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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597194010.1371/journal.pmed.0020157Correspondence and Other CommunicationsOtherMedical EthicsClinical TrialsEthicsThe Debate over Placebo-Controlled Trials CorrespondenceMiller Frankllin National Institutes of HealthBethesda, MarylandUnited States of AmericaE-mail: [email protected]
Competing Interests: The author has declared that no competing interests exist.
6 2005 28 6 2005 2 6 e157Copyright: © Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Is It Always Unethical to Use a Placebo in a Clinical Trial?
Authors' Reply
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Turner and Tramèr provide a cogent argument in favor of the ethical use of placebo controls despite “proven effective treatment” [1]. However, they are wide of the mark citing the APPROVe trial in support of their position. Because there is no established treatment to prevent adenomatous polyps, few commentators would have any objections to the use of placebo controls in this study. Nevertheless, they are right to suggest that it would have been desirable to have included a placebo control in the VIGOR study to provide a more rigorous assessment of safety. Whether, all things considered, a placebo control would have been ethical in this study of treatment for rheumatoid arthritis is debatable.
Another issue not discussed in this PLoS Medicine Debate is the value of placebo controls in early “proof of concept” efficacy trials, despite the existence of established treatment. The efficiency of seeking a rigorous efficacy signal before moving on to larger-scale trials (and exposing as few subjects as possible to drugs that might not work or turn out to be toxic) is a valid ethical reason for using placebo controls, provided subjects are not exposed to undue risks of harm from withholding established treatment [2].
Citation: Miller F (2005) The debate over placebo-controlled trials. PLoS Med 2(6): e157.
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References
Stang A Hense H Jöckel K Turner EH Tramèr MR Is it always unethical to use a placebo in a clinical trial? PLoS Med 2005 2 e72 10.1371/journal.pmed.0020072 15783259
Emanuel EJ Miller FG The ethics of placebo-controlled trials—A middle ground N Engl J Med 2001 345 915 919 11565527
| 15971940 | PMC1160572 | CC BY | 2021-01-05 10:39:53 | no | PLoS Med. 2005 Jun 28; 2(6):e157 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020157 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597480510.1371/journal.pmed.0020160Research ArticleImmunologyEpidemiology/Public HealthGeneral MedicinePublic HealthSmokingImmunology and allergySmoking Cessation and Cardiovascular Disease Risk Factors: Results from the Third National Health and Nutrition Examination Survey Smoking and Cardiovascular Disease RiskBakhru Arvind
1
2
*Erlinger Thomas P.
3
4
5
1University of Rochester Medical Center, School of MedicineRochester, New YorkUnited States of America2Department of Epidemiology and Public Health, Yale University School of MedicineNew Haven, ConnecticutUnited States of America3Department of Epidemiology, Johns Hopkins University Bloomberg School of Public HealthBaltimore, MarylandUnited States of America4Department of Medicine, Johns Hopkins University School of MedicineBaltimore, MarylandUnited States of America5Welch Center for Prevention, Epidemiologyand Clinical Research, Johns Hopkins Medical Institutions, Baltimore, MarylandUnited States of AmericaLopez Alan D Academic EditorUniversity of QueenslandAustralia
Competing Interests: The authors have declared that no competing interests exist.
Author Contributions: AB and TPE designed the study. AB analyzed the data. AB and TPE contributed to writing the paper.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e16029 10 2004 13 4 2005 Copyright: © 2005 Bakhru and Erlinger.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Smoking and Inflammation
Background
Cigarette smoking is a major risk factor for the development and progression of cardiovascular disease. While smoking is associated with increased levels of inflammatory markers and accelerated atherosclerosis, few studies have examined the impact of smoking cessation on levels of inflammatory markers. The degree and rate at which inflammation subsides after smoking cessation are uncertain. It also remains unclear as to whether traditional risk factors can adequately explain the observed decline in cardiovascular risk following smoking cessation.
Methods and Findings
Using data from 15,489 individuals who participated in the Third National Health and Nutrition Examination Survey (NHANES III), we analyzed the association between smoking and smoking cessation on levels of inflammatory markers and traditional cardiovascular risk factors. In particular, we examined changes in C-reactive protein, white blood cell count, albumin, and fibrinogen. Inflammatory markers demonstrated a dose-dependent and temporal relationship to smoking and smoking cessation. Both inflammatory and traditional risk factors improved with decreased intensity of smoking. With increased time since smoking cessation, inflammatory markers resolved more slowly than traditional cardiovascular risk factors.
Conclusion
Inflammatory markers may be more accurate indicators of atherosclerotic disease. Inflammatory markers returned to baseline levels 5 y after smoking cessation, consistent with the time frame associated with cardiovascular risk reduction observed in both the MONICA and Northwick Park Heart studies. Our results suggest that the inflammatory component of cardiovascular disease resulting from smoking is reversible with reduced tobacco exposure and smoking cessation.
Analyzing inflammatory markers quantifies the cardiovascular risk of smoking, and suggests that inflammation induced by smoking is reversible, with markers returning to baseline by 5 years after cessation.
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Introduction
In addition to the role of smoking in cancer initiation and promotion, cigarette smoking accelerates atherogenic cardiovascular disease in both a dose- and a duration-dependent manner through several concurrent pathways. Smoking incites an immunologic response to vascular injury, described as oxidative stress leading to lipid peroxidation, endothelial cell dysfunction, and foam cell proliferation in the tunica media [1,2]. Smoking also enhances platelet aggregation, impairs lipoprotein metabolism, depresses high-density lipoprotein (HDL) cholesterol, and reduces distensibility of vessel walls [3,4].
Cigarette smoking is associated with increased levels of inflammatory markers. During the acute phase of inflammatory states, there are quantifiable increases in C-reactive protein, white blood cell count, and fibrinogen, and decreases in serum albumin [5–8]. Acute inflammatory markers have been shown to be both prognostic and predictive of future cardiovascular events in several populations. For example, C-reactive protein has been discussed extensively as a marker of cardiovascular disease risk [9–16].
C-reactive protein in particular is also increasingly being implicated in the pathogenesis of atherosclerosis [17–19]. C-reactive protein is a pentaxin, highly conserved across species [17,20,21] and stimulated synergistically by both IL-6 and IL-1β [22–25]. Previously believed to be synthesized by the liver, recent evidence suggests that C-reactive protein is also produced at the site of atherosclerosis by smooth muscle cells [17]. In the vessel wall, it induces expression of adhesion molecules on endothelial cells and increases monocyte chemotactic protein-1, which attracts monocytes and T cells into the vessel wall [26–28]. Additionally, C-reactive protein reduces endothelial nitric oxide synthase, upregulates the proatherosclerotic NF-κB pathway, enhances low-density lipoprotein (LDL) uptake by macrophages, which become foam cells, and facilitates T-cell- and complement-mediated destruction and apoptosis of the endothelium [2,26,29–31]. Thus, C-reactive protein is increasingly described not only as a marker of the inflammatory response, but also as a mediator in the pathogenesis of atherosclerotic cardiovascular disease [2,27]. Other inflammatory markers may also have causal properties, but are not as well understood. The inflammatory response therefore not only indicates atherosclerotic potential, but may accelerate atherosclerosis.
Several studies have described a prevalent inflammatory state in smokers. Despite the known impact of smoking on cardiovascular disease progression, few studies have examined the impact of smoking cessation on levels of inflammatory markers or on cardiovascular risk reduction [6,32]. The level to which the inflammatory response subsides following smoking cessation and the rate at which the inflammatory response subsides are uncertain. Furthermore, it remains unclear whether traditional risk factors can adequately explain the decline in cardiovascular risk following smoking cessation.
Using the Third National Health and Nutrition Examination Survey (NHANES III), a population-based representative sample of United States adults, we investigated the association between smoking and smoking cessation and levels of inflammatory markers and traditional cardiovascular risk factors. We examined the association between changes in the inflammatory markers—C-reactive protein, white blood cell count, albumin, and fibrinogen—and the traditional risk factors—total cholesterol, HDL cholesterol, triglycerides, systolic blood pressure, and diabetes—with decreased smoking intensity and increased time since smoking cessation. Our primary objective was to investigate changes in C-reactive protein in an effort to characterize the excess cardiovascular risk associated with smoking and any associated decline in risk with smoking cessation. We also aimed to characterize whether the inflammatory markers or traditional risk factors explained observed cardiac risk reduction following smoking cessation.
Methods
Study Population
The NHANES III survey and data collection procedures have been described in detail elsewhere [33,34]. Briefly, NHANES III was a national probability survey conducted between 1988 and 1994. This survey used a complex, multi-stage, stratified cluster sampling design to obtain a representative sample of the non-institutionalized civilian United States population. Participation included a home examination and a visit to a mobile examination center in one of 89 locations. For those unable to travel to a mobile examination center, a special home visit was arranged.
Of the 19,618 persons aged 18 and over included in the NHANES III population, we excluded 3,021 persons for whom C-reactive protein levels were missing, given that this was our primary marker of interest. Another 292 persons were excluded because they were pregnant, and 816 people were excluded because they reported cigar, pipe, snuff, or chewing tobacco use. Data for 15,489 persons were analyzed.
Measures
Self-reported race/ethnicity was categorized as non-Hispanic white, non-Hispanic black, Mexican-American, or other. The self-reported poverty to income ratio, a marker of socioeconomic status used in multiple prior studies [35,36], was classified as <1.5, 1.5–3, or >3.
Self-reported clinical factors were dichotomized for analysis. Participants reported on use of non-steroidal anti-inflammatory drugs, estrogen replacement therapy, and vitamin supplements. Use of any alcohol in the past 24 h was measured by dietary recall questionnaire. Prior physician-diagnosed angina, myocardial infarction, or stroke was defined as prevalent atherosclerotic cardiovascular disease (ASCVD). Prevalent inflammatory disease was defined as the presence of rheumatoid arthritis, asthma, emphysema, or chronic bronchitis. Presence of acute illness was indicated by a positive answer to the question: “In the past few days have you had a cough, cold, or other acute illness?”
Additional clinical factors were measured by a combination of participant self-report and clinical exam and were also dichotomized for analysis. Hypertension was indicated by use of anti-hypertensive medication, an average systolic blood pressure over 140 mm Hg, or an average diastolic blood pressure over 90 mm Hg, over four readings. Diabetes mellitus was considered present if the participant reported physician diagnosis of diabetes mellitus (excluding diagnosis during pregnancy), was taking diabetes medications, had fasting plasma glucose over 7 mmol/l (126 mg/dl), or had non-fasting glucose levels over 11.1 mmol/l (200 mg/dl). Finally, serum cholesterol and triglyceride levels were measured enzymatically (Hitachi 704 analyzer, Boehringer Mannheim, Mannheim, Germany). LDL was calculated using the Friedewald equation and measured fasting triglycerides, total cholesterol, and HDL cholesterol. LDL cholesterol and triglycerides were included as continuous variables and were logarithmically transformed to approximate the normal curve.
Smoking history was categorized based on both self-report and serum cotinine levels. Persons who gave a history of current smoking and/or had serum cotinine levels greater than 56.8 nmol/l (10 ng/ml) were considered current smokers. Current smokers were categorized into four roughly equal groups, based on number of cigarettes per day: 1–9, 10–19, 20–29, and over 30 cigarettes per day. Former smokers were defined by self-report as having smoked over 100 cigarettes in their lifetime and as having quit smoking. Former smokers were categorized by years since smoking cessation. Since the zero year since cessation point is subject to misclassification bias, the following levels were used in analyses: <1, 1–3, 3–5, 5–7, 7–9, and >9 ysince cessation. Never smokers were defined by self-report and as having serum cotinine levels under 56.8 nmol/l (<10 ng/ml). Passive smokers with cotinine levels over 56.8 nmol/l (10 ng/ml) were categorized as smokers at the lowest dose-intensity level.
Our main outcome variable, C-reactive protein, was measured using a latex-enhanced Behring Nephelometer Analyzer System (Dade Behring, Deerfield, Illinois, United States). Because the presence or absence, rather than the absolute level, of C-reactive protein appears to be associated with cardiovascular risk, we compared undetectable versus detectable levels of this marker. This is consistent with previous research, given the nonlinearity of C-reactive protein interpretation [37,38]. Since the limit of detection for the latex-enhanced nephelometry assay was 2.1 mg/l, we defined undetectable as less than 2.1 mg/l. Other outcome variables were treated continuously. White blood cell count was determined using a fully automated Coulter S-PLUS JR hematology analyzer (Beckman Coulter, Fullerton, California, United States). Albumin was measured using the bromocresol purple method. Fibrinogen was measured using a quantitative assay of clotting time compared to a known standard.
Statistical Methods
The distributions of traditional risk factors, inflammatory markers, and clinical and sociodemographic factors were examined by smoking status. Chi-square and analysis of variance tests, as appropriate, were performed to test for statistically significant differences. Variables identified as potential confounders by these bivariate analyses were included in multivariate models, as were the cofactors believed to be clinically relevant.
For multivariate analyses, our primary independent variables were smoking intensity and time since smoking cessation. Our primary dependent variables were the inflammatory markers—C-reactive protein, fibrinogen, white blood cell count, and serum albumin—and the traditional risk factors—systolic blood pressure, total cholesterol, triglycerides, HDL, LDL, diabetes, and alcohol use. Two sets of multivariate models were developed. The first set of analyses (minimally adjusted) were adjusted for age, sex, and race, given previously reported differences in the distribution of inflammatory markers in those groups. The second set of analyses (fully adjusted) were adjusted for all covariates and potential confounders: age, sex, race, poverty to income ratio, body mass index, prevalent cardiovascular disease, prevalent diabetes, prevalent chronic inflammatory condition, current acute illness, and use of alcohol, non-steroidal anti-inflammatory medication, aspirin, and estrogen replacement.
For inflammatory markers and traditional risk factors that were measured continuously, linear regressions were used to model the relationship to smoking intensity and time since smoking cessation. Least square means were calculated. C-reactive protein (a dichotomous variable) was modeled with logistic regression, using never smokers as the reference category. Odds ratios, however, do not approximate risk ratios, given that the prevalence of elevated C-reactive protein is over 10%.
The dose–response relationship between both smoking intensity and time since smoking cessation and each of the outcomes was assessed using Mantel tests for trend. Three sets of tests were performed, evaluating the trends: (1) among current smokers by cigarettes per day; (2) among former smokers by time since cessation; and (3) among smokers, former smokers, and non-smokers, despite the potential nonlinearity between groups. To further explain the relationship between smoking exposure and C-reactive protein, to increase the power to detect a trend, and to reduce residual confounding, C-reactive protein was categorized into three levels: under 2.1 mg/l (undetectable), 2.1–9.9 mg/l (mildly elevated), and ≥10.0 mg/l (clinically significant).
Lastly, blocked group comparisons were done using adjusted chi-square tests. Inflammatory and traditional risk factors were compared across current, former, and never smokers to validate findings from previous studies.
All analyses were weighted to represent the total civilian, non-institutionalized United States population; they are, therefore, population prevalence estimates. Analyses were conducted in SAS (version 8.02; SAS Institute, Cary, North Carolina, United States) and SUDAAN (version 9.0; Research Triangle Institute, Research Triangle Park, North, Carolina, United States), incorporating sampling weights to account for nonresponse and oversampling. All analyses were conducted using two-sided tests with alpha set to 0.05.
Results
Of the 15,489 persons in our sample, 7,665 were classified as never smokers, 3,459 were classified as former smokers, and 4,365 were classified as current smokers. The average time since smoking cessation for former smokers was 13 y. The average cotinine level for current smokers was 1,255 nmol/l (221 ng/ml); for both former and never smokers the average cotinine level was <3 nmol/l (<0.5 ng/ml). Weighted descriptive statistics are given in Table 1.
Table 1 Descriptive Statistics of the NHANES III Sample Weighted to Reflect the United States Population
aGeometric mean.CRP, C-reactive protein; NSAID, non-steroidal anti-inflammatory drug; SD, standard deviation; SE, standard error.
Bivariate analyses of smoking status showed that among the inflammatory risk factors, unadjusted C-reactive protein, white blood cell count, and fibrinogen were all significantly and positively associated with smoking status (p < 0.01). Albumin levels were similar across smoking categories. Among traditional risk factors, smokers had higher total cholesterol and triglycerides, but were otherwise younger, had lower body mass index, and had lower blood pressure, and a smaller proportion were diabetic. Former smokers were most likely to have prevalent atherosclerotic cardiovascular disease.
Bivariate analyses of the inflammatory markers showed that C-reactive protein was significantly associated with older age (p-trend < 0.01 by 10-y age group), female sex (p < 0.01), and black race (p < 0.01). Serum albumin and serum fibrinogen showed similarly significant associations. White cell count was associated with race (p < 0.01), but not with age or sex. Among traditional risk factors, analyses showed that total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and systolic blood pressure were each associated with age, sex, and race (all p < 0.01). Diabetes was associated with age and race only (p < 0.01). Alcohol use was associated with age and sex only (p < 0.01). All models, therefore, were adjusted for age, race, and sex.
