5; P<0.01), as did those who received payment for their work (n=8) than the informal caregivers (n=139) (28.3, 95% CI 25.6–30.9 versus 23.9, 95% CI 22.9–25.0; P<0.05).\nIn terms of the treatment administered, correlation was observed between the degree of satisfaction and the number of daily administrations of the blood glucose-lowering medication (r=−0.21, P<0.05).\nOverall satisfaction in each of the groups of global treatment is shown in Figure 1. Caregivers of patients treated with insulin plus an oral antidiabetic agent (n=23) had lower average satisfaction levels than the rest of the caregivers (n=196) (20.0, 95% CI 17.0–23.0 versus 25.3, 95% CI 24.5–26.1; P<0.001). This was also the case with caregivers of patients treated with rapid-acting insulin or analog (n=14) (20.6, 95% CI 16.5–24.8 versus 25.1, 95% CI 24.2–25.9; P<0.01) or intermediate-acting insulin or analog (n=11) (21.1, 95% CI 17.2–25.0 versus 25.0, 95% CI 24.1–25.8; P<0.05). On the other hand, caregivers of patients treated only with a long-acting insulin analog (n=52) had higher average satisfaction levels than the rest of the caregivers (n=167) (26.4, 95% CI 25.0–27.8 versus 24.3, 95% CI 23.3–25.2; P<0.01).\nThe STCD2-a questionnaire was administered to 219 caregivers. The average HbA1c levels of the patients they looked after were 7.4% (95% CI 7.2–7.5). A total of 101 (46.1%) cases were on insulin treatment, of which 14 (6.4%) were receiving treatment with rapid-acting insulin or analog, eleven (5.0%) with intermediate-acting insulin or analog, 24 (11.0%) with preloaded mixtures of rapid-acting insulin or analog plus intermediate-acting insulin or analog, and 69 (31.5%) with long-acting analog. A total of 141 (64.4%) were receiving an oral antidiabetic agent, and 23 (10.5%) cases insulin plus an oral antidiabetic agent.\nThe median number of daily administrations for the antidiabetic medication (insulin and/or oral antidiabetics) was two (one to six). The median caregivers’ satisfaction with the blood glucose levels was three (one to five), with a mean overall satisfaction of 24.8 points (95% CI 24.0–25.6). Caregivers were “very” or “somewhat” satisfied with what they knew about the treatment of diabetes in 32 (39.4%) cases. The caregivers with the highest scores for overall satisfaction were those who looked after the patients in a residential care setting (26.0, 95% CI 24.6–27.5), as opposed to those who did so at home (24.2, 95% CI 23.2–25.2; P<0.05). Among those caring at home, there was a correlation between the satisfaction score and the number of hours dedicated to caring for the patient (r=0.29, P<0.001). The caregivers who had no help at home (n=38) were more satisfied than those who received help (n=109) (26.4, 95% CI 22.3–24.5 versus 23.4, 95% CI 2.3–24.5; P<0.01), as did those who received payment for their work (n=8) than the informal caregivers (n=139) (28.3, 95% CI 25.6–30.9 versus 23.9, 95% CI 22.9–25.0; P<0.05).\nIn terms of the treatment administered, correlation was observed between the degree of satisfaction and the number of daily administrations of the blood glucose-lowering medication (r=−0.21, P<0.05).\nOverall satisfaction in each of the groups of global treatment is shown in Figure 1. Caregivers of patients treated with insulin plus an oral antidiabetic agent (n=23) had lower average satisfaction levels than the rest of the caregivers (n=196) (20.0, 95% CI 17.0–23.0 versus 25.3, 95% CI 24.5–26.1; P<0.001). This was also the case with caregivers of patients treated with rapid-acting insulin or analog (n=14) (20.6, 95% CI 16.5–24.8 versus 25.1, 95% CI 24.2–25.9; P<0.01) or intermediate-acting insulin or analog (n=11) (21.1, 95% CI 17.2–25.0 versus 25.0, 95% CI 24.1–25.8; P<0.05). On the other hand, caregivers of patients treated only with a long-acting insulin analog (n=52) had higher average satisfaction levels than the rest of the caregivers (n=167) (26.4, 95% CI 25.0–27.8 versus 24.3, 95% CI 23.3–25.2; P<0.01).\n Administration of the change version of the questionnaire The STCD2-c questionnaire was given to 127 caregivers of patients in whom the blood glucose-lowering treatment had been modified as described earlier. These caregivers had a lower average satisfaction score in the STCD2-c than the caregivers of patients in whom no treatment changes had been made (22.9, 95% CI 21.8–24.0 versus 27.3, 95% CI 26.3–28.3; P<0.001) and lower satisfaction scores for blood glucose levels (3.0, 95% CI 2.8–3.2 versus 3.7, 95% CI 3.5–3.9; P<0.001). There were no statistically significant differences between the two groups in HbA1c levels during the hospital stay.\nThe changes made to treatment were as follows: change in dosage, 39 cases (30.7%); switch from oral antidiabetic drug to insulin therapy, 25 cases (19.7%); discontinuation of all blood glucose-lowering medication, 19 cases (15%); change of oral antidiabetic drug, 16 cases (12.6%); change of type of insulin, 14 cases (11.0%); discontinuation of oral antidiabetic drug in an insulin-plus-antidiabetic regimen, 12 cases (9.4%); addition of a new insulin to the previous insulin regimen, one case (0.8%); and discontinuation of insulin therapy in an insulin-plus-antidiabetic regimen, one case (0.8%). Of the 40 cases in which insulin was added to the treatment, this was a long-acting insulin analog in 31 cases.\nThe median number of daily administrations for the anti-diabetic medication (insulin and/or oral antidiabetic) was one (zero to four). The degree of satisfaction with the change (the sum of scores for each of the items in the change questionnaire) was 27.3 (95% CI 26.4–28.2). Statistically significant differences in the questionnaire score were found based on the change in treatment between those with a change in the dosage and those with no dosage change (25.1, 95% CI 23.8–26.4 versus 28.3, 95% CI 27.1–29.5; P<0.01) and between those in whom all blood glucose-lowering medication was discontinued and all other changes (30.8, 95% CI 27.8–33.8 versus 26.6, 95% CI 25.7–27.5; P<0.01). Additionally, the cases in which a long-acting insulin analog was included in the new treatment had higher scores for the item referring to greater satisfaction with the patient’s blood glucose levels (4.6, 95% CI 4.3–4.9 versus 3.9, 95% CI 3.7–4.1; P<0.001) and for the item asking about greater satisfaction in terms of continuing with the treatment after the change (4.5, 95% CI 4.2–4.9 versus 4.0, 95% CI 3.8–4.2; P<0.01).\nCaregivers of patients receiving more frequent administration of their antidiabetic medication prior to the change were more satisfied with the change (r=0.24, P<0.001). Similarly, correlation was found between the number of daily administrations for blood glucose-lowering medication after the change and the degree of satisfaction (r=−0.43, P<0.001).\nThe STCD2-c questionnaire was given to 127 caregivers of patients in whom the blood glucose-lowering treatment had been modified as described earlier. These caregivers had a lower average satisfaction score in the STCD2-c than the caregivers of patients in whom no treatment changes had been made (22.9, 95% CI 21.8–24.0 versus 27.3, 95% CI 26.3–28.3; P<0.001) and lower satisfaction scores for blood glucose levels (3.0, 95% CI 2.8–3.2 versus 3.7, 95% CI 3.5–3.9; P<0.001). There were no statistically significant differences between the two groups in HbA1c levels during the hospital stay.\nThe changes made to treatment were as follows: change in dosage, 39 cases (30.7%); switch from oral antidiabetic drug to insulin therapy, 25 cases (19.7%); discontinuation of all blood glucose-lowering medication, 19 cases (15%); change of oral antidiabetic drug, 16 cases (12.6%); change of type of insulin, 14 cases (11.0%); discontinuation of oral antidiabetic drug in an insulin-plus-antidiabetic regimen, 12 cases (9.4%); addition of a new insulin to the previous insulin regimen, one case (0.8%); and discontinuation of insulin therapy in an insulin-plus-antidiabetic regimen, one case (0.8%). Of the 40 cases in which insulin was added to the treatment, this was a long-acting insulin analog in 31 cases.