Table 2 displays abatement of the inflammatory response with (1) reduced intensity of smoking and (2) increased time since smoking cessation, adjusted for age, sex, and race. C-reactive protein, white blood cell count, and fibrinogen were all positively associated with increased smoking intensity (p ≤ 0.01) and were all significantly negatively associated with time since smoking cessation (p < 0.05). Similarly, albumin showed a negative association with intensity of smoking (p ≤ 0.01), but no relationship after cessation. All acute phase reactant inflammatory markers had significant trends overall, from current to former to never smokers (p ≤ 0.01) (Figure 1).
Figure 1 Trends in Inflammatory Markers with Smoking Intensity and Time since Cessation Are Given, Adjusted for Age, Sex, and Race
Table 2 Levels of Inflammatory Markers by Smoking Status and Smoking Intensity
Changes in inflammatory markers with smoking intensity and time since smoking cessation, adjusted for age, sex, and race.
*Significantly different from referent value.
LS mean, least square mean.
Differences in the traditional cardiovascular risk factors are shown in Table 3, adjusted for age, sex, and race. Total and HDL cholesterol, triglycerides, alcohol usage, and systolic blood pressure all showed a dose-dependent association with smoking intensity (p < 0.05). Triglycerides and alcohol use showed time-dependent associations with smoking cessation (p ≤ 0.01). Overall trends (from current to former to never smokers) were present for alcohol use, triglycerides, total cholesterol, and HDL cholesterol, as shown in Figure 2. In models fully adjusted for all covariates (not shown), dose-dependent associations persisted for HDL cholesterol and triglycerides (p ≤ 0.01). Both total cholesterol and triglycerides showed an association with time since smoking cessation (p = 0.03 for both) and an overall trend from current to never smokers in fully adjusted models.
Figure 2 Trends in Traditional Risk Factors with Smoking Intensity and Time since Cessation Are Given, Adjusted for Age, Sex, and Race
Table 3 Physiologic Cardiovascular Measures by Smoking Status and Smoking Intensity
Changes in traditional cardiovascular risk factors with smoking intensity and time since smoking cessation, adjusted for age, sex, and race.
aLog base ten transformation.
*Significantly different from referent value.
LS mean, least square mean.
Lastly, the inflammatory markers were examined in fully adjusted models. C-reactive protein continued to show abatement of the acute phase response with reduced smoking intensity and increased time since cessation despite adjustment for covariates (Table 4). White blood cell count and albumin were associated with smoking intensity but not with time since cessation. All positive acute phase reactants showed an associated decline from current to former to never smokers in fully adjusted models (p ≤ 0.01); albumin, likewise showed a significant increase (p-trend ≤ 0.01).
Table 4 Levels of Inflammatory Markers Fully Adjusted for All Covariates
*Significantly different from referent value.
LS mean, least square mean.
Analyses using pack-years were also done (not shown); the results were unchanged using this measure. Additionally, data were reanalyzed excluding those individuals with prevalent cardiovascular disease (not shown); results were again unchanged and measures of association were even slightly stronger. Overall, we observed the following associations: (1) improvement in both inflammatory markers and traditional risk factors with decreased intensity of smoking, and (2) improvement in inflammatory markers with increased time since smoking cessation.
Discussion
In this cross-sectional population-based study, we were able to demonstrate dose effects from smoking and temporal effects from cigarette smoking cessation. The NHANES III dataset allowed us to utilize both self-reported smoking and serum cotinine levels to minimize misclassification error. It also allowed us to control for a wide range of confounders and assess consistency across measures by analyzing four acute phase proteins and several traditional risk factors. By examining both traditional risk factors and inflammatory markers of cardiovascular risk, this study contributes to the literature by providing a more complete analysis with regard to the effects of smoking and smoking cessation.
It is known that after smokers give up smoking, their risk of mortality and future cardiac events declines [32,39], but there is little data quantifying the rate of risk reduction and when, or even whether, cardiovascular risk for former smokers reaches that of never smokers [12,40,41]. We found that the smoking-associated inflammatory response subsides within 5 y after smoking cessation. This suggests that the vascular effects are reversible and that cardiovascular risk subsides gradually with reduced exposure. The time to risk factor “correction” was longest with C-reactive protein; only one traditional risk factor—triglycerides—showed a similar pattern. Our findings are consistent with prior studies, suggesting that the full benefit of smoking cessation may be achieved gradually [39,42,43].
Our estimates of the time to risk factor “correction” are shorter than those for the decline in mortality risk and risk for specific cardiac events reported in some prospective studies [42,44], but on par with those in other prospective [45], case-control [46,47], and cross-sectional [48] studies. Discrepancies may be due to smoking recidivism in prospective studies, where former smokers recommence smoking and increase their risk, such that the reported time since cessation is longer than the true time, or due to a lag between changes in cardiovascular risk factors and regression of disease. Additionally, one would expect morbidity risk to subside first, since mortality is a more distant endpoint. Prior studies also did not control for known cardiovascular risk factors, did not use serum cotinine levels, did not examine the associated change in inflammatory markers post-cessation, or grouped participants into broad categories of current, former, and never smokers, increasing residual confounding while failing to explore the relative effects within each group [9,12,49]. Nevertheless, it remains unclear whether C-reactive protein's ongoing decline in former smokers simply indicates a prevalent underlying burden of atherosclerotic plaque, or “simmering” ongoing disease processes. The direct associations of C-reactive protein with mortality decrement seen in these other studies—and the dose–response seen in our own study—suggest the latter.
Three studies have systematically examined inflammatory markers following smoking: the MONICA Study (Monitoring Trends and Determinants in Cardiovascular Disease) [46], the Cardiovascular Health Study (CHS) [48], and the Northwick Park Heart Study [45]. The Cardiovascular Health Study found no association between inflammatory markers and smoking status itself, although C-reactive protein was strongly related to lifetime smoking exposure as measured by pack-years. Our results, while based on more complete modeling, support findings from the Northwick Park Heart Study and MONICA Study, both of which found that fibrinogen levels (adjusted only for age) reached normal levels within 5 y.
Our study supports the hypothesis that cardiovascular risk falls with inflammatory abatement, and that inflammatory markers are better indicators of such risk reduction. In this analysis, triglycerides showed the strongest dose-intensity and temporal trends of the traditional cardiovascular risk factors. This is both consistent with prior research and not surprising since the strongest association between C-reactive protein and lipid measures is for triglycerides. Despite the colinearity, the inflammatory markers studied appear to have a much clearer trend and longer lasting effect after smoking cessation than traditional risk factors. This would suggest their greater utility in being more accurate markers of disease, particularly given C-reactive protein's increasingly apparent role in the pathogenesis of atherosclerosis. Alternative explanations include (1) the presence of multiple independent causal pathways leading to cardiovascular disease and (2) traditional risk factors correlating more closely with mortality than morbidity or underlying pathophysiology. The relative value of novel inflammatory markers versus traditional risk factors remains of much debate [50,51].
Limitations of this study include measurement error from self-reporting, residual confounding from use of indicator variables, smoking recidivism, lack of newer measures such as interleukin-6 and high-sensitivity C-reactive protein [52], and lack of data on secondhand smoke. Recent studies have suggested that toxin exposures from secondhand smoke may impact the biomarkers used in this study [53,54]. We attempted to adjust for bias in self-reporting and secondhand exposure by using serum cotinine levels, but residual confounding may exist. It has also been suggested that circulating C-reactive protein levels may not reflect all relevant inflammatory effectors, owing to post-transcriptional regulation of C-reactive protein [22]. We chose C-reactive protein, however, for its clear role in the inflammatory response and for its increasing clinical relevance. Lastly, it is noteworthy that we are not using a linear scale, but measures of both intensity and duration. Time since cessation and cigarettes per day are distinctly different measures, and a discontinuity arises at cessation.
Further research should explore the acute phase response in the months following smoking cessation [53,55–57]. Time intervals of less than a year since cessation have not been assessed adequately in other studies nor in this study because of sample size limitations of the NHANES III data. Additionally, if cardiovascular risk subsides gradually upon smoking cessation, do vessel wall damage and pro-aggregatory cascades all completely reverse course upon cessation? Future research should continue to explore the relationship between the inflammatory cascade and alterations in the vessel wall [7,58].
This and other similar studies suggest that smoking cessation should play a larger role in public policy. Linked to poverty, decreased productivity, and premature death, tobacco remains the second major cause of preventable death in the world and fourth most common risk factor for disease worldwide [59]. While tobacco is clearly a worldwide concern, states and localities are left responsible for addressing tobacco use and smoking in a comprehensive manner. Our results suggest that policy-makers faced with escalating health-care costs should look to smoking cessation as an opportunity to achieve both long- and short-term health-care cost savings through cardiovascular risk reduction [60]. If cardiovascular benefits are, in fact, readily attainable, there exists an opportunity for short-term gain from smoking cessation. This may be sufficient to induce policy change. Larger scale action is also needed. Arguments persist in the developing world regarding the forced opening of tobacco markets as a result of the Global Agreements on Tariffs and Trade. Guidance may be forthcoming, however, from the World Health Organization Framework Convention on Tobacco Control, which began operation in February 2005 as an international treaty aimed at controlling tobacco packaging, marketing, and use [61]. Over 60 nation-states are currently parties to the Framework Convention on Tobacco Control, including many European nations and Japan, although the United States has yet to approve this treaty. Opportunities for tobacco control remain available, and it is becoming increasingly clear that smoking cessation models deserve high priority both in research and in any preventive health care system.
Patient Summary
Background
Smoking is associated with an increased chance of getting cardiovascular abnormalities, such as atherosclerosis, which can lead to heart attacks and other illnesses. One of the ways in which smoking causes damage is by causing inflammation; this inflammation can be measured by looking at the levels of various substances (such as C-reactive protein, white blood cells, albumin, and fibrinogen) in the blood. Stopping smoking decreases the chances of developing cardiovascular disease, and this decreased risk may be related to the levels of the inflammatory markers in the blood.
What Did the Researchers Do?
They compared the levels of the markers of inflammation measured in the blood of around 15,000 people who had taken part in a National lifestyle study in the United States with information about whether these people had ever smoked and whether they smoked currently.
The researchers found that the markers of inflammation were lowest in those people who had never smoked, and decreased as the time since stopping smoking increased. It took five years after stopping smoking for the inflammatory makers to return to normal.
What Do These Findings Mean?
Smoking is the second most important cause of death worldwide, and one of the main ways it causes death is through cardiovascular disease. This work confirms that the risk of cardiovascular disease does decrease after stopping smoking, and is marked by changes in the levels of inflammatory markers. Encouraging people to give up smoking is very important to reduce the amount of cardiovascular disease caused by smoking, but the markers of inflammation, and hence the risk of getting cardiovascular disease, may take several years to return to normal.
Where Can I Find More Information?
MedlinePlus has a Web page on smoking and other related health issues: http://www.nlm.nih.gov/medlineplus/smoking.html
The World Health Organization has a section on tobacco control: http://www.who.int/tobacco/en/
Citation: Bakhru A, Erlinger TP (2005) Smoking cessation and cardiovascular disease risk factors: Results from the Third National Health and Nutrition Examination survey. PLoS Med 2(6): e160.
Abbreviations
HDLhigh-density lipoprotein
LDLlow-density lipoprotein
NHANES IIIThird National Health and Nutrition Examination Survey
==== Refs
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| 15974805 | PMC1160573 | CC BY | 2021-01-05 11:13:38 | no | PLoS Med. 2005 Jun 28; 2(6):e160 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020160 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597194110.1371/journal.pmed.0020161Research ArticleBiotechnologyDevelopmentPathologyDerivation of Multipotent Mesenchymal Precursors from Human Embryonic Stem Cells Human ES Cell-Derived Mesenchymal PrecursorsBarberi Tiziano
1
Willis Lucy M
1
Socci Nicholas D
2
Studer Lorenz
1
*1Laboratory of Stem Cell and Tumor Biology, Division of Neurosurgery and Developmental Biology ProgramSloan-Kettering Institute, New York, New YorkUnited States of America2Computational Biology Center, Sloan-Kettering InstituteNew York, New YorkUnited States of AmericaTemple Sally Academic EditorAlbany Medical CollegeUnited States of America
Competing Interests: The authors have declared that no competing interests exist.
Author Contributions: TB and LS designed the study. TB and LMW performed the experiments. TB, LMW, NDS, and LS analyzed the data. TB, NDS, and LS contributed to writing the paper.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e16113 10 2004 15 4 2005 Copyright: © 2005 Barberi et al.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Turning Stem Cells into Mesenchymal Tissues
Background
Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors.
Methods and Findings
Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells.
Conclusion
Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications.
Lorenz Studer and colleagues describe the use of embryonic stem cells to derive mesenchymal precursors and then fat, cartilage, bone, and skeletal muscle cells.
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Introduction
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the blastocyst that can be maintained in culture for an extended period of time without losing differentiation potential. The successful isolation of human ES cells (hESCs) has raised the hope that these cells may provide a universal tissue source to treat many human diseases. However, directed differentiation of hESCs into specific tissue types poses a formidable challenge. Protocols are currently available for only a few cell types, mostly of neural identity [1–3], and differentiation into many of the cell types derived from the paraxial mesoderm has not been reported, with the exception of a recent study indicating osteoblastic differentiation [4]. Mesenchymal stem cells (MSCs) have been isolated from the adult bone marrow [5], adipose tissue [6], and dermis and other connective tissues [7]. Harvesting MSCs from any of these sources requires invasive procedures and the availability of a suitable donor. The number of MSCs that can be obtained from a single donor is limited, and the capacity of these cells for long-term proliferation is rather poor. In contrast, hESCs could provide an unlimited number of specialized cells. In this study, we present techniques for the generation and purification of mesenchymal precursors from hESCs and their directed differentiation in vitro into various mesenchymal derivatives, including skeletal myoblasts. Our isolation method for mesenchymal precursors is the first example, to our knowledge, of efficiently deriving structures of the paraxial mesoderm from ES cells, and further highlights the potential of hESCs for basic biology and regenerative medicine.
Methods
Cell Culture and FACS
Undifferentiated hESCs, H1 (WA-01, XY, passages 40–65) and H9 (WA-09, XX, passages 35–45), were cultured on mitotically inactivated mouse embryonic fibroblasts (Specialty Media, Phillipsburg, New Jersey, United States) and maintained under growth conditions and passaging techniques described previously [3]. OP9 cells were maintained in alpha MEM medium containing 20% fetal bovine serum (FBS) and 2 mM L-glutamine. Mesenchymal differentiation was induced by plating 10 × 103 to 25 × 103 cells/cm2 on a monolayer of OP9 cells in the presence of 20% heat-inactivated FBS in alpha MEM medium. Flow-activated cell sorting (FACS) (CD73-PE; PharMingen, San Diego, California, United States) was performed on a MoFlo (Cytomation, Fort Collins, Colorado, United States). All human ES cell–derived mesenchymal precursor cell (hESMPC) lines in this study are of polyclonal origin. Primary human bone marrow–derived MSCs and primary human foreskin fibroblasts (both from Poietics, Cambrex, East Rutherford, New Jersey, United States) were grown in alpha MEM medium containing 10% FBS and 2 mM L-glutamine.
Adipocytic Differentiation
hESMPCs are grown to confluence followed by exposure to 1 mM dexamethasone, 10 μg/ml insulin, and 0.5 mM isobutylxanthine (all from Sigma, St. Louis, Missouri, United States) in alpha MEM medium containing 10% FBS for 2–4 wk. Data were confirmed in hESMPC-H1.1, -H1.2, -H1.3, and -H9.1 (hESMPC-H1.4 was not tested).
Chondrocytic Differentiation
Differentiation of hESMPCs was induced in pellet culture [5] by exposure to 10 ng/ml TGF-β3 (R & D Systems, Minneapolis, Minnesota, United States) and 200 μM ascorbic acid (Sigma) in alpha MEM medium containing 10% FBS for 3–4 wk. Data were confirmed in hESMPC-H1.1, -H1.3, and -H9.1 (hESMPC-H1.2 and -H1.4 were not tested).
Osteogenic Differentiation
hESMPCs were plated at low density (1 × 103 to 2.5 × 103 cells/cm2) on tissue-culture-treated dishes in the presence of 10 mM β-glycerol phosphate (Sigma), 0.1 μM dexamethasone, and 200 μM ascorbic acid in alpha MEM medium containing 10% FBS for 3–4 wk. Data were confirmed in hESMPC-H1.1, -H1.3, and -H9.1 (hESMPC-H1.2 and -H1.4 were not tested).
Myogenic Differentiation
Confluent hESMPCs were maintained for 2–3 wk in alpha MEM medium with 20% heat-inactivated FBS. More rapid induction was observed in the presence of medium conditioned for 24 h by differentiated C2C12 cells. Coculture of hESMPCs and C2C12 cells was carried out in alpha MEM with 3% horse serum and 1% FBS [8]. Data were confirmed in hESMPC-H1.3, -H1.4, and -H9.1 (hESMPC-H1.1 and -H1.2 were not tested).
Cytochemistry
Immunocytochemistry for all surface markers was performed on live cells. Monoclonal antibodies VCAM, STRO-1, ICAM-1(CD54), CD105, CD29, and MF20 were from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, Iowa, United States); CD73, CD44, and ALCAM(CD166) were from BD Biosciences Pharmingen (San Diego, California, United States). All other immunocytochemical analyses were performed after fixation in 4% paraformaldehyde and 0.15% picric acid, followed by permeabilization in 0.3% Triton X100. Polyclonal antibodies used were MyoD (Santa Cruz Biotechnology, Santa Cruz, California, United States) and nestin (gift from R. McKay); monoclonal antibodies were vimentin, alpha smooth muscle actin, fast-switch myosin, pan-cytokeratin (all from Sigma), and human nuclear antigen (Chemicon, Temecula, California, United States).
Alkaline phosphatase reaction was performed using a commercially available kit (Kit-86; Sigma) and the mineral was stained with silver nitrate according to the von Kossa method. Fat granules were visualized by Oil Red O staining solution (Sigma). Alcian Blue (Sigma) was used to detect extracellular matrix proteoglycans in chondrogenic cultures.
Gene-Expression Analyses
RT-PCR analysis
Total RNA was extracted by using the RNeasy kit and DNase I treatment (Qiagen, Valencia, California, United States). Total RNA (2 μg each) was reverse transcribed (SuperScript; Invitrogen, Carlsbad, California, United States). PCR conditions were optimized and linear amplification range was determined for each primer by varying annealing temperature and cycle number. PCR products were identified by size, and identity was confirmed by DNA sequencing. Primer sequences, cycle numbers, and annealing temperatures are provided in Table S1.
Affymetrix analysis
Total RNA (5 μg) from primary MSCs, from hESMPC-H9.1, hESMPC-H1.2, and three samples of undifferentiated hESCs (H1; passages 42–46), were processed by the Memorial Sloan-Kettering Cancer Center Genomics Core Facility and hybridized on Affymetrix (Santa Clara, California, United States) U133A human oligonucleotide arrays. Data were analyzed using MAS5.0 (Affymetrix) software. Transcripts selectively expressed in each of the mesenchymal cell populations (MSC, hESMPC-H9.1, and hESMPC-H1.2) were defined as those called “increased” by the MAS5.0 algorithm in each of three comparisons with independent samples of undifferentiated hESCs. A Venn diagram was generated to visualize overlap in gene expression. Further statistical analyses were performed as described below.
Results
Mesenchymal differentiation of hESCs (lines H1 [WA-01] and H9 [WA-09]) [9] was induced by plating undifferentiated hESCs on a monolayer of murine OP9 stromal cells [10], in the presence of 20% heat-inactivated FBS in alpha MEM medium. OP9 cells have been previously shown to induce blood cell differentiation from mouse ES cells [11]. After 40 d of coculture, cells were harvested and sorted by FACS for CD73, a surface marker expressed in adult MSCs [5] (Figure 1A). An average of 5% CD73+ cells was obtained from the mixed culture of OP9 and differentiated hESC progeny. CD73+ cells were replated in the absence of stromal feeders on tissue culture plates and expanded in alpha MEM medium with 20% FBS for 7–14 d. We next established the membrane antigen profile of the resulting population of flat spindle-like cells. The H1- and H9-derived CD73+ cells expressed a comprehensive set of markers that are considered to define adult MSCs, including CD105(SH2), STRO-1, VCAM (CD106), CD29(integrin β1), CD44, ICAM -1(CD54), ALCAM(CD166), vimentin, and alpha smooth muscle actin (Figure 1B and 1C). The cells were negative for hematopoietic markers such as CD34, CD45, and CD14. They were also negative for neuroectodermal, epithelial, and muscle cell markers including nestin, pancytokeratin, and desmin (data not shown). The human identity of these presumed mesenchymal cells (termed hESMPC-H1.1, -H1.2, -H1.3, -H1.4, and -H9.1) was confirmed for all experiments by immunocytochemistry for human nuclear antigen to rule out the possibility of contamination with OP9 cells (Figure S1).
Figure 1 Isolation and Characterization of hESMPCs
(A) FACS (MoFlo, Cytomation) for the isolation of CD73+ precursors (right) and isotype control (left).
(B) Flow cytometry analysis of the CD73+ hESMPC population for various markers characteristic of MSCs, including CD44, CD73, CD105, CD166, VCAM, ICAM-1, CD29, and STRO-1.
(C) Immunocytochemistry of hESMPCs for MSC markers (VCAM, STRO-1, CD73, and CD105). The cells also express vimentin and alpha smooth muscle actin. Scale bar = 50 μm.
(D) Venn diagram presenting the overlap among transcripts selectively expressed in hESMPC-H1.2, hESMPC-H9.1, and primary adult human MSCs.
To further characterize hESMPCs, we performed genome-wide expression analysis using oligonucleotide arrays (Affymetrix U133A). The expression profiles of hESMPC-H1.2 and hESMPC-H9.1 were compared with that of human primary adult MSCs. Housekeeping genes for each of the mesenchymal cell populations were eliminated by subtracting those transcripts also expressed in at least one of three independent samples of undifferentiated hESCs. Based on this analysis, 1,280 transcripts were selectively expressed in hESMPC-H1.2, 932 transcripts in hESMPC-H9.1, and 1,218 transcripts in primary adult MSCs. A remarkable overlap of 579 transcripts shared among the three mesenchymal populations was observed (Figure 1D). Using the genes that were selected in the initial filter, we performed a statistical analysis on the expression levels to determine whether the genes were expressed significantly differently in the two cell types. We used a Bayesian extension to the standard t-test [12] to assess this difference. Of the 579 genes, 412 of them were significantly different, at a false discovery rate cutoff of 0.05. The relative fold changes were also extremely large in many of the cases. We also looked at the variance of the expression levels within the cell types. For the MSCs, 94% had a coefficient of variation less than 20% for the expression (log transformed); for the ES-derived cells, 72% had a coefficient of variation less than 20%. Numerous known MSC markers were included in the list of 412 genes, such as the mesenchymal stem cell protein DSC54 (13.9-fold increase, p < 0.001), neuropilin 1 (30.4-fold increase, p < 0.001), hepatocyte growth factor (48.1-fold increase, p < 0.001), forkhead box D1 (14.8-fold increase, p < 0.001), and notch homolog 2 (2.9-fold increase, p < 0.001) . Table S2 lists the p-values from the test, the mean and standard deviation of the expression levels, and the relative fold change of all 412 genes between the two types.
Known markers of MSCs, such as mesenchymal stem cell protein DSC54, were all included within the 579 shared transcripts. These findings support the immunocytochemical data and suggest that hESMPCs and primary MSCs are highly related.
MSCs are characterized functionally by their ability to differentiate into mesenchymal tissues, such as fat, cartilage, and bone. Therefore, we tested whether hESMPCs have the same potential (Figure 2).
Figure 2 Selective Differentiation of hESMPCs into Various Mesenchymal Derivatives
(A) Adipocytic differentiation in the presence of dexamethasone, insulin, and isobutylxanthine. Adipocytic characterization by Oil Red O staining and RT-PCR analysis for PPARγ.
(B) Chondrocytic differentiation in the presence of TGF-β3 and ascorbic acid. Chondrocytic characterization by Alcian Blue staining and RT-PCR for aggrecan and collagen II.
(C) Osteogenic differentiation in the presence of β-glycerolphosphate, dexamethasone, and ascorbic acid. Osteocytic characterization by von Kossa staining and RT-PCR for bone-specific alkaline phosphatase (ALP) and bone sialoprotein (BSP).
(D) Phase-contrast image of hESMPCs and RT-PCR for the ES cell markers Nanog and Oct-4 in hESMPC-H1.1 and -H9.1 compared with undifferentiated H1 hESCs.
Scale bar = 50 μm for all panels.
Adipocytic differentiation of hESMPCs was induced under conditions described previously for primary adult MSCs [5]. Appearance of cells harboring fat granules was observed after 10–14 d in culture. After 3 wk of induction, more than 70% of the cells displayed Oil Red O+ fat granules, and PPARγ, a marker of adipocytic differentiation, was detected by RT-PCR. (Figure 2A).
Chondrocytic differentiation was achieved using the pellet culture system [5]. After 28 d in culture, more than 50% of all cells exhibited robust staining for Alcian Blue, a marker specific for extracellular matrix proteoglycans. Chondrocytic differentiation was confirmed by the gene expression of collagen II and aggrecan, two components of extracellular matrix selectively expressed by chondrocytes, using RT-PCR (Figure 2B).
Osteogenic differentiation was induced in the presence of β-glycerolphosphate [5]. Osteogenesis was demonstrated by specific staining for calcium deposition in the matrix (von Kossa, Figure 2C; or Alizarin Red, Figure S2A) and increased expression of bone-specific alkaline phosphatase and bone sialoprotein at day 28 of treatment (Figures 2C and S2B). At day 28, Alizarin Red staining was detected in approximately 70% of all cells. Throughout these studies, human adult MSCs and foreskin fibroblasts were used as positive and negative controls, respectively.
In addition to adipocytic, chondrocytic, and osteogenic differentiation, reports suggested that adult MSCs can form skeletal muscle [13]. Although generation of skeletal muscle cells from adult MSCs remains controversial, we tested whether hESMPCs exhibit this potential. Under the conditions previously described [13], hESMPC-H1.1 and -H9.1 did not yield significant numbers of MyoD+ cells after 15–20 d in culture. However, when confluent cells were maintained in culture in the presence or absence of 5-AzaC without passage for more than 21 d, expression of specific skeletal muscle markers such as MyoD and fast-switch myosin was observed (Figure 3A). More rapid myogenic differentiation was obtained in the presence of 24-h-conditioned medium from the murine myoblastic cell line C2C12 previously induced to form myotubes [14]. Direct coculture of hESMPCs with C2C12 cells led to the formation of hESMPC-derived myotubes, as visualized by expression of human nuclear antigen (Figure 3B), similar to those formed by host C2C12 cells. After 1 wk of coculture, myotubes composed of human nuclei accounted for more than 10% of the total number of human cells present, and each human myotube was composed of up to ten human nuclei. Human cell contribution to myotubes in coculture was confirmed by expression of human muscle-specific transcripts such as MyoD, myosin heavy chain IIa, and myogenin (data not shown). These data demonstrate that hESMPCs can give rise to mesenchymal derivatives typically obtained from primary adult MSCs, as well as to cells expressing markers of skeletal muscle.
Figure 3 Myogenic Differentiation of hESMPCs
(A) Immunocytochemistry for MyoD (red) and fast-switch myosin (green). RT-PCR for MyoD in human skeletal muscle as a positive control (hSM), and in hESMPC-H9.1 cells differentiated for 10 d in the presence of C2C12-conditioned medium (hESMPC).
(B) Myotube formation induced at high cell densities in the presence of C2C12 cells. Myotube characterization by immunocytochemistry for MF20 against sarcomeric myosin (green) and human nuclear antigen (hNA, red). Left panel: Control undifferentiated hESCs (H9) do not fuse with C2C12. Right panel: Under identical culture conditions, hESMPCs (line 9.1) efficiently fuse with C2C12 cells, forming myotubes containing human nuclei. RT-PCR for human specific muscle transcripts myosin heavy chain IIa (MYHC-2) and MyoD in C2C12 cells, in human skeletal muscle as positive control (huSM), and in hESMPC-H9.1 cells cocultured with C2C12 cells.
One concern for the clinical application of hESC-derived progeny in regenerative medicine is the risk of teratoma formation due to the presence of residual undifferentiated ES cells among the differentiated progeny. We did not detect markers of undifferentiated hESCs, such as Nanog [15] or Oct-4 [16], in any of the hESMPCs by RT-PCR (see Figure 2D) and immunocytochemistry (data not shown), suggesting the lack of any undifferentiated ES cells in hESMPC cultures. However, future in vivo studies are required to rule out the potential of these cells for teratoma formation.
Discussion
Previous studies have demonstrated the derivation of neural cells [1–3], hematopoietic [17] and endothelial lineages [18], and cardiomyocytes [19] from hESCs. This study presents the induction of paraxial mesoderm with the generation of multipotent mesenchymal precursors. We calculate that under these conditions a single undifferentiated hESC yields an average of one CD73+ cell at day 40 of differentiation, suggesting a balance between cell proliferation and cell selection. There were no obvious differences in marker and gene-expression profile or in differentiation behavior among the five hESMPC lines generated. However, some of the lines (e.g., hESMPC9.1) exhibited a tendency of spontaneous osteogenic differentiation after long-term propagation. Directed differentiation of hESCs into somatic stem-cell-like precursors represents a substantial advancement in harnessing the developmental potential of hESCs. The high purity, unlimited availability, and multipotentiality of hESMPCs will provide the basis for future therapeutic efforts using these cells in preclinical animal models of disease. Such in vivo studies will also be required to properly assess the safety profile of these cells. Furthermore, our system also offers a novel platform to study basic mechanisms of mesodermal induction and differentiation during early human development.
Supporting Information
Figure S1 Human Identity of CD73+ Cells after FACS
All cells as visualized by DAPI+ nuclei express human nuclear antigen (hNA) confirming the absence of any contaminating OP9 cells. Scale bar = 50 μm.
(148 KB PDF).
Click here for additional data file.
Figure S2 Additional Markers of Bone Differentiation
(A) Alizarin Red staining for calcium deposition in the matrix in hESMPCs untreated (left panel) or treated in the presence of β-glycerolphosphate (right panel; compare to Figure 2C).
(B) Increasing alkaline phosphatase reactivity during osteogenic differentiation of hESMPC-H1.1. Scale bar = 250 μm for main panels, 50 μm for insets.
(278 KB PDF).
Click here for additional data file.
Table S1 All Primers Used in This Study
(22 KB PDF).
Click here for additional data file.
Table S2 List of Shared Genes
List of 421 genes that are shared between primary and hESC-derived mesenchymal precursors but significantly different from undifferentiated hESCs (see main text for details).
(107 KB XLS).
Click here for additional data file.
Accession Numbers
The Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) accession number for all raw microarray data used in this study is GSE2248.
The Unigene (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene) accession numbers for the gene products discussed in this paper are aggrecan (Hs.2159 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=2159; bone sialoprotein (Hs.518726 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=518726; bone-specific alkaline phosphatase (Hs.75431 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=75431; collagen II (Hs.408182 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=408182; forkhead box D1 (Hs.519385 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=519385; hepatocyte growth factor (Hs.396530 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=396530; mesenchymal stem cell protein (DSC54, Hs.157461 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=157461; MyoD (Hs.520119 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=520119; myogenin (Hs.2830 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=2830; myosin heavy chain IIa (Hs.513941 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=513941; Nanog (Hs.329296 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=329296]) [15]; neuropilin 1 (Hs.131704 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=131704; notch homolog 2 (Hs.549056 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=549056; Oct-4 (Hs.504658 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=504658; and PPARγ (Hs.162646 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=162646]).
Patient Summary
Background
The discovery and isolation of human embryonic stem cells (cells that are capable of renewing themselves and turning into the many different cell types that make up the human body) has the potential to revolutionize the treatment of many diseases that require the replacement of abnormal or missing cells. In particular, it would be very valuable to be able to replace tissues that are derived from one particular tissue type—mesenchyme—which bone, cartilage, fat and muscle develop from. However, before such treatments can happen, it will be necessary to work out exactly how embryonic cells become other cells, and whether it is possible to make these changes happen in the laboratory.
What Did the Researchers Do?
They took two lines of completely undifferentiated human embryonic stem cells and by culturing them in the presence of mouse cells stimulated them to turn into mesenchymal cells. They then treated these cells with compounds to make them change into specialized bone, cartilage, fat, and muscle cells. They were able to confirm that these cells were all human (important because the early part of the experiment is done in the presence of mouse cells) and also that there was no evidence that the cells became cancerous.
What Do These Findings Mean?
It is theoretically possible to produce lines of bone, cartilage, fat, and muscle cells from human embryonic stem cells. However, the process will need more refinement before the cell lines could be used for treatment; ideally, for example, all the culturing would be done without any mouse cells.
Where Can I Get More Information?
The United States National Institutes of Health has a group of Web pages on stem cells: http://stemcells.nih.gov/info/faqs.asp
The International Society for Stem Cell Research has a list of frequently asked questions about stem cells: http://www.isscr.org/science/faq.htm
We thank R. McKay for nestin antibody; P. Song and the Sloan-Kettering Genomics and Flow Cytometry Core Facilities for technical assistance; and R. Stan, V. Tabar, M. Tomishima, Y. Elkabetz, and S. Desbordes for critical review of the manuscript. This work was supported in part by the Kinetics Foundation. The funder had no role in the study design, data analysis, decision to publish, or manuscript preparation and content.
Citation: Barberi T, Willis LM, Socci ND, Studer L (2005) Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med 2(6): e161.