\nThe median number of daily administrations for the anti-diabetic medication (insulin and/or oral antidiabetic) was one (zero to four). The degree of satisfaction with the change (the sum of scores for each of the items in the change questionnaire) was 27.3 (95% CI 26.4–28.2). Statistically significant differences in the questionnaire score were found based on the change in treatment between those with a change in the dosage and those with no dosage change (25.1, 95% CI 23.8–26.4 versus 28.3, 95% CI 27.1–29.5; P<0.01) and between those in whom all blood glucose-lowering medication was discontinued and all other changes (30.8, 95% CI 27.8–33.8 versus 26.6, 95% CI 25.7–27.5; P<0.01). Additionally, the cases in which a long-acting insulin analog was included in the new treatment had higher scores for the item referring to greater satisfaction with the patient’s blood glucose levels (4.6, 95% CI 4.3–4.9 versus 3.9, 95% CI 3.7–4.1; P<0.001) and for the item asking about greater satisfaction in terms of continuing with the treatment after the change (4.5, 95% CI 4.2–4.9 versus 4.0, 95% CI 3.8–4.2; P<0.01).\nCaregivers of patients receiving more frequent administration of their antidiabetic medication prior to the change were more satisfied with the change (r=0.24, P<0.001). Similarly, correlation was found between the number of daily administrations for blood glucose-lowering medication after the change and the degree of satisfaction (r=−0.43, P<0.001).", "Two versions of the questionnaire were created (Tables 1 and 2): the first (STCD2-a) to assess satisfaction with current treatment (“over the last few weeks”), and the second (STCD2-c) to assess satisfaction after a change in treatment (“over the last few months”). Both consist of seven questions with a five-response scale, from “Very satisfied” to “Very dissatisfied” in the first six items and from “I would definitely recommend it” to “I wouldn’t recommend it at all” in the last item. The first item refers to overall treatment satisfaction, the next five assess satisfaction in specific areas, and the final item concerns whether or not they would recommend the patient’s current treatment.", "A summary of the clinical and demographic data for the 219 patients included in the study and their caregivers is shown in Tables 3 and 4.", "The internal consistency of the STCD2-a was assessed in the entire sample (219 cases), with Cronbach’s α-coefficient being 0.93 (values above 0.7 are considered adequate).15 All the items correlated with each other (r=0.36–0.91, P<0.001). Test–retest reliability was analyzed in 88 cases interviewed by telephone after the hospital stay. The intraclass correlation coefficient was 0.96 (95% confidence interval [CI] 0.94–0.97, P<0.001).\nConstruct validity was demonstrated in the correlation matrix: all the items, as well as the overall satisfaction score, were correlated with the degree of satisfaction and with the blood glucose levels (r=0.40–0.86, P<0.001). All the items were correlated with the HbA1c figures (r=−0.24 to −0.35, P<0.001), except for satisfaction with their knowledge about diabetes (r=−0.07, P<0.05).\nIn the analysis of longitudinal validity, a significant correlation was demonstrated in the increase in the overall satisfaction index and the increased satisfaction with the blood glucose levels between the initial survey and the one carried out to assess the change (r=0.77, P<0.001). In four cases (1.8%) and 0 cases, respectively, extreme overall satisfaction scores were achieved (floor/ceiling effect).", "The validation of the change survey was carried out in 127 cases. Cronbach’s α-coefficient was 0.92. Correlation was demonstrated between all the items (r=0.33–0.79, P<0.001). In the test–retest reliability analysis, the intraclass correlation coefficient was 0.96 (95% CI 0.95–0.97, P<0.001).\nThe scores for all the questionnaire items and the overall satisfaction score were correlated with the satisfaction scores for the blood glucose levels (r=0.44–0.78, P<0.001). In eight cases (6.2%) and 0 cases, respectively, extreme scores were achieved (floor/ceiling effect).", "The STCD2-a questionnaire was administered to 219 caregivers. The average HbA1c levels of the patients they looked after were 7.4% (95% CI 7.2–7.5). A total of 101 (46.1%) cases were on insulin treatment, of which 14 (6.4%) were receiving treatment with rapid-acting insulin or analog, eleven (5.0%) with intermediate-acting insulin or analog, 24 (11.0%) with preloaded mixtures of rapid-acting insulin or analog plus intermediate-acting insulin or analog, and 69 (31.5%) with long-acting analog. A total of 141 (64.4%) were receiving an oral antidiabetic agent, and 23 (10.5%) cases insulin plus an oral antidiabetic agent.