Abbreviations
ESembryonic stem
FACSflow-activated cell sorting
FBSfetal bovine serum
hESChuman embryonic stem cell
hESMPChuman embryonic stem cell–derived mesenchymal precursor cell
MSCmesenchymal stem cell
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References
Perrier AL Tabar V Barberi T Rubio ME Bruses J Derivation of midbrain dopamine neurons from human embryonic stem cells Proc Natl Acad Sci U S A 2004 101 12543 12548 15310843
Reubinoff BE Itsykson P Turetsky T Pera MF Reinhartz E Neural progenitors from human embryonic stem cells Nat Biotechnol 2001 19 1134 1140 11731782
Zhang SC Wernig M Duncan ID Brustle O Thomson JA In vitro differentiation of transplantable neural precursors from human embryonic stem cells Nat Biotechnol 2001 19 1129 1133 11731781
Sottile V Thomson A McWhir J In vitro osteogenic differentiation of human ES cells Cloning Stem Cells 2003 5 149 155 12930627
Pittenger MF Mackay AM Beck SC Jaiswal RK Douglas R Multilineage potential of adult human mesenchymal stem cells Science 1999 284 143 147 10102814
Zuk PA Zhu M Mizuno H Huang J Futrell JW Multilineage cells from human adipose tissue: Implications for cell-based therapies Tissue Eng 2001 7 211 228 11304456
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Shi D Reinecke H Murry CE Torok-Storb B Myogenic fusion of human bone marrow stromal cells, but not hematopoietic cells Blood 2004 104 290 294 15010375
Thomson JA Itskovitz-Eldor J Shapiro SS Waknitz MA Swiergiel JJ Embryonic stem cell lines derived from human blastocysts Science 1998 282 1145 1147 9804556
Kodama H Nose M Niida S Nishikawa S Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells Exp Hematol 1994 22 979 984 7522185
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Wakitani S Saito T Caplan AI Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine Muscle Nerve 1995 18 1417 1426 7477065
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Scholer HR Ciesiolka T Gruss P A nexus between Oct-4 and E1A: Implications for gene regulation in embryonic stem cells Cell 1991 66 291 304 1830243
Kaufman DS Hanson ET Lewis RL Auerbach R Thomson JA Hematopoietic colony-forming cells derived from human embryonic stem cells Proc Natl Acad Sci U S A 2001 98 10716 10721 11535826
Levenberg S Golub JS Amit M Itskovitz-Eldor J Langer R Endothelial cells derived from human embryonic stem cells Proc Natl Acad Sci U S A 2002 99 4391 4396 11917100
Mummery C Ward-van Oostwaard D Doevendans P Spijker R van den Brink S Differentiation of human embryonic stem cells to cardiomyocytes: Role of coculture with visceral endoderm-like cells Circulation 2003 107 2733 2740 12742992
| 15971941 | PMC1160574 | CC BY | 2021-01-05 10:39:51 | no | PLoS Med. 2005 Jun 28; 2(6):e161 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020161 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597194110.1371/journal.pmed.0020161Research ArticleBiotechnologyDevelopmentPathologyDerivation of Multipotent Mesenchymal Precursors from Human Embryonic Stem Cells Human ES Cell-Derived Mesenchymal PrecursorsBarberi Tiziano
1
Willis Lucy M
1
Socci Nicholas D
2
Studer Lorenz
1
*1Laboratory of Stem Cell and Tumor Biology, Division of Neurosurgery and Developmental Biology ProgramSloan-Kettering Institute, New York, New YorkUnited States of America2Computational Biology Center, Sloan-Kettering InstituteNew York, New YorkUnited States of AmericaTemple Sally Academic EditorAlbany Medical CollegeUnited States of America
Competing Interests: The authors have declared that no competing interests exist.
Author Contributions: TB and LS designed the study. TB and LMW performed the experiments. TB, LMW, NDS, and LS analyzed the data. TB, NDS, and LS contributed to writing the paper.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e16113 10 2004 15 4 2005 Copyright: © 2005 Barberi et al.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Turning Stem Cells into Mesenchymal Tissues
Background
Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors.
Methods and Findings
Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells.
Conclusion
Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications.
Lorenz Studer and colleagues describe the use of embryonic stem cells to derive mesenchymal precursors and then fat, cartilage, bone, and skeletal muscle cells.
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Introduction
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the blastocyst that can be maintained in culture for an extended period of time without losing differentiation potential. The successful isolation of human ES cells (hESCs) has raised the hope that these cells may provide a universal tissue source to treat many human diseases. However, directed differentiation of hESCs into specific tissue types poses a formidable challenge. Protocols are currently available for only a few cell types, mostly of neural identity [1–3], and differentiation into many of the cell types derived from the paraxial mesoderm has not been reported, with the exception of a recent study indicating osteoblastic differentiation [4]. Mesenchymal stem cells (MSCs) have been isolated from the adult bone marrow [5], adipose tissue [6], and dermis and other connective tissues [7]. Harvesting MSCs from any of these sources requires invasive procedures and the availability of a suitable donor. The number of MSCs that can be obtained from a single donor is limited, and the capacity of these cells for long-term proliferation is rather poor. In contrast, hESCs could provide an unlimited number of specialized cells. In this study, we present techniques for the generation and purification of mesenchymal precursors from hESCs and their directed differentiation in vitro into various mesenchymal derivatives, including skeletal myoblasts. Our isolation method for mesenchymal precursors is the first example, to our knowledge, of efficiently deriving structures of the paraxial mesoderm from ES cells, and further highlights the potential of hESCs for basic biology and regenerative medicine.
Methods
Cell Culture and FACS
Undifferentiated hESCs, H1 (WA-01, XY, passages 40–65) and H9 (WA-09, XX, passages 35–45), were cultured on mitotically inactivated mouse embryonic fibroblasts (Specialty Media, Phillipsburg, New Jersey, United States) and maintained under growth conditions and passaging techniques described previously [3]. OP9 cells were maintained in alpha MEM medium containing 20% fetal bovine serum (FBS) and 2 mM L-glutamine. Mesenchymal differentiation was induced by plating 10 × 103 to 25 × 103 cells/cm2 on a monolayer of OP9 cells in the presence of 20% heat-inactivated FBS in alpha MEM medium. Flow-activated cell sorting (FACS) (CD73-PE; PharMingen, San Diego, California, United States) was performed on a MoFlo (Cytomation, Fort Collins, Colorado, United States). All human ES cell–derived mesenchymal precursor cell (hESMPC) lines in this study are of polyclonal origin. Primary human bone marrow–derived MSCs and primary human foreskin fibroblasts (both from Poietics, Cambrex, East Rutherford, New Jersey, United States) were grown in alpha MEM medium containing 10% FBS and 2 mM L-glutamine.
Adipocytic Differentiation
hESMPCs are grown to confluence followed by exposure to 1 mM dexamethasone, 10 μg/ml insulin, and 0.5 mM isobutylxanthine (all from Sigma, St. Louis, Missouri, United States) in alpha MEM medium containing 10% FBS for 2–4 wk. Data were confirmed in hESMPC-H1.1, -H1.2, -H1.3, and -H9.1 (hESMPC-H1.4 was not tested).
Chondrocytic Differentiation
Differentiation of hESMPCs was induced in pellet culture [5] by exposure to 10 ng/ml TGF-β3 (R & D Systems, Minneapolis, Minnesota, United States) and 200 μM ascorbic acid (Sigma) in alpha MEM medium containing 10% FBS for 3–4 wk. Data were confirmed in hESMPC-H1.1, -H1.3, and -H9.1 (hESMPC-H1.2 and -H1.4 were not tested).
Osteogenic Differentiation
hESMPCs were plated at low density (1 × 103 to 2.5 × 103 cells/cm2) on tissue-culture-treated dishes in the presence of 10 mM β-glycerol phosphate (Sigma), 0.1 μM dexamethasone, and 200 μM ascorbic acid in alpha MEM medium containing 10% FBS for 3–4 wk. Data were confirmed in hESMPC-H1.1, -H1.3, and -H9.1 (hESMPC-H1.2 and -H1.4 were not tested).
Myogenic Differentiation
Confluent hESMPCs were maintained for 2–3 wk in alpha MEM medium with 20% heat-inactivated FBS. More rapid induction was observed in the presence of medium conditioned for 24 h by differentiated C2C12 cells. Coculture of hESMPCs and C2C12 cells was carried out in alpha MEM with 3% horse serum and 1% FBS [8]. Data were confirmed in hESMPC-H1.3, -H1.4, and -H9.1 (hESMPC-H1.1 and -H1.2 were not tested).
Cytochemistry
Immunocytochemistry for all surface markers was performed on live cells. Monoclonal antibodies VCAM, STRO-1, ICAM-1(CD54), CD105, CD29, and MF20 were from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, Iowa, United States); CD73, CD44, and ALCAM(CD166) were from BD Biosciences Pharmingen (San Diego, California, United States). All other immunocytochemical analyses were performed after fixation in 4% paraformaldehyde and 0.15% picric acid, followed by permeabilization in 0.3% Triton X100. Polyclonal antibodies used were MyoD (Santa Cruz Biotechnology, Santa Cruz, California, United States) and nestin (gift from R. McKay); monoclonal antibodies were vimentin, alpha smooth muscle actin, fast-switch myosin, pan-cytokeratin (all from Sigma), and human nuclear antigen (Chemicon, Temecula, California, United States).
Alkaline phosphatase reaction was performed using a commercially available kit (Kit-86; Sigma) and the mineral was stained with silver nitrate according to the von Kossa method. Fat granules were visualized by Oil Red O staining solution (Sigma). Alcian Blue (Sigma) was used to detect extracellular matrix proteoglycans in chondrogenic cultures.
Gene-Expression Analyses
RT-PCR analysis
Total RNA was extracted by using the RNeasy kit and DNase I treatment (Qiagen, Valencia, California, United States). Total RNA (2 μg each) was reverse transcribed (SuperScript; Invitrogen, Carlsbad, California, United States). PCR conditions were optimized and linear amplification range was determined for each primer by varying annealing temperature and cycle number. PCR products were identified by size, and identity was confirmed by DNA sequencing. Primer sequences, cycle numbers, and annealing temperatures are provided in Table S1.
Affymetrix analysis
Total RNA (5 μg) from primary MSCs, from hESMPC-H9.1, hESMPC-H1.2, and three samples of undifferentiated hESCs (H1; passages 42–46), were processed by the Memorial Sloan-Kettering Cancer Center Genomics Core Facility and hybridized on Affymetrix (Santa Clara, California, United States) U133A human oligonucleotide arrays. Data were analyzed using MAS5.0 (Affymetrix) software. Transcripts selectively expressed in each of the mesenchymal cell populations (MSC, hESMPC-H9.1, and hESMPC-H1.2) were defined as those called “increased” by the MAS5.0 algorithm in each of three comparisons with independent samples of undifferentiated hESCs. A Venn diagram was generated to visualize overlap in gene expression. Further statistical analyses were performed as described below.
Results
Mesenchymal differentiation of hESCs (lines H1 [WA-01] and H9 [WA-09]) [9] was induced by plating undifferentiated hESCs on a monolayer of murine OP9 stromal cells [10], in the presence of 20% heat-inactivated FBS in alpha MEM medium. OP9 cells have been previously shown to induce blood cell differentiation from mouse ES cells [11]. After 40 d of coculture, cells were harvested and sorted by FACS for CD73, a surface marker expressed in adult MSCs [5] (Figure 1A). An average of 5% CD73+ cells was obtained from the mixed culture of OP9 and differentiated hESC progeny. CD73+ cells were replated in the absence of stromal feeders on tissue culture plates and expanded in alpha MEM medium with 20% FBS for 7–14 d. We next established the membrane antigen profile of the resulting population of flat spindle-like cells. The H1- and H9-derived CD73+ cells expressed a comprehensive set of markers that are considered to define adult MSCs, including CD105(SH2), STRO-1, VCAM (CD106), CD29(integrin β1), CD44, ICAM -1(CD54), ALCAM(CD166), vimentin, and alpha smooth muscle actin (Figure 1B and 1C). The cells were negative for hematopoietic markers such as CD34, CD45, and CD14. They were also negative for neuroectodermal, epithelial, and muscle cell markers including nestin, pancytokeratin, and desmin (data not shown). The human identity of these presumed mesenchymal cells (termed hESMPC-H1.1, -H1.2, -H1.3, -H1.4, and -H9.1) was confirmed for all experiments by immunocytochemistry for human nuclear antigen to rule out the possibility of contamination with OP9 cells (Figure S1).
Figure 1 Isolation and Characterization of hESMPCs
(A) FACS (MoFlo, Cytomation) for the isolation of CD73+ precursors (right) and isotype control (left).
(B) Flow cytometry analysis of the CD73+ hESMPC population for various markers characteristic of MSCs, including CD44, CD73, CD105, CD166, VCAM, ICAM-1, CD29, and STRO-1.
(C) Immunocytochemistry of hESMPCs for MSC markers (VCAM, STRO-1, CD73, and CD105). The cells also express vimentin and alpha smooth muscle actin. Scale bar = 50 μm.
(D) Venn diagram presenting the overlap among transcripts selectively expressed in hESMPC-H1.2, hESMPC-H9.1, and primary adult human MSCs.
To further characterize hESMPCs, we performed genome-wide expression analysis using oligonucleotide arrays (Affymetrix U133A). The expression profiles of hESMPC-H1.2 and hESMPC-H9.1 were compared with that of human primary adult MSCs. Housekeeping genes for each of the mesenchymal cell populations were eliminated by subtracting those transcripts also expressed in at least one of three independent samples of undifferentiated hESCs. Based on this analysis, 1,280 transcripts were selectively expressed in hESMPC-H1.2, 932 transcripts in hESMPC-H9.1, and 1,218 transcripts in primary adult MSCs. A remarkable overlap of 579 transcripts shared among the three mesenchymal populations was observed (Figure 1D). Using the genes that were selected in the initial filter, we performed a statistical analysis on the expression levels to determine whether the genes were expressed significantly differently in the two cell types. We used a Bayesian extension to the standard t-test [12] to assess this difference. Of the 579 genes, 412 of them were significantly different, at a false discovery rate cutoff of 0.05. The relative fold changes were also extremely large in many of the cases. We also looked at the variance of the expression levels within the cell types. For the MSCs, 94% had a coefficient of variation less than 20% for the expression (log transformed); for the ES-derived cells, 72% had a coefficient of variation less than 20%. Numerous known MSC markers were included in the list of 412 genes, such as the mesenchymal stem cell protein DSC54 (13.9-fold increase, p < 0.001), neuropilin 1 (30.4-fold increase, p < 0.001), hepatocyte growth factor (48.1-fold increase, p < 0.001), forkhead box D1 (14.8-fold increase, p < 0.001), and notch homolog 2 (2.9-fold increase, p < 0.001) . Table S2 lists the p-values from the test, the mean and standard deviation of the expression levels, and the relative fold change of all 412 genes between the two types.
Known markers of MSCs, such as mesenchymal stem cell protein DSC54, were all included within the 579 shared transcripts. These findings support the immunocytochemical data and suggest that hESMPCs and primary MSCs are highly related.
MSCs are characterized functionally by their ability to differentiate into mesenchymal tissues, such as fat, cartilage, and bone. Therefore, we tested whether hESMPCs have the same potential (Figure 2).
Figure 2 Selective Differentiation of hESMPCs into Various Mesenchymal Derivatives
(A) Adipocytic differentiation in the presence of dexamethasone, insulin, and isobutylxanthine. Adipocytic characterization by Oil Red O staining and RT-PCR analysis for PPARγ.
(B) Chondrocytic differentiation in the presence of TGF-β3 and ascorbic acid. Chondrocytic characterization by Alcian Blue staining and RT-PCR for aggrecan and collagen II.
(C) Osteogenic differentiation in the presence of β-glycerolphosphate, dexamethasone, and ascorbic acid. Osteocytic characterization by von Kossa staining and RT-PCR for bone-specific alkaline phosphatase (ALP) and bone sialoprotein (BSP).
(D) Phase-contrast image of hESMPCs and RT-PCR for the ES cell markers Nanog and Oct-4 in hESMPC-H1.1 and -H9.1 compared with undifferentiated H1 hESCs.
Scale bar = 50 μm for all panels.
Adipocytic differentiation of hESMPCs was induced under conditions described previously for primary adult MSCs [5]. Appearance of cells harboring fat granules was observed after 10–14 d in culture. After 3 wk of induction, more than 70% of the cells displayed Oil Red O+ fat granules, and PPARγ, a marker of adipocytic differentiation, was detected by RT-PCR. (Figure 2A).
Chondrocytic differentiation was achieved using the pellet culture system [5]. After 28 d in culture, more than 50% of all cells exhibited robust staining for Alcian Blue, a marker specific for extracellular matrix proteoglycans. Chondrocytic differentiation was confirmed by the gene expression of collagen II and aggrecan, two components of extracellular matrix selectively expressed by chondrocytes, using RT-PCR (Figure 2B).
Osteogenic differentiation was induced in the presence of β-glycerolphosphate [5]. Osteogenesis was demonstrated by specific staining for calcium deposition in the matrix (von Kossa, Figure 2C; or Alizarin Red, Figure S2A) and increased expression of bone-specific alkaline phosphatase and bone sialoprotein at day 28 of treatment (Figures 2C and S2B). At day 28, Alizarin Red staining was detected in approximately 70% of all cells. Throughout these studies, human adult MSCs and foreskin fibroblasts were used as positive and negative controls, respectively.