\nThe median number of daily administrations for the antidiabetic medication (insulin and/or oral antidiabetics) was two (one to six). The median caregivers’ satisfaction with the blood glucose levels was three (one to five), with a mean overall satisfaction of 24.8 points (95% CI 24.0–25.6). Caregivers were “very” or “somewhat” satisfied with what they knew about the treatment of diabetes in 32 (39.4%) cases. The caregivers with the highest scores for overall satisfaction were those who looked after the patients in a residential care setting (26.0, 95% CI 24.6–27.5), as opposed to those who did so at home (24.2, 95% CI 23.2–25.2; P<0.05). Among those caring at home, there was a correlation between the satisfaction score and the number of hours dedicated to caring for the patient (r=0.29, P<0.001). The caregivers who had no help at home (n=38) were more satisfied than those who received help (n=109) (26.4, 95% CI 22.3–24.5 versus 23.4, 95% CI 2.3–24.5; P<0.01), as did those who received payment for their work (n=8) than the informal caregivers (n=139) (28.3, 95% CI 25.6–30.9 versus 23.9, 95% CI 22.9–25.0; P<0.05).\nIn terms of the treatment administered, correlation was observed between the degree of satisfaction and the number of daily administrations of the blood glucose-lowering medication (r=−0.21, P<0.05).\nOverall satisfaction in each of the groups of global treatment is shown in Figure 1. Caregivers of patients treated with insulin plus an oral antidiabetic agent (n=23) had lower average satisfaction levels than the rest of the caregivers (n=196) (20.0, 95% CI 17.0–23.0 versus 25.3, 95% CI 24.5–26.1; P<0.001). This was also the case with caregivers of patients treated with rapid-acting insulin or analog (n=14) (20.6, 95% CI 16.5–24.8 versus 25.1, 95% CI 24.2–25.9; P<0.01) or intermediate-acting insulin or analog (n=11) (21.1, 95% CI 17.2–25.0 versus 25.0, 95% CI 24.1–25.8; P<0.05). On the other hand, caregivers of patients treated only with a long-acting insulin analog (n=52) had higher average satisfaction levels than the rest of the caregivers (n=167) (26.4, 95% CI 25.0–27.8 versus 24.3, 95% CI 23.3–25.2; P<0.01).", "The STCD2-c questionnaire was given to 127 caregivers of patients in whom the blood glucose-lowering treatment had been modified as described earlier. These caregivers had a lower average satisfaction score in the STCD2-c than the caregivers of patients in whom no treatment changes had been made (22.9, 95% CI 21.8–24.0 versus 27.3, 95% CI 26.3–28.3; P<0.001) and lower satisfaction scores for blood glucose levels (3.0, 95% CI 2.8–3.2 versus 3.7, 95% CI 3.5–3.9; P<0.001). There were no statistically significant differences between the two groups in HbA1c levels during the hospital stay.\nThe changes made to treatment were as follows: change in dosage, 39 cases (30.7%); switch from oral antidiabetic drug to insulin therapy, 25 cases (19.7%); discontinuation of all blood glucose-lowering medication, 19 cases (15%); change of oral antidiabetic drug, 16 cases (12.6%); change of type of insulin, 14 cases (11.0%); discontinuation of oral antidiabetic drug in an insulin-plus-antidiabetic regimen, 12 cases (9.4%); addition of a new insulin to the previous insulin regimen, one case (0.8%); and discontinuation of insulin therapy in an insulin-plus-antidiabetic regimen, one case (0.8%). Of the 40 cases in which insulin was added to the treatment, this was a long-acting insulin analog in 31 cases.\nThe median number of daily administrations for the anti-diabetic medication (insulin and/or oral antidiabetic) was one (zero to four). The degree of satisfaction with the change (the sum of scores for each of the items in the change questionnaire) was 27.3 (95% CI 26.4–28.2). Statistically significant differences in the questionnaire score were found based on the change in treatment between those with a change in the dosage and those with no dosage change (25.1, 95% CI 23.8–26.4 versus 28.3, 95% CI 27.1–29.5; P<0.01) and between those in whom all blood glucose-lowering medication was discontinued and all other changes (30.8, 95% CI 27.8–33.8 versus 26.6, 95% CI 25.7–27.5; P<0.01). Additionally, the cases in which a long-acting insulin analog was included in the new treatment had higher scores for the item referring to greater satisfaction with the patient’s blood glucose levels (4.6, 95% CI 4.3–4.9 versus 3.9, 95% CI 3.7–4.1; P<0.001) and for the item asking about greater satisfaction in terms of continuing with the treatment after the change (4.5, 95% CI 4.2–4.9 versus 4.0, 95% CI 3.8–4.2; P<0.01).\nCaregivers of patients receiving more frequent administration of their antidiabetic medication prior to the change were more satisfied with the change (r=0.24, P<0.001). Similarly, correlation was found between the number of daily administrations for blood glucose-lowering medication after the change and the degree of satisfaction (r=−0.43, P<0.001).", "This paper describes how the first questionnaires on satisfaction with blood glucose-lowering treatment for caregivers of dependent type 2 diabetic patients were designed, validated, and then implemented. The research team considers that the degree of satisfaction felt by caregivers for the different aspects of the blood glucose-lowering treatment they administer to patients should be considered when planning the treatment.16 Implicit in the satisfaction is the degree of caregiver involvement, adherence to treatment, and lastly better care of these patients.17\nSatisfaction with health care services is a complex concept that is related to a great variety of factors, such as way of life, previous experiences, expectations of the future, and values of the individual and of society.18 It is not surprising, therefore, that in our individual talks with the investigated caregivers, we were listening to different criteria to justify their degree of satisfaction with the treatment that they were administering to the patients. Some of these criteria (acceptance of the treatment by the patient, easiness of administration, number of doses) were specifically investigated in the tests. Other questions, such as the perception of the evolution of the disease, may have also had a variable weight (which is not quantified in this study) in the degree of expressed satisfaction of the surveyed caregivers.\nThe average age of the patients included in the study was over 84 years. Most patients came from their homes, not residential care, and there was a predominance of informal, unpaid caregivers (in our case, they were family caregivers, since there were no voluntary workers). These characteristics are similar to those in published studies on dependent patients,19 although this is often a result of the selection criteria applied. Some studies based on surveys of caregivers on different aspects exclude institutionalized patients, in order to avoid a positive bias (better training of the caregivers, various caregivers involved, easier access to information through residential care staff, etc).20 Other studies prefer informal caregivers when analyzing satisfaction aspects, because they have been shown to have a greater degree of involvement in the care of the patient and a greater degree of responsibility.9\nCaregivers who work in residences for the elderly have a greater degree of overall satisfaction than caregivers in the home setting, possibly because the residences have more suitable material and human resources at their disposal in terms of providing health care. The paid caregivers also had higher overall satisfaction scores than the informal caregivers. This has been observed in other studies on caregiver satisfaction and stress, with stress levels always lower in the paid workers.5 However, although informal caregivers generally had lower satisfaction scores, it was found that the more time they spent caring for the patient (often because there was no other caregiver to share the work), the higher their degree of satisfaction. This highlights a greater degree of involvement and dedication among informal caregivers.16 This major implication and satisfaction with the administration of treatment can be explained in the context of the traditional concept of the Spanish family, probably still firmly rooted in a significant number of caregivers in the study, in which the self-sacrificing care of the older ones on the part of their direct relatives is considered to be a social value and a privilege.