In addition to adipocytic, chondrocytic, and osteogenic differentiation, reports suggested that adult MSCs can form skeletal muscle [13]. Although generation of skeletal muscle cells from adult MSCs remains controversial, we tested whether hESMPCs exhibit this potential. Under the conditions previously described [13], hESMPC-H1.1 and -H9.1 did not yield significant numbers of MyoD+ cells after 15–20 d in culture. However, when confluent cells were maintained in culture in the presence or absence of 5-AzaC without passage for more than 21 d, expression of specific skeletal muscle markers such as MyoD and fast-switch myosin was observed (Figure 3A). More rapid myogenic differentiation was obtained in the presence of 24-h-conditioned medium from the murine myoblastic cell line C2C12 previously induced to form myotubes [14]. Direct coculture of hESMPCs with C2C12 cells led to the formation of hESMPC-derived myotubes, as visualized by expression of human nuclear antigen (Figure 3B), similar to those formed by host C2C12 cells. After 1 wk of coculture, myotubes composed of human nuclei accounted for more than 10% of the total number of human cells present, and each human myotube was composed of up to ten human nuclei. Human cell contribution to myotubes in coculture was confirmed by expression of human muscle-specific transcripts such as MyoD, myosin heavy chain IIa, and myogenin (data not shown). These data demonstrate that hESMPCs can give rise to mesenchymal derivatives typically obtained from primary adult MSCs, as well as to cells expressing markers of skeletal muscle.
Figure 3 Myogenic Differentiation of hESMPCs
(A) Immunocytochemistry for MyoD (red) and fast-switch myosin (green). RT-PCR for MyoD in human skeletal muscle as a positive control (hSM), and in hESMPC-H9.1 cells differentiated for 10 d in the presence of C2C12-conditioned medium (hESMPC).
(B) Myotube formation induced at high cell densities in the presence of C2C12 cells. Myotube characterization by immunocytochemistry for MF20 against sarcomeric myosin (green) and human nuclear antigen (hNA, red). Left panel: Control undifferentiated hESCs (H9) do not fuse with C2C12. Right panel: Under identical culture conditions, hESMPCs (line 9.1) efficiently fuse with C2C12 cells, forming myotubes containing human nuclei. RT-PCR for human specific muscle transcripts myosin heavy chain IIa (MYHC-2) and MyoD in C2C12 cells, in human skeletal muscle as positive control (huSM), and in hESMPC-H9.1 cells cocultured with C2C12 cells.
One concern for the clinical application of hESC-derived progeny in regenerative medicine is the risk of teratoma formation due to the presence of residual undifferentiated ES cells among the differentiated progeny. We did not detect markers of undifferentiated hESCs, such as Nanog [15] or Oct-4 [16], in any of the hESMPCs by RT-PCR (see Figure 2D) and immunocytochemistry (data not shown), suggesting the lack of any undifferentiated ES cells in hESMPC cultures. However, future in vivo studies are required to rule out the potential of these cells for teratoma formation.
Discussion
Previous studies have demonstrated the derivation of neural cells [1–3], hematopoietic [17] and endothelial lineages [18], and cardiomyocytes [19] from hESCs. This study presents the induction of paraxial mesoderm with the generation of multipotent mesenchymal precursors. We calculate that under these conditions a single undifferentiated hESC yields an average of one CD73+ cell at day 40 of differentiation, suggesting a balance between cell proliferation and cell selection. There were no obvious differences in marker and gene-expression profile or in differentiation behavior among the five hESMPC lines generated. However, some of the lines (e.g., hESMPC9.1) exhibited a tendency of spontaneous osteogenic differentiation after long-term propagation. Directed differentiation of hESCs into somatic stem-cell-like precursors represents a substantial advancement in harnessing the developmental potential of hESCs. The high purity, unlimited availability, and multipotentiality of hESMPCs will provide the basis for future therapeutic efforts using these cells in preclinical animal models of disease. Such in vivo studies will also be required to properly assess the safety profile of these cells. Furthermore, our system also offers a novel platform to study basic mechanisms of mesodermal induction and differentiation during early human development.
Supporting Information
Figure S1 Human Identity of CD73+ Cells after FACS
All cells as visualized by DAPI+ nuclei express human nuclear antigen (hNA) confirming the absence of any contaminating OP9 cells. Scale bar = 50 μm.
(148 KB PDF).
Click here for additional data file.
Figure S2 Additional Markers of Bone Differentiation
(A) Alizarin Red staining for calcium deposition in the matrix in hESMPCs untreated (left panel) or treated in the presence of β-glycerolphosphate (right panel; compare to Figure 2C).
(B) Increasing alkaline phosphatase reactivity during osteogenic differentiation of hESMPC-H1.1. Scale bar = 250 μm for main panels, 50 μm for insets.
(278 KB PDF).
Click here for additional data file.
Table S1 All Primers Used in This Study
(22 KB PDF).
Click here for additional data file.
Table S2 List of Shared Genes
List of 421 genes that are shared between primary and hESC-derived mesenchymal precursors but significantly different from undifferentiated hESCs (see main text for details).
(107 KB XLS).
Click here for additional data file.
Accession Numbers
The Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) accession number for all raw microarray data used in this study is GSE2248.
The Unigene (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene) accession numbers for the gene products discussed in this paper are aggrecan (Hs.2159 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=2159; bone sialoprotein (Hs.518726 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=518726; bone-specific alkaline phosphatase (Hs.75431 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=75431; collagen II (Hs.408182 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=408182; forkhead box D1 (Hs.519385 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=519385; hepatocyte growth factor (Hs.396530 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=396530; mesenchymal stem cell protein (DSC54, Hs.157461 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=157461; MyoD (Hs.520119 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=520119; myogenin (Hs.2830 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=2830; myosin heavy chain IIa (Hs.513941 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=513941; Nanog (Hs.329296 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=329296]) [15]; neuropilin 1 (Hs.131704 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=131704; notch homolog 2 (Hs.549056 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=549056; Oct-4 (Hs.504658 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=504658; and PPARγ (Hs.162646 [http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=162646]).
Patient Summary
Background
The discovery and isolation of human embryonic stem cells (cells that are capable of renewing themselves and turning into the many different cell types that make up the human body) has the potential to revolutionize the treatment of many diseases that require the replacement of abnormal or missing cells. In particular, it would be very valuable to be able to replace tissues that are derived from one particular tissue type—mesenchyme—which bone, cartilage, fat and muscle develop from. However, before such treatments can happen, it will be necessary to work out exactly how embryonic cells become other cells, and whether it is possible to make these changes happen in the laboratory.
What Did the Researchers Do?
They took two lines of completely undifferentiated human embryonic stem cells and by culturing them in the presence of mouse cells stimulated them to turn into mesenchymal cells. They then treated these cells with compounds to make them change into specialized bone, cartilage, fat, and muscle cells. They were able to confirm that these cells were all human (important because the early part of the experiment is done in the presence of mouse cells) and also that there was no evidence that the cells became cancerous.
What Do These Findings Mean?
It is theoretically possible to produce lines of bone, cartilage, fat, and muscle cells from human embryonic stem cells. However, the process will need more refinement before the cell lines could be used for treatment; ideally, for example, all the culturing would be done without any mouse cells.
Where Can I Get More Information?
The United States National Institutes of Health has a group of Web pages on stem cells: http://stemcells.nih.gov/info/faqs.asp
The International Society for Stem Cell Research has a list of frequently asked questions about stem cells: http://www.isscr.org/science/faq.htm
We thank R. McKay for nestin antibody; P. Song and the Sloan-Kettering Genomics and Flow Cytometry Core Facilities for technical assistance; and R. Stan, V. Tabar, M. Tomishima, Y. Elkabetz, and S. Desbordes for critical review of the manuscript. This work was supported in part by the Kinetics Foundation. The funder had no role in the study design, data analysis, decision to publish, or manuscript preparation and content.
Citation: Barberi T, Willis LM, Socci ND, Studer L (2005) Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med 2(6): e161.
Abbreviations
ESembryonic stem
FACSflow-activated cell sorting
FBSfetal bovine serum
hESChuman embryonic stem cell
hESMPChuman embryonic stem cell–derived mesenchymal precursor cell
MSCmesenchymal stem cell
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References
Perrier AL Tabar V Barberi T Rubio ME Bruses J Derivation of midbrain dopamine neurons from human embryonic stem cells Proc Natl Acad Sci U S A 2004 101 12543 12548 15310843
Reubinoff BE Itsykson P Turetsky T Pera MF Reinhartz E Neural progenitors from human embryonic stem cells Nat Biotechnol 2001 19 1134 1140 11731782
Zhang SC Wernig M Duncan ID Brustle O Thomson JA In vitro differentiation of transplantable neural precursors from human embryonic stem cells Nat Biotechnol 2001 19 1129 1133 11731781
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| 0 | PMC1160575 | CC BY | 2021-01-05 10:39:53 | no | PLoS Med. 2005 Jun 28; 2(6):e165 | latin-1 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020165 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597194310.1371/journal.pmed.0020167Research in TranslationInfectious DiseasesAnesthesiologyIntensive CareCritical Care / Intensive CareAnaesthesiaTreating Critical Illness: The Importance of First Doing No Harm Research in TranslationSinger Mervyn *Glynne Paul Mervyn Singer is Professor of Intensive Care Medicine, and Paul Glynne is Senior Lecturer in Intensive Care Medicine, at the Bloomsbury Institute of Intensive Care Medicine, University College London, United Kingdom.
Competing Interests: MS is on the editorial board of PLoS Medicine. PG declares that he has no competing interests.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e167Copyright: © 2005 Singer and Glynne.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Singer and Glynne present evidence to suggest that the short- term benefits of many interventions for treating critical illness may camouflage an underlying tendency to cause harm.
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The Battle of Trafalgar was a short-lasting but bloody affair. By the end of the afternoon of 21 October 1805, the French/Spanish fleet had lost 4,408 men, and a further 2,545 were wounded while the victorious British force suffered 455 dead and 1,242 wounded [1]. The British flagship HMS Victory, where Admiral Horatio Nelson lost his life to a sniper's bullet, sustained 56 other deaths. The surgeon on board, Dr. William Beatty, also listed 102 wounded sailors who survived the battle or its immediate aftermath [2]. He needed to perform ten amputations, mainly involving the leg, and reported instances of death from gangrene and tetanus. Yet only six of the 102 wounded sailors subsequently died.
This remarkably low mortality rate was mirrored by that sustained by the 13th Light Dragoons during the Battle of Waterloo in 1815. Of 52 privates reported as wounded, only three later died of their wounds [3].
The United States Civil War five decades later was a more protracted but equally bitter conflict, with over 550,000 deaths in the four years of battle [4]. Of note, twice as many troops died from disease, privation, and accidents than died of injuries sustained on the battlefield. Typhus, typhoid fever, mumps, and measles were rife in army camps where poor sanitation, hygiene, and an inadequate diet were the norm. The surgeons in these camps often held their instruments between their teeth, and these tools were only cleaned at the end of the day's operating. At the Battle of Antietam, maize husks were used as bandages. Yet despite the prevailing conditions and lack of aseptic technique, mortality rates were remarkably good. The Union forces kept detailed records and reported a directly attributable mortality rate from battlefield injuries of 14%. The overall mortality rate following amputation was only about 25%, (Table 1) with some patients even surviving hindquarter amputation [5].
Table 1 Mortality Rates from Amputations During the US Civil War
Data are taken from [5]
Then and Now
These survival rates do appear impressive, especially when one considers that they were obtained without antisepsis, antibiotics, blood transfusion, oxygen, and the other paraphernalia of modern medicine, and that the surgeons relied upon rudimentary surgical techniques performed without the assistance or comforts of sophisticated anaesthesia or mechanical ventilation.
Clearly, a direct analogy should not be made to the mortality rates for today's battlefield injuries, which involve far more destructive weaponry, or to those of modern hospital populations, where many patients are elderly and/or immunosuppressed. Nevertheless, it is valid to wonder why even greater improvements in outcome have not been achieved in the last two to three decades despite the huge advances made in medical technology, treatments, and understanding of underlying pathological mechanisms. Barring the 1919 influenza pandemic, Armstrong et al. reported an impressive 22-fold fall in crude mortality rates for infectious diseases in the US between 1900 and 1980 [6]. Yet they also showed how mortality rates (to 1996) have since increased—by 50%—with the septicaemia rate nearly doubling.
While it is true that people are living longer, with an increase in life expectancy from 1980 to 1996 of 2.4 years (from 73.7 to 76.1) [7], these gains are unlikely to be due to advances in hospital medicine. They are far more likely to be due to the contribution of better public health and education, including reductions in environmental pollution, altered eating and smoking habits, and increased exercise.
The Poor Evidence Base for Many Interventions
An important but generally overlooked consideration is the possibility that superficially attractive, short-term benefits may camouflage an underlying tendency to cause harm. There are high-profile instances where injury is belatedly recognised. A recent example is the increased risk in serious cardiovascular thrombotic events seen in patients taking the anti-inflammatory COX II inhibitor rofecoxib [8]. Clearly, some individuals have suffered, yet the target patient group as a whole has been protected by the much greater surveillance given to a new pharmaceutical compound and the improved likelihood of detecting a major complication. How many long-standing medications, devices, or treatment regimens have been scrutinised to a remotely similar extent?
A fundamental tenet of medical teaching is to first do no harm to our patients. Every decision affecting patient management should thus be judged on the basis of the ratio of likely risks to benefits. Alas, large chunks of perceived wisdom rely on expert opinion, historical practice, and blind acceptance, rather than on an adequate evidence base, to vindicate continued use of a drug, device, or management strategy.
For example, more than half the 50-plus recommendations made in the recent Surviving Sepsis guidelines [9], which have been endorsed and are now being heavily promoted by the US and European critical care societies as a standard of care, were based solely on expert opinion. Many of the other, more highly graded, recommendations relied upon studies with small patient numbers and/or methodological flaws. Of only four Grade A recommendations (i.e., those supported by at least two large, randomised trials with clear-cut results), deep vein thrombosis prophylaxis and ventilator weaning are generic issues for all critically ill patients, while the other two (avoidance of high-dose corticosteroids, and not striving to achieve specified target values of oxygen delivery/consumption when resuscitating patients with fluids and inotropes) were based upon studies performed over a decade ago. The two latter recommendations arose from “negative” studies in which the standards of care at the time were shown to be ineffective [10] or even harmful [11]. This recognition of harm also applies to many of the Grade B recommendations (i.e., supported by one large, randomised trial), for example, the lower threshold of haemoglobin used to trigger blood transfusion [12], or the reduced tidal volumes delivered during mechanical ventilation [13].
Fashionable Treatments for Critical Illness: Are They Harming Patients?
The major advances of intensive care medicine in the last 20 years have been related more to the recognition and removal of harmful practices rather than to any novel pharmacological or mechanical interventions. It is thus reasonable to question how many currently fashionable strategies may actually prove injurious when submitted to critical examination (Table 2)? This assumes, of course, that the inclination to challenge dogma exists.
Table 2 Examples of Fashionable Treatments for Critical Illness That May Cause Harm
A perfect example of this unwavering adherence to an article of faith is the flawed reliance upon furosemide as first-line therapy in the management of patients with acute heart failure. Such patients rarely have intravascular volume overload, yet they are often given a potent loop diuretic, which will frequently result in hypovolaemia, vasoconstriction, increased ventricular strain, and a decrease in cardiac output [14]. This effect is not apparent at the end of the needle, where the initial but short-lived vasodilatation produces symptomatic relief and a transient improvement in haemodynamics. Thus, it is highly convenient to blame the patient's failing heart for any subsequent deterioration without recognition and acceptance of any iatrogenic component.
Recent European Society of Cardiology guidelines for the management of acute heart failure [15] make repeated references to the harmful consequences of diuretic use, emphasising that “secondary effects are frequent and may be life-threatening.” Yet the Task Force of experts still proceeded to make a Class I recommendation for their continued use (i.e., evidence and/or general agreement that a given diagnostic procedure/treatment is beneficial, useful, and effective), with a “B” level of evidence (i.e., data derived from a single, randomised clinical trial or large, nonrandomised studies). Their justification for this grading—which was not actually underpinned by any trial data, either large or randomised—was that “The symptomatic benefits (of diuretics) and their universal clinical acceptance have precluded a formal evaluation in large-scale randomised clinical trials.” Indeed, the single prospective randomised outcome study performed to date (and cited by the guidelines) actually showed the superiority of nitrates over furosemide [16].
The key phrase used above is “universal clinical acceptance”—that is to say, “we believe diuretics work, as we've used them unquestioningly throughout our medical careers, so we can't possibly question this particular shibboleth in a critically objective fashion.” Has there been any consideration of the possibility that the long-term outcome benefits derived from ACE inhibitors may be, at least in part, related to the necessary decrease in diuretic dosing?
In more severe heart failure and other forms of shock associated with low blood pressures and/or low cardiac outputs, there is a conventional reliance upon catecholamines such as dobutamine, norepinephrine, and epinephrine. Yet these inotrope and pressor agents have many effects distant from their cardiovascular actions. They have metabolic effects including increased ß-oxidation of fats; they are pro-arrhythmogenic; they have pro- and anti-inflammatory effects; and they can alter both immunity and mitochondrial function [17–21]. Lyte et al. showed that the use of catecholamine inotropes was associated with significant increases in bacterial growth of Gram-positive [22] and Gram-negative [23] bacteria and in the formation of biofilms [22].
Indeed, all large randomised studies performed to date in patients with chronic heart failure that have compared catecholamines or phosphodiesterase inhibitors (both of which increase cyclic adenosine monophosphate [AMP] levels) against either placebo or another treatment have also shown detriment. Even short-term (1–2 day) infusions of the phosphodiesterase inhibitors milrinone and vesnanrinone significantly worsened six-month outcomes [24,25]. A similar effect has been reported with dobutamine in comparison to the calcium sensitiser levosimendan [26,27].
Intensive care physicians also use antibiotics, sedatives, inotropes, and blood products extensively. While necessary in many cases, there is an increasingly strong feeling that these agents are being overused, as many problems and complications are directly attributable to them. For example, excessive antibiotic use is related to the development of bacterial resistance and fungal overgrowth [28], while overuse of sedatives delays weaning from mechanical ventilation [29]. However, less consideration is paid to other effects of these drugs that could arguably be just as injurious, if not more so.
Underlying Mechanisms for Why Our Treatments May Cause Harm
How can we explain, at the molecular level, the covert harm to the patient from standard drugs such as antibiotics, sedatives, and inotropes? The answer may lie in understanding the pathophysiological mechanisms underlying multiple organ failure, such as changes in immune and hormonal status and the role of the mitochondrion.
Over a billion years ago, a bacterium containing the oxygen-consuming respiratory chain is likely to have invaded the early eukaryotic cell. Most of the bacterial genetic information was subsequently transferred to the nucleus, transforming these bacterial symbionts into “slave” mitochondrial organelles. This provided a far more efficient system for using available energy sources and also protected the cell against the potentially toxic effects of oxygen. More than 90% of total body oxygen consumption is used to generate adenosine triphosphate (ATP) by the mitochondrial electron transport chain, and this, in turn, provides more than 90% of the body's power, the remainder coming from glycolysis.
An attractive hypothesis to explain the pathophysiology of multiple organ failure following infection and other inflammatory insults is a mitochondrial shutdown leading to “energy failure” and a consequent inability to drive the various metabolic processes that maintain normal cellular functioning (Figure 1). Inflammatory mediators released in considerable excess in sepsis, such as tumor necrosis factor and nitric oxide, are known to directly inhibit mitochondrial respiration. We and others have demonstrated this mechanism in septic patients and laboratory models [30–32]. The down-regulation of endocrine function seen in established sepsis, for example, the sick euthyroid syndrome, insulin resistance, and hypoleptinaemia, will also impinge on mitochondrial activity [33]. If the cell attempts to continue to function normally despite inadequate energy production, the resulting fall in adenosine triphosphate will trigger necrotic and apoptotic death pathways.
Figure 1 Hypothesis Explaining the Pathophysiology of Multiple Organ Failure Following Infection and Other Inflammatory Insults
Antibiotics, sedatives, and inotropes may cause harm through inhibition of mitochondrial function. Antibiotics may also delay recovery by impeding mitochondrial regeneration.
However, as this process is not immediate (it takes hours to days to develop fully) the cell has time to potentially adapt to this prolonged, life-threatening insult. It is likely to do so by entering a hibernation-like state. The impressive and almost total absence of cell death seen in organs that have “failed” biochemically and/or physiologically [34] lends credence to this hypothesis. Restoration of cellular function, and thus recovery from organ failure, must therefore depend upon repair of damaged mitochondria and/or production of new organelles, a process known as mitochondrial biogenesis.
Harm from Antibiotics
The reason for this preamble is to emphasise the role of the systemic inflammatory response and the likely fundamental importance of the mitochondrion in the development of multiple organ failure, and also the mitochondrion's distant lineage but existing genetic linkage to bacteria. We use antibiotics to fight bacteria, and they are undoubtedly successful in many instances. Many of the antibiotic classes, such as the penicillins and cephalosporins, are bactericidal through cell-wall disruption, whereas other classes, such as chloramphenicol and aminoglycosides, act in a bacteriostatic manner by inhibiting protein synthesis. However, by virtue of their action, the cell-wall disrupters—in particular the cephalosporins—cause increased levels of endotoxin release from Gram-negative bacteria [35,36] and lipoteichoic acid and peptidoglycan release from Gram-positive bacteria [37,38]. This enhanced toxin release leads to significantly higher inflammatory mediator production. This may well explain the rapid clinical deterioration often seen in patients with sepsis after the first dose of cidal antibiotics, though long-term consequences remain unknown.
A delayed and potentially significant effect of antibiotics may be seen through their inhibition of mitochondrial activity and biogenesis. This inhibition has been shown in numerous in vitro studies, in which cell lines or isolated mitochondria have been incubated with antibiotics at concentrations equivalent to therapeutic blood levels. Significant decreases in respiratory enzyme activity and protein turnover have been found across a wide range of antibiotic classes [39–42]. Could our antibiotic therapy be thus accentuating the degree of sepsis and multiple organ dysfunction through increased inflammatory mediator release and mitochondrial depression? Could such therapy also be delaying recovery by impeding mitochondrial regeneration?
Harm from Sedatives
Continuing on this theme, the major classes of sedative/anesthetic agents (opiates, benzodiazepines, propofol, barbiturates, and volatile anaesthetic agents) routinely used to enable mechanical ventilation in the operating theatre or intensive care unit all have effects on mitochondrial function in vitro [43–48]. In the case of propofol, these effects appear to be significantly enhanced in the presence of nitric oxide through formation of nitrosopropofol [45]. Thus, sepsis may potentially amplify the effects of this sedative agent on mitochondrial inhibition. This mechanism may explain the severe metabolic and physiological deterioration reported in children and adults receiving propofol [48].
All classes of sedative agents have also been shown to alter immune function in neutrophils, monocytes, and lymphocytes in vitro and to affect rates of apoptosis [49–54]. Immunosuppression (for example, assessed in monocytes by HLA-DR status) is well recognised in established sepsis and is related to worse outcomes [55,56]. The clinical significance of sedative drug actions on the immune response to critical illness remains unknown.
Certain sedatives are also known to affect hormonal status. The most striking example is the classic study by Watt and Ledingham, who sought an explanation for the sudden jump in mortality rates in their critically ill trauma patients: from 28% in those receiving opiates and benzodiazepines to 77% of those sedated with etomidate [57]. They showed a significant etomidate-induced depression of adrenal function that led to withdrawal of its use for medium- to long-term sedation in intensive care. Etomidate is, however, still frequently used as an induction agent for anaesthesia because of its cardiovascular stability. Unfortunately, this practice continues despite the fact that Absalom et al. have shown that a single dose of etomidate given before surgery in critically ill patients was sufficient to compromise adrenal function 24 hours later [58].
Harm from Other Drugs and Interventions
Other drugs are known to affect hormone levels. Low-dose dopamine, which was a popular and subsequently disproved therapy for maintaining renal function, rapidly reduces serum prolactin levels [59]. Prolactin has immunostimulatory effects, and a low prolactin level has been associated with a worse outcome in septic mice [60]. The recognition that impaired adrenal function, as assessed by a subnormal rise in plasma cortisol to synthetic adrenocorticotropic hormone, was related to poor outcomes in septic shock [61] led to a multicentre trial that revealed survival benefit from early administration of hydrocortisone 50 mg four times daily [62]. However, the debate continues surrounding its contribution to the development of critical illness neuromyopathy and delayed weaning [63]. The sick euthyroid syndrome is likewise associated with worse outcomes [64] yet several drugs that affect thyroid function, such as amiodarone, are frequently used in critically ill patients.
These concerns can be replicated across virtually every therapy area in the critically ill.
Mechanical ventilation.
A strategy of delivering low tidal volumes rather than the previously fashionable high tidal volumes during mechanical ventilation reduced mortality from 39% to 31% [13]; this change in strategy has been separately shown to also reduce both the local and systemic inflammatory response, presumably from lowering shear stresses within the lung [65].
Immunonutrition.
A trial of immunonutrition had to be prematurely terminated after an interim analysis revealed a significant mortality increase in septic patients [66].
Drotrecogin-alpha.
The Canadian Department of Health recently issued a safety alert on Drotrecogin-alpha (Xigris), the first licensed therapy for severe sepsis, after post-hoc analyses of trial data revealed an excess mortality in patients with single organ dysfunction who had received surgery within 30 days prior to study treatment [67].
Blood transfusion.
Lowering the threshold for blood transfusion from 10 g/dl to 7 g/dl, and thus reducing transfusion requirements from an average of 5.6 ± 5.3 red-cell units per patient to 2.6 ± 4.1 units, reduced 30-day mortality rates from 23.3% to 18.7% [12]. In a recent retrospective analysis of three large trials of patients with acute coronary syndromes, Rao et al. reported a 3-fold increase in death and myocardial infarction rates in those who received a blood transfusion [68]. There may be an immunological reason underlying this apparent harm. Hebert et al. found significant reductions in mortality rates, post-transfusion fevers, and antibiotic use in patients who received leukoreduced blood transfusions, compared to historical controls who received “normal” blood [69]. It remains to be seen whether remaining blood constituents in leukocyte-depleted blood are able to also affect the immune response.
Proton pump inhibitors.
A recent meta-analysis [70] comparing the use of proton pump inhibitors against either placebo or an H2-antagonist found a significant reduction in rebleeding and the need for surgical intervention. Yet despite this clear benefit, the trend in mortality was actually in the opposite direction. For studies of intravenous therapy, as is given to critically ill patients, the odds ratio for mortality was 1.22 (95% confidence interval 0.84–1.78).
Conclusions
It should be immediately acknowledged that most of the above findings have been derived from relatively small patient studies or extrapolated from in vivo and in vitro laboratory studies. As with most aspects of medicine, there are contradictory results. Yet sufficient data exist to suggest that the possibility of insidious harm should not be lightly dismissed. The above litany of problems should also not be used as a reason to abandon current practices, but instead to stimulate discussion, refine their use, and to encourage trials designed to confirm or refute detriment.
Our concern is that neither the inclination nor the funding will be generally available to revisit accepted dogma. We will thus have to rely on a slowly evolving approach, where new therapies are compared with conventional treatments, or where a media-highlighted concern propels a certain strategy into the spotlight. This was the case, for example, with the use of albumin for fluid administration. A Cochrane meta-analysis suggested a 4% increase in mortality with albumin over crystalloid solutions [70]. The subsequent prospective randomised trial of 6,997 patients revealed no overall difference in mortality; intriguingly, subset analysis suggested benefit when used in sepsis but harm in head-injured patients [71].
Citation: Singer M, Glynne P (2005) Treating critical illness: The importance of first doing no harm. PLoS Med 2(6): e167.
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Delogu G Antonucci A Moretti S Marandola M Tellan G Oxidative stress and mitochondrial glutathione in human lymphocytes exposed to clinically relevant anesthetic drug concentrations J Clin Anesth 2004 16 189 194 15217658
Docke WD Randow F Syrbe U Krausch D Asadullah K Monocyte deactivation in septic patients: Restoration by IFN-gamma treatment Nat Med 1997 3 678 681 9176497
Lekkou A Karakantza M Mouzaki A Kalfarentzos F Gogos CA Cytokine production and monocyte HLA-DR expression as predictors of outcome for patients with community-acquired severe infections Clin Diagn Lab Immunol 2004 11 161 167 14715564
Watt I Ledingham IM Mortality amongst multiple trauma patients admitted to an intensive therapy unit Anaesthesia 1984 39 973 981 6496912
Absalom A Pledger D Kong A Adrenocortical function in critically ill patients 24 h after a single dose of etomidate Anaesthesia 1999 54 861 867 10460557
Bailey AR Burchett KR Effect of low-dose dopamine on serum concentrations of prolactin in critically ill patients Br J Anaesth 1997 78 97 99 9059216
Zellweger R Zhu XH Wichmann MW Ayala A DeMaso CM Prolactin administration following hemorrhagic shock improves macrophage cytokine release capacity and decreases mortality from subsequent sepsis J Immunol 1996 157 5748 5754 8955229
Annane D Sebille V Troche G Raphael JC Gajdos P A 3-level prognostic classification in septic shock based on cortisol levels and cortisol response to corticotropin JAMA 2000 283 1038 1045 10697064
Annane D Sebille V Charpentier C Bollaert PE Francois B Effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock JAMA 288 862 871
De Jonghe B Sharshar T Lefaucheur JP Paresis acquired in the intensive care unit: A prospective multicenter study JAMA 2002 288 2859 2867 12472328
Leon-Sanz M Lorente JA Larrodera L Ros P Alvarez J Pituitary-thyroid function in patients with septic shock and its relation with outcome Eur J Med Res 1997 2 477 482 9385118
Ranieri VM Suter PM Tortorella C De Tullio R Dayer JM Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: A randomized controlled trial JAMA 1999 282 54 61 10404912
Bertolini G Iapichino G Radrizzani D Facchini R Simini B Early enteral immunonutrition in patients with severe sepsis: Results of an interim analysis of a randomised multicentre clinical trial Intensive Care Med 2003 29 834 840 12684745
Health Canada Notice to hospitals: Health Canada endorsed important safety information on Xigris 2005 Available: http://www.hc-sc.gc.ca/hpfb-dgpsa/tpd-dpt/xigris_nth_e.pdf . Accessed 22 April 2005
Rao SV Jollis JG Harrington RA Granger CB Newby LK Relationship of blood transfusion and clinical outcomes in patients with acute coronary syndromes JAMA 2004 292 1555 1562 15467057
Hebert PC Fergusson D Blajchman MA Clinical outcomes following institution of the Canadian universal leukoreduction program for red blood cell transfusions JAMA 2003 289 1941 1949 12697796
Leontiadis GI Sharma VK Howden CW Systematic review and meta-analysis of proton pump inhibitor therapy in peptic ulcer bleeding BMJ 2005 330 568 15684023
Human albumin administration in critically ill patients: Systematic review of randomised controlled trials. Cochrane Injuries Group Albumin Reviewers BMJ 317 235 240 [No authors listed]
Finfer S Bellomo R Boyce N French J Myburgh J A comparison of albumin and saline for fluid resuscitation in the intensive care unit N Engl J Med 2004 350 2247 2256 15163774
| 15971943 | PMC1160576 | CC BY | 2021-01-05 10:39:52 | no | PLoS Med. 2005 Jun 28; 2(6):e167 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020167 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597194410.1371/journal.pmed.0020168Best PracticeInfectious DiseasesPrimary CareInfectious DiseasesGeneral Practice/Family Practice/Primary CareVitamin C for Preventing and Treating the Common Cold Best PracticeDouglas Robert M *Hemilä Harri Robert M. Douglas is at the National Centre for Epidemiology and Population Health, Australian National University, Canberra, Australia. Harri Hemilä is at the Department of Public Health, University of Helsinki, Finland.
Competing Interests: RMD was an organising author of one of the papers considered in the review. HH declares that he has no competing interests.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e168Copyright: © 2005 Douglas and Hemilä.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Whether vitamin C has an effect on the common cold has been a subject of controversy for at least 60 years. What does the evidence show?
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The role of vitamin C in the prevention and treatment of the common cold has been a subject of controversy for at least 60 years. Public interest in the subject, stimulated originally by the vigorous advocacy of Nobel laureate Linus Pauling during the 1970s [1], continues to be high. We have recently updated the Cochrane Review [2] on this topic (Text S1), incorporating 55 comparative studies that have been carried out over a period of 65 years.
The Updated Review
We sought to discover whether vitamin C in doses of 200 mg or more daily (Figure 1) reduces the incidence, duration, or severity of the common cold when used either as continuous prophylaxis or after the onset of cold symptoms. Criteria for inclusion were placebo-controlled trials to prevent or treat the common cold using oral doses of vitamin C of 200 mg/day or more. Literature from 1940 to 2004 was methodically screened.
Figure 1 500-mg Vitamin C Tablets and Paprikas
Vitamin C was identified in the 1930s by Albert Szent-Györgyi, who received his Nobel Prize partly for this work. He found that paprika is a particularly rich source of the vitamin, which made it possible to produce kilograms of it for research purposes ([1963] Annu Rev Biochem 32: 1–14). Nowadays, the most convenient way to increase vitamin C intake is by way of 500-mg tablets, but further research is needed to explore the conditions in which supplementation may be beneficial.
An overview of the results of the three meta-analyses is shown in Table 1. Incidence was not altered in the subgroup of 23 community studies where prophylactic doses as high as 2 g daily were used. But a subgroup of six studies of marathon runners, skiers, and soldiers exposed to significant cold and/or physical stress experienced, on average, 50% reduction in common cold incidence.
Table 1 Results of the Three Meta-Analyses
These meta-analyses were of placebo-controlled trials that provided data on incidence and duration of respiratory episodes during continuous oral prophylaxis, or on duration of respiratory episodes following therapy at onset of colds using doses in excess of 200 mg of vitamin C.
Prophylaxis indicates vitamin C supplementation over the entire study period. All combined results were pooled using the random effects model.
a Heterogeneity between trial outcomes in the group: the 2 test for heterogeneity with p > 0.05 indicates lack of heterogeneity, and the I2 test with I2 = 0% indicates no evidence of heterogeneity, with 100% being the maximum in the I2 scale [5].
b CI, confidence interval; RED, relative effect on duration of colds (e.g., if a seven-day cold is shortened by one day, that corresponds to RED = -14% [= -1/7]); RR, relative risk of colds.
c Statistically significant benefit favours those receiving vitamin C with p < 0.0001.
d Statistically significant benefit favours those receiving vitamin C with p < 0.002.