\nIn terms of overall satisfaction, the most satisfied caregivers were those who had to administer medication less frequently. This greater degree of satisfaction also correlated with lower HbA1c levels. Consequently, in our study, caregiver satisfaction was associated with better metabolic control in the dependent diabetic patients, which has also been observed indirectly in other studies on satisfaction with diabetes treatment.3\nThe study was designed so that patients would be treated according to the routine clinical criteria of their internal medicine specialist. The treatment changes applied were not subject to any other norms. Taking the patient type into account (elderly, dependent, multiple medications, and multiple disorders), the objective of the treatment was not only to control HbA1c levels, but as in other studies conducted with elderly patients, it was also considered equally important to simplify the treatment, make administration easier, and above all prevent hypoglycemia.21\nThe mean scores for overall satisfaction and satisfaction with blood glucose levels in the STCD2-c were lower for the caregivers of patients whose treatment was changed than for the caregivers of patients in whom no treatment change was made. It would therefore seem that the caregivers and the physicians had similar criteria in terms of assessing the need for a change in treatment.\nComparison of the questionnaires showed that where changes were made, the degree of satisfaction increased, with the most appreciated change being discontinuation of all blood glucose-lowering medication. Significant differences were also observed in the increase in overall satisfaction as the number of medication administration times reduced after the change. This once more highlights the fact that simplifying the treatment is one of the most influential factors on satisfaction.22\nCaregivers of patients receiving only a long-acting insulin analog showed an overall satisfaction index score higher than those of the patients of the remaining therapeutic groups. Also, when introduced in the treatment change, once-a-day administration of long-acting insulin or analog made the caregivers more likely to feel satisfied with the blood glucose levels and to recommend the treatment to others. Similarly to other studies on satisfaction and diabetes, despite the fact that many of these patients changed from taking tablets to needing medication by injection, the good blood glucose control and the ease of administration were highly appreciated by the caregivers.23\nSeveral studies have addressed satisfaction with treatment in the case of the caregivers, eg, on parents of type 1 diabetic children in which special attention is paid in evaluating the perception that the parents have about the incidence and managing of hyper- and hypoglycemias.24 Nevertheless, to the best of our knowledge, there are no previous studies measuring satisfaction with treatment in caregivers of dependent type 2 diabetes patients in whom mainly the results of the application of quality-of-life questionnaires have been published.25 For this reason, we cannot compare our results with similar investigations in the field of diabetes.\nIn our opinion, knowing how satisfied caregivers of type 2 diabetic patients are with the treatment is an essential factor when planning the treatment. Having administered our innovative questionnaire to a group of Spanish patients, results suggest that the simplicity of the antidiabetic treatment must be taken into account when planning treatment for dependent type 2 diabetic patients when caregiver satisfaction is an additional objective. The validation and application of questionnaires of similar structure might be also useful in other therapeutic areas (eg, arterial hypertension, hyperlipidemias)." ]

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[ "caregivers type diabetic", "satisfaction knowledge diabetes", "diabetic patients caregiver", "diabetes specific quality", "studies satisfaction diabetes" ]