Duration of cold episodes that occurred during prophylaxis was significantly reduced in both children and adults. For children this represented an average reduction of 14% in symptom days, while in adults the reduction was 8%.
For the seven trials that evaluated the therapeutic impact of vitamin C used at the onset of symptoms (all in adults), benefits were not observed for duration of episodes, although one of the large trials recorded a statistically significant reduction in the duration of colds among participants administered a single vitamin C dose of 8 g on the day of symptom onset [3].
Implications of the Review
The lack of effect of prophylactic vitamin C supplementation on the incidence of common cold in normal populations throws doubt on the utility of this wide practice. The clinical significance of the minor reduction in duration of common cold episodes experienced during prophylaxis is questionable, although the consistency of these findings points to a genuine biological effect.
In special circumstances, where people used prophylaxis prior to extreme physical exertion and/or exposure to significant cold stress, the collective evidence indicates that vitamin C supplementation may have a considerable beneficial effect; it was the results of one of these six trials, with schoolchildren in a skiing school [4], that particularly impressed Pauling [1]. However, great caution should be exercised in generalizing from this finding, which is based mainly on marathon runners.
No benefits have been observed from therapeutic use of doses totalling 10 g that was divided for the first three days of illness. The equivocal findings of the large study, which used 8 g only on the day of onset of respiratory symptoms [3], are tantalising and deserve further assessment.
None of the therapeutic trials carried out so far has examined the effect of vitamin C on children, even though the prophylaxis trials have shown substantially greater effect on episode duration in children.
Study quality for the trials included in these three meta-analyses was variable, but sensitivity analysis, where we excluded studies from the analysis that were less adequately blinded or randomized, did not change the general conclusions of the Cochrane Review.
Future work on this topic should explore the value of high dose therapy—in particular, in children—and the mechanisms underlying the observed prophylaxis benefits in those exposed to substantial physical and/or cold stress.
Supporting Information
Text S1 Updated Cochrane Review
Douglas RM, Hemilä H, D'Souza R, Chalker EB, Treacy B (2004) Vitamin C for preventing and treating the common cold. Cochrane Database Syst Rev 4: CD000980.pub2.
Date of most recent substantive amendment: 10 August 2004.
This data supplement can be freely accessed on the PLoS Medicine Web site, but it is not published under the Creative Commons Attribution License.
Copyright © 2004 The Cochrane Collaboration. Published by John Wiley and Sons. All rights reserved.
(1.3 MB PDF).
Click here for additional data file.
Citation: Douglas RM, Hemilä H (2005) Vitamin C for preventing and treating the common cold. PLoS Med 2(6): e168.
==== Refs
References
Pauling L The significance of the evidence about ascorbic acid and the common cold Proc Natl Acad Sci U S A 1971 68 2678 2681 4941984
Douglas RM Hemilä H D'Souza R Chalker EB Treacy B Vitamin C for preventing and treating the common cold Cochrane Database Syst Rev 2004 2004 CD000980.pub2
Anderson TW Suranyi G Beaton GH The effect on winter illness of large doses of vitamin C Can Med Assoc J 1974 111 31 36 4601508
Ritzel G Kritische Beurteilung des Vitamins C als Prophylacticum und Therapeuticum der Erkältungskrankheiten Helv Med Acta 1961 28 63 68 13741912
Higgins JPT Thompson SG Deeks JJ Altman DG Measuring inconsistency in meta-analyses BMJ 2003 327 557 560 12958120
| 15971944 | PMC1160577 | CC BY | 2021-01-05 10:39:51 | no | PLoS Med. 2005 Jun 28; 2(6):e168 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020168 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597194510.1371/journal.pmed.0020169Health in ActionBioethicsOtherEmergency MedicineEpidemiology/Public HealthWomen's HealthPublic HealthInternational healthEmergency MedicineMedicine in Developing CountriesConfidentialityEthicsMedical journalsPrivacyHow the Cochrane Collaboration Is Responding to the Asian Tsunami Health in ActionTharyan Prathap *Clarke Mike Green Sally Prathap Tharyan is Professor of Psychiatry at the Christian Medical College in Vellore, Tamil Nadu, India, and Coordinator of the South Asian Cochrane Network. Mike Clarke is Director of the UK Cochrane Centre in Oxford, United Kingdom. Sally Green is Director of the Australasian Cochrane Centre at Monash Institute of Health Services Research, Monash University, Clayton, Australia. The views expressed in this article are those of the authors and are not necessarily the views of the Cochrane Collaboration.
Competing Interests: Prathap Tharyan is an editor with the Cochrane Schizophrenia Group, a reviewer and co-reviewer with other Cochrane collaborative review groups, and Coordinator of the South Asian Cochrane Network. These are not paid posts, but he has received funding from the Cochrane Collaboration and from John Wiley and Sons to host a meeting of the South Asian Cochrane Network and from the Cochrane Collaboration to attend annual colloquia. Mike Clarke is employed as Director of the UK Cochrane Centre and to work on systematic reviews. This employment depends on the value placed on the work of the Cochrane Collaboration and systematic reviews. Sally Green is employed as Director of the Australasian Cochrane Centre and is a member of the Cochrane Collaboration Steering Committee.
*To whom correspondence should be addressed: E-mail: [email protected] 2005 28 6 2005 2 6 e169Copyright: © 2005 Tharyan et al.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Addressing Psychosocial Needs in the Aftermath of the Tsunami
Meeting the Health Needs of Migrant Workers Affected by the Tsunami
Is the filming and photography of patients and dead bodies in hospitals during disasters ethically permissible?
How Women Were Affected by the Tsunami: A Perspective from Oxfam
Revisiting the South Asia Tsunami: Health Consequences of Flooding
After The Tsunami: Legal Implications of Mass Burials of Unidentified Victims in Sri Lanka
Tharyan and colleagues describe why the Cochrane collaboration made its library of systematic reviews freely available to affected countries.
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The powerful earthquake-triggered tsunami that devastated the coasts of many countries in two continents bordering the Indian Ocean on 26 December 2004 killed more than 280,000 people, displaced more than 1 million, and affected the lives of around 5 million more [1]. Unprecedented media coverage, in turn, triggered a worldwide outpouring of empathy, financial aid, and pledges of aid; the mobilisation of resources; and concerted action from governmental and nongovernmental organisations and international agencies such as the United Nations (Figure 1) and the World Health Organization. However, some of the well-meaning responses were not without drawbacks. There are concerns that the unregulated, uncoordinated, and poorly sustained activities of independent visiting health-care teams or individuals will undermine local health-care efforts [2].
Figure 1 The United Nations Population Fund Sends Aid to Those Affected by the Tsunami
Dr. Sanantha Wajewardena, the chief pharmacist at the Training Hospital in Galle, Sri Lanka, and two other pharmacists (background) unpack a shipment of essential safe-birthing supplies and surgical equipment supplied by the United Nations Population Fund to replace equipment that the hospital lost in the tsunami on 26 December 2004. (Photo: Joanne Ornag)
Six months after the tsunami, the attention of the media has largely shifted to other more pressing issues, leaving many unanswered questions about the appropriate response to such natural disasters. Is there a comprehensive list, prioritised and organised, of the health and social consequences of disasters? Do people have ready access to regularly updated evidence-based resources about the interventions relevant to such disasters? Is there a mechanism by which these resources could be made available to policy makers making decisions about the allocation of resources and interventions, as well as to people planning, providing, and receiving care in affected regions? Would these resources prove useful and, if so, which of the groups involved in disaster management should make use of them?
The Need for Evidence-Based Interventions in Disaster Management
A commonly used strategy in the wake of traumatic events is brief “debriefing”, both voluntary and mandatory. The aim of debriefing is to reduce immediate psychological distress as well as to prevent the development of psychological disorders, notably post-traumatic stress disorder (PTSD).
In the wake of the tsunami, many teams rushed to the Nagapattinam district, one of the worst hit areas of Tamil Nadu, the state in India with the largest number of casualties from the tsunami. These teams offered forms of brief debriefing to survivors in each village before rushing on to the next of the 73 tsunami-affected villages in the district.
Prathap Tharyan was part of a team summoned by the government of Tamil Nadu to provide psychosocial support. The team checked the evidence and found a relevant Cochrane systematic review on the effects of debriefing [3]. The Cochrane review had not found evidence that brief single-session debriefing reduced psychological morbidity but showed, instead, that limited evidence from one trial indicated a significantly increased risk of PTSD at one year in those receiving debriefing (odds ratio 2.88 [95% confidence interval 1.11–7.53]) [3]. Because of this review, we urged officials and nongovernmental organisations to desist from offering brief, single-session debriefing.
This message about debriefing was incorporated into the content of counsellor training workshops along with evidence for interventions that were supported by the results of systematic reviews and randomised controlled trials [4–6]. Recent surveys of parts of the Nagapattinam district suggest that PTSD is not a significant mental-health problem among adult survivors of the tsunami.
Similarly, other evidence-based interventions, such as the distribution of insecticide-treated bed-nets [7], have helped in the prevention of outbreaks of malaria and dengue. Well-meaning but misdirected and sometimes harmful interventions could be prevented if those making decisions about the nature of responses had access to reliable and up-to-date evidence of what works and what does not.
The Response of the Cochrane Collaboration
Shortly after the tsunami, it was felt that the Cochrane Collaboration, as the world's largest international organisation committed to providing good evidence about health care and with many members working in the region, had a moral duty to help in the global-relief and rehabilitation efforts. A working party was convened in early January 2005 of people in the region and elsewhere, with an E-mail discussion list and regular teleconferences aiding discussion and planning of initiatives. Further details, including a full list of the members of the working party, are available at http://www.cochrane.org/docs/asiancrisis.htm#response.
Prioritisation of reviews of relevant health-care interventions.
A disaster of this magnitude raises the spectre of epidemics of infectious diseases and many other potential health-care problems. The working party, in consultation with all Cochrane entities and around 200 individuals from affected countries listed as contributors to the work of the Cochrane Collaboration, and members of other agencies such as the World Health Organization, Oxfam, and the publishers of BMJ's Clinical Evidence, drew up a list of over 200 interventions considered relevant to health care in the aftermath of the tsunami.
These topics were further prioritised and grouped to ascertain which interventions currently had an up-to-date Cochrane review and which would need Cochrane reviews to be updated or even commissioned. This list will be modified as further input from other sources becomes available, and it may become a valuable resource for coping with the aftermath of other disasters and health-care emergencies. The relevant reviews should provide a valuable one-stop resource for people making decisions about health care in the future.
Disseminating the evidence.
The tsunami affected many countries where access to the Cochrane Library, which is available by individual or national subscription through Wiley InterScience (http://www.interscience.wiley.com/cochrane), is limited. The Cochrane Collaboration and John Wiley and Sons, the publishers, recognised the need to make Cochrane reviews more available and, so, agreed to provide free “one-click” access to all contents of the Cochrane Library for people in affected countries (Box 1) for a six-month period from February to July 2005 (http://www.thecochranelibrary.org). Governmental and nongovernmental agencies and institutions as well as individuals involved in health planning and health care in these countries now have access via the Internet to one of the best single sources of evidence on the effects of interventions likely to be useful in their efforts at no cost.
Box 1. Countries Affected by the Tsunami That Qualified for Free Access to the Cochrane Library
Bangladesh
India
Indonesia
Kenya
The Maldives
Malaysia
Myanmar (Burma)
The Seychelles
Somalia
Sri Lanka
Tanzania
Thailand
Evidence aid: Summaries of evidence-based interventions.
Members of the working party, aided by others in the Cochrane Collaboration, are preparing concise evidence summaries of systematic reviews of topics of high priority. These summaries cover interventions relevant to infectious diseases, injuries and wounds, rebuilding of communities and infrastructures, mental health, nutrition, rehabilitation, and pregnancy and childbirth. They are available at http://www.cochrane.org/docs/tsunamiresponse. If a summary is not currently available but there is a relevant Cochrane review in the Cochrane Library, a link takes people straight to that review. If a suitable Cochrane review is not available, links are included to other identified sources of evidence, in particular, to topics in the BMJ's Clinical Evidence (http://clinicalevidence.com).
Do We Know Enough to Deal Effectively with the Consequences of Disasters?
Sadly, the answer is “No, not nearly enough”. Of the topics in the list of the 200 or more interventions that are thought to be relevant to health care after a disaster such as the tsunami, there is an up-to-date, good-quality systematic review available for only a quarter of them. And, of these, not all have conclusions that can guide practice now because of a lack of relevant good-quality studies.
How, then, do we get the required evidence? The tsunami was a reminder that the divisions within and between nations as well as attempts to close our eyes and borders to problems abroad flounder in the face of the challenges posed by nature [8]. As the world prepares to debate strategies for global equity in health care and the millennium development goals at the G8 summit in July 2005 [9], the lessons learned from the tsunami should not be forgotten. Good-quality systematic reviews form the basis on which interventions should be implemented and on which new interventions should be planned and evaluated [10]. These reviews, however, are only as good as the studies they review. Adequate funding coupled with the necessary volunteers to prepare and maintain systematic reviews of relevant interventions, as well as pragmatic randomised controlled trials to fill the gaps indicated by these reviews, would complete the process initiated by the Cochrane Collaboration, and could well be one of the lasting legacies of the tsunami.
Citation: Tharyan P, Clarke M, Green S (2005) How the Cochrane Collaboration is responding to the Asian tsunami. PLoS Med 2(6): e169.
Abbreviation
PTSDpost-traumatic stress disorder
==== Refs
References
World Health Organization Three months after the Indian Ocean earthquake-tsunami: Health consequences and WHO's response 2005 Available: http://www.who.int/hac/crises/international/asia_tsunami/3months/en/index.html . Accessed 8 April 2005
Lee ACK The tsunami and the dangers of goodwill BMJ 2005 330 261
Rose S Bisson J Wessely S Psychological debriefing for preventing post traumatic stress disorder (PTSD) Cochrane Database Syst Rev 2002 2002 CD000560
National Institute for Clinical Excellence Post-traumatic stress disorder (PTSD): The management of PTSD in adults and children in primary and secondary care 2004 Available: http://www.nice.org.uk/pdf/PTSD_2ndcons_niceguideline.pdf . Accessed 27 April 2005
Ursano RJ Bell C Eth S Friedman M Norwood A Practice guideline for the treatment of patients with acute stress disorder and posttraumatic stress disorder Am J Psychiatry 2004 161 Suppl 11 3 31
Stein DJ Zungu-Dirwayi N van der Linden GJH Seedat S Pharmacotherapy for post traumatic stress disorder (PTSD) Cochrane Database Syst Rev 2000 2000 CD002795
Lengeler C Insecticide-treated bed nets and curtains for preventing malaria Cochrane Database Syst Rev 2004 2004 CD000363
Abbasi K Death by tsunami and poverty BMJ 2005 330 10.1136/bmj.330.7482.0-f
Labonte R Schrecker T Gupta AS A global health equity agenda for the G8 summit BMJ 2005 330 533 536 15746137
Clarke M Doing new research? Don't forget the old PLoS Med 2004 1 e35 10.1371/journal.pmed.0010035 15578106
| 15971945 | PMC1160578 | CC BY | 2021-01-05 11:13:38 | no | PLoS Med. 2005 Jun 28; 2(6):e169 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020169 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597194610.1371/journal.pmed.0020171Research ArticleNutritionNutrition and MetabolismObesityIntention to Lose Weight, Weight Changes, and 18-y Mortality in Overweight Individuals without Co-Morbidities Weight Loss and MortalitySørensen Thorkild I.A
1
Rissanen Aila
2
Korkeila Maarit
3
Kaprio Jaakko
3
4
*1Danish Epidemiology Science Centre, Institute of Preventive MedicineCopenhagen University Hospital, CopenhagenDenmark2Obesity Research Unit, Helsinki University Central HospitalHelsinkiFinland3Department of Public HealthUniversity of HelsinkiFinland4Department of Mental Health, National Public Health InstituteHelsinkiFinlandLudwig David Academic EditorChildren's Hospital BostonUnited States of America
Competing Interests: See Acknowledgments.
Author Contributions: TIAS, AR, and JK designed the study. JK analyzed the data and enrolled participants. TIAS, AR, MK, and JK contributed to writing the paper.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e17121 4 2004 25 4 2005 Copyright: © 2005 Sørensen et al.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Weight Loss and Mortality: What Does the Evidence Show?
Weight Loss and Mortality
Background
Weight loss in the obese improves risk factors for cardiovascular diseases and diabetes. However, several studies have shown inconsistent long-term effects of weight loss on mortality. We investigated the influence on mortality of intention to lose weight and subsequent weight changes among overweight individuals without known co-morbidities.
Methods and Findings
In 1975, a cohort of individuals reported height, weight, and current attempts (defined as “intention”) to lose weight, and in 1981, they reported current weight. Mortality of the 2,957 participants with body mass index ≥ 25 kg/m2 in 1975 and without pre-existing or current diseases was followed from 1982 through 1999, and 268 participants died. The association of intention to lose weight in 1975 and actual weight change until 1981 with mortality was analysed while controlling for behavioural and psychosocial risk factors and hypertension as possible confounders. Compared with the group not intending to lose and able to maintain stable weight, the hazard ratios (with 95% confidence intervals) in the group intending to lose weight were 0.84 (0.49–1.48) for those with stable weight, 1.86 (1.22–2.87) for those losing weight, and 0.93 (0.55–1.56) for those gaining weight. In the group not intending to lose weight, hazard ratios were 1.17 (0.82–1.66) for those who did lose weight, and 1.57 (1.08–2.30) for those gaining weight.
Conclusion
Deliberate weight loss in overweight individuals without known co-morbidities may be hazardous in the long term. The health effects of weight loss are complex, possibly composed of oppositely acting processes, and need more research.
Deliberate weight loss in overweight individuals without known illness is not obviously beneficial, and may be hazardous in the long term.
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Introduction
Weight loss in overweight and obese individuals leads to rapid improvement of the cardiovascular risk factor profile and reduced risk of developing type 2 diabetes. These observations have emerged from several studies of the general population [1–5] as well as from planned intervention studies [5–9]. In contrast, weight gain is associated with a worsened cardiovascular risk profile, increased risk of cardiovascular diseases and type 2 diabetes, and greater mortality from cardiovascular diseases and from all causes [5,10–13]. It would therefore be expected that overweight or obese individuals who lose weight would benefit from the effects leading to reduction or elimination of the excess mortality associated with overweight and obesity, which is largely attributable to cardiovascular disease and type 2 diabetes [5]. However, several prospective, long-term population-based studies have shown that weight loss, compared with stable weight in overweight or obese participants, is associated with future excess or unchanged mortality [10–13]. Moreover, cardiovascular diseases remain the main cause of mortality, even in studies that have taken other pertinent risk factors into account and eliminated confounding by diseases known to cause both weight loss and increased mortality [10–13].
The prevailing hypothesis to account for this apparent paradox is that the epidemiological studies have been unable to remove important confounding from several possible sources [14–18]. The excess mortality could be due to admixture of participants in the study populations with clinically manifest but undiagnosed diseases, sub-clinical diseases, or high-risk conditions or behaviours associated with both unintentional weight loss and excess mortality. Similarly, there may be participants with diseases or with high-risk conditions who have undertaken intentional weight loss in the hope of improving their health but who failed to eliminate the excess mortality, leading to so-called confounding by indication. A number of recent studies have included retrospective questions about whether an achieved weight loss was intentional or not [15,19–22]. The morbidity and mortality following intentional weight loss in these studies are equivocal and inconsistent, and they may also suffer from a variety of biases and confounding by indication [17,18]. One study from Israel [12], which included information on dieting for medical or slimming purposes prior to weight changes, found excess mortality associated with weight loss throughout the range of initial weight, although less excess mortality in the obese range. There was excess mortality in those who lost weight irrespective of whether it had been intended, but the study did not address this aspect specifically in the healthy overweight and obese groups. It is noteworthy that, contrary to its original aim, the large-scale, long-term clinical trial “Swedish obesity subjects study” comparing bariatric surgery to conventional treatment of obesity, in spite of considerable weight loss, improvement of risk factors, and reduction of diabetes incidence, has not yet found a reduced mortality [6].
In view of the rapidly rising prevalence of overweight and obesity almost everywhere, it is of paramount public health importance to know the long-term effects on mortality of the prevailing attempts to lose weight [5,17,18]. Randomised trials may be the best study design to address the effects of intended weight loss in selected groups such as the very obese, and among overweight or obese individuals with major co-morbidities or other high-risk conditions. However, such trials may not be feasible for addressing the problem of long-term effects in overweight and obese individuals who try to manage their body weight and who are still otherwise healthy and therefore have a much lower mortality in later life.
In the present population-based cohort study, we investigated whether intention to lose weight, as judged from actual attempts to lose weight at one point in time, and subsequent weight change among overweight or obese individuals were associated with subsequent long-term excess mortality. We selected individuals for whom there was no available evidence of diseases possibly linked to excess mortality or who were otherwise at increased risk of death, and we took into account lifestyle-related risk factors and any changes in them during the period of weight change.
Methods
The study was based on The Finnish Twin Cohort, which was composed of all same-sex twin pairs born in Finland before 1958 in which both twins were alive in 1967 [23]. The participants received questionnaires in 1975 and in 1981 addressing height, weight, lifestyle issues, and physician-diagnosed diseases, and the 1975 questionnaire included additional items about current attempts to lose weight. The response rate in the 1975 survey was 89%. Among those still alive at the 1981 survey and who had responded in 1975, the response rate was 91%. The present study sample was derived from the 19,993 participants who were alive at the end of 1981, when ages ranged from 24 y through 60 y. The further delineation of the study sample is illustrated in Figure 1.
Figure 1 Flowchart Shows the Delineation of the Study Sample by Various Exclusions
MI, myocardial infarction; COPD, chronic obstructive pulmonary disease
We aimed to exclude from the study sample participants suffering from any recognized chronic disease that could induce weight loss during 1975–1982 and subsequent increase in mortality. Thus, we excluded from the analyses participants who in the 1981 survey reported physician-diagnosed angina pectoris (n = 555), myocardial infarction (n = 170), or diabetes (n = 269), or who had angina pectoris according to standard chest pain history questions (n = 879). In addition, the cohort was linked to the national hospital discharge register, and participants with inpatient admissions for diabetes (n = 183), cardiovascular disease (n = 542) (except hypertension and venous disease), or chronic obstructive lung disease (n = 244) between 1972 and the end of 1982 were excluded. In addition, linking the cohort to a national drug prescription register allowed us to exclude participants who, based on medical certificates prior to 1983, had been granted reimbursable medication for somatic and psychiatric diseases. We excluded participants who had received prescriptions for all major chronic diseases (n = 1,119) except for hypertension. Presence of drug-treated arterial hypertension, as either reported in the 1981 survey or according to the medication reimbursement register, was rather common and therefore included as a covariate rather than used as an exclusion criterion. Participants who according to the Finnish Cancer Registry had diagnosed cancers before 1983 were excluded (n = 177). A total of 2,804 persons were excluded on the basis of known diseases. Finally, in order to take into account possible ill health not recorded otherwise, we excluded 1,274 participants who were not working in 1981 (“working” being defined as gainfully employed, working at home, or studying, and “not working” as being on early or disability pension (n = 356), unemployed (n = 461), or other (n = 457)), which is known to be associated with increased mortality.
Body mass index (BMI = weight/height2, kg/m2) was calculated from the reported height in the 1975 survey and from weight in both the surveys. Self-reported current height and weight generally have high accuracy [24,25], and the accuracy of the reports in the present study is supported by an analysis of another sub-study of The Finnish Twin Cohort [26]. The present analysis included only the participants who in 1975 were overweight or obese, defined as BMI ≥ 25 kg/m2, forming a total of 4,466 out of the 19,993 participants. These numbers were reduced to 3,152 out of 15,764 participants (with missing data in 151 participants) by the exclusions described above. Within this group, the median BMI was 26.7 kg/m2, the maximum BMI was 47.0 kg/m2, and 313 (9.9%) were obese (BMI ≥ 30 kg/m2). Recognizing that a change of 1.00 kg in body weight may have different implications for tall and short people, in analogy with body weight as such, we analysed weight changes as changes in BMI units rather than in kg. Weight loss and weight gain were analysed as separate continuous variables as well as categorical variables (the category weight loss was defined as any decline in BMI between 1975 and 1981, weight gain as an increase in BMI ≥ 1 kg/m2, and stable weight as no change in BMI or a gain less than 1 kg/m2).
In the 1975 survey, participants were asked whether they were currently trying to lose weight because of overweight, which was interpreted as “intention to lose weight,” and coded as yes/no. Then, if they were trying to lose weight, they were asked if they were doing so by voluntary restricting their food intake or changing their diet, increasing exercise, or by medication (coded yes/no in each case). No further details were requested, and these questions were not asked in the 1981 survey.
The lifestyle factors were recorded at both surveys and used in the analysis both as originally coded and recoded in simpler categories in order to avoid overloading the multivariate models. Smoking habits were originally coded as never smoker, occasional smoker (i.e., more than 10 packs of cigarettes in the lifetime, but never daily), former regular smoker, and current smoker. This variable was recoded as current smoker, yes/no. For alcohol drinking, the original question was used: five bottles of beer, a bottle of wine, or half a bottle of spirits on a single occasion at least once a month, yes/no; average alcohol consumption in g/d was also estimated. Physical activity was primarily assessed via an item on physical activity during leisure time with four response levels (i.e., equivalent to walking, alternating between walking and jogging, jogging, or running), but was recoded as engaging in physical activity more intense than walking, yes/no, which has previously proven adequate in relation to mortality analysis [27,28]. Life satisfaction was coded as being dissatisfied, yes/no, using a cut-point on a four-item dissatisfaction scale (range four to 20 points): yes if 12 or more, no if four to 11 [29]. Each of these risk factors were then categorized as being present both in 1975 and 1981, only in 1975, only in 1981, or absent at both surveys, which was coded by three dummy variables [28]. Work status (coded as gainfully employed, working at home, unemployed, studying, or other) and income (coded as eight levels) in 1975 were also included as potential confounders. Some of this information was missing in 195 participants, which led to the final sample size being reduced to 2,957 participants (Figure 1).
The participants were followed up until death, or the end of 1999, and 268 died. Underlying causes of death were available from Statistics Finland (http://www.stat.fi/index_en.html). The ascertainment of causes of death was based on forensic autopsy in 40% of cases. The age-sex adjusted mortality during follow-up of those included in the analysis was significantly lower than of all overweight participants replying to the two questionnaires (hazard ratio [HR] = 0.42, 95% confidence 0.36–0.49).
Twin analyses of the genetic and environmental contributions to BMI, change in body weight, intention to lose weight, and mortality have been carried out using the same cohort data [27,28,30,31]. The selection of the study sample for the present study implied that 2,067 participants out of the final sample of 2,957 did not have the co-twin included in the sample. Only six pairs were concordant for death, i.e., both died during the follow-up period. The within-pair correlations of phenotypes were adjusted for in the statistical analyses (see below).
Statistical analysis
Total mortality from 1982 through 1999 was analysed by the Cox proportional hazards regression model with the number of days since the 1982 survey as the time scale using Stata software [32]. The estimated regression coefficients and standard errors were converted to HRs with 95% confidence intervals (CIs). Two types of models were analysed: a basic and a fully adjusted model. The basic model included sex and age (in 1981), an indication of whether the participants had attempted to lose weight (in 1975), the weight-change variables, and the BMI (in 1975). The validity of the model was assessed by running separate analyses, in which the covariates for sex, age, initial BMI (1975), and current smoking (1981) were each used for stratification; these analyses showed that the estimated effects of the variables of interest were reasonably consistent across the strata. In another series of analyses, we kept the covariates in the model together with the interaction terms between the variables of interest and the covariates, which showed that such interaction terms were not needed. The fully adjusted model additionally included the covariates for hypertension, smoking, alcohol drinking, physical activity, life satisfaction, work status, and income. For each of the covariates for which simplified recoding of the original variable was performed, models with the original and with the recoded version were estimated to assess if the simplified recoding showed indications of residual confounding of the effects of variables of interest, which was not the case. Therefore, only results from the analysis of the simplified, recoded covariates are presented. In both the basic and the fully adjusted models, age in 1981 was also used as a built-in stratification variable (age intervals 24–29, 30–34, 35–44, ≥45 y), allowing age to be modelled as a continuous variable within these age intervals as well.
In both types of models, we estimated HR for the main effects on mortality of intention to lose weight, and of weight change, and the separate effects of the 2 × 3 combinations of intention to lose weight and weight change, using the group not intending to lose weight and remaining stable in weight as the reference group (HR = 1.0). The intention to lose weight and the actual weight changes were then tested for interaction between them in their association to subsequent mortality (tested by the difference in global likelihood ratios of the models with and without the interaction terms). Thereafter, we estimated the differences in effects of weight changes within each of the groups intending and not intending to lose weight, and the differences in effects of intention versus no intention within each of the weight-change groups. The influences of the methods used to the weight-loss attempts were assessed by estimating the effects of weight loss versus stable weight among those who stated that they used diet as the method and among those using exercise as the method.
In order to evaluate the effects of sex, age, and follow-up time on the HR of interest, separate fully adjusted models were analysed within the two sexes, age strata < 45 and ≥ 45 y, the combination of sex and these age strata, and for the first 5 y of follow-up and the period thereafter.
The proportionality of hazards and linearity of effect of the continuous variables were checked. Using the available “cluster” option in the Stata program, correlated observations in twin pairs included in the final sample were adjusted for in the estimation of the standard errors of the HR and the p values. Two-tailed p-values below 0.05 were considered statistically significant. Using the Stata program, survival functions were estimated from the Cox regression models for the groups of interest while adjusting for relevant co-variables (sex, median age, and median BMI).
Results
The numbers of participants were distributed by weight-loss intention in 1975 and weight change between 1975 and 1981 as shown in Table 1, which also shows their mean age at the start of mortality follow-up in 1981. Baseline BMI and the median and range of weight changes (in BMI units) in each group are shown in Table 2 for all participants and the 268 among the 2,957 participants who died during the follow-up. The weight changes were about the same irrespective of intention to lose weight and whether the participants died or survived the follow-up period.
Table 1 Number of Participants and Mean Age by Sex and Intention to Lose Weight in 1975 and Weight Change between 1975 and 1981 among 2,957 Overweight or Obese Participants (BMI greater than or equal to 25 kg/m2) Aged 24-60 y in 1981
Table 2 Median and Range of Baseline BMI by Intention to Lose Weight in 1975 and Weight Change between 1975 and 1981, and Median and Range of Weight Change (BMI units kg/m2) for Each Group among Overweight or Obese Participants (BMI greater than or equal to 25 kg/m2) Aged 24-60 y in 1981
Note that 1 kg/m2 equals 2.25 kg in a person 1.5 m tall and 3.80 kg in a person 1.95 m tall. Numbers of deaths by group are given in Table 4.
Analysis of Main Effects of Intention to Lose Weight and of Weight Changes
Intention to lose weight as such had no influence on mortality in the follow-up period, the HR in the basic and fully adjusted models being 0.86 (CI 0.66–1.12) and 1.00 (CI 0.75–1.32), respectively, compared with no intention to lose weight (HR = 1.0).
Both those who gained weight and those who lost weight had increased mortality compared with the weight-stable group (HR = 1.0). In the basic and fully adjusted models, the respective HR for the weight losers was 1.43 (CI 1.06–1.92) and 1.40 (CI 1.04–1.90). For the weight gainers, the HRs were 1.46 (CI 1.06–2.02) and 1.38 (CI 1.00–1.92), respectively. Weight changes were also analysed as continuous variables (divided in weight loss and weight gain, the latter including the category defined as stable weight, i.e., with a gain of less than 1.00 kg/m2). The HR for weight loss per BMI units estimated in the two models was 1.12 (CI 1.00–1.26) and 1.11 (CI 0.99–1.24), respectively. For weight gain, the HRs were 1.13 (CI 1.02–1.25) and 1.11 (CI 1.00–1.23).
Combined Analysis of Intention to Lose Weight and Actual Weight Changes
Table 3 combines intention to lose weight with the actual weight-change variable and compares each group with the group not intending to lose weight and able to maintain stable weight (HR = 1.0) during the 6 y from 1975 through 1981. There was significant excess mortality only in the group intending to lose weight in 1975 and who lost weight by 1981, and in the group not intending to lose weight and then gaining weight.
Table 3 HRs with 95% Confidence Intervals of Total Mortality between 1982 and 1999 by Intention to Lose Weight in 1975 and Weight Change between 1975 and 1981
The HRs were estimated by a basic and a fully adjusted Cox proportional hazards regression model, and the mortality of the group of participants not intending to lose weight and with subsequent stable weight served as reference for comparison.
The basic model included variables for sex, age (in 1981), and BMI (in 1975).
The fully adjusted model included, in addition, variables for hypertension, smoking, alcohol drinking, physical activity, life satisfaction, work status, and income.
The group “No, stable” was defined as the reference group assigned a HR = 1.00; NA, not applicable.
The basic and fully adjusted models produced approximately the same estimates, except for the similar increases by adjustments in HR for the groups intending to lose weight and actually losing weight or maintaining it. The HR for mortality following intended weight loss increased from 1.49 to 1.87 and became statistically significant (p decreased from 0.06 to 0.004) with the adjustments of the fully adjusted model. In stepwise rebuilding of the model, it was found that adjustment for smoking increased the HR from 1.49 to 1.66, and further adjustment for physical activity increased the HR from 1.66 to 1.85, indicating the importance of these two variables as confounders; the group with intent to lose weight and actual weight loss smoked less and exercised more than other groups.
Except for this difference between the basic and the fully adjusted models, the two models produced essentially the same results, wherefore only the results from the fully adjusted models are presented in the following.
The lowest mortality was found in the group intending to lose weight but who maintained stable weight, the HR being 0.84, although this was not statistically significantly different from the reference group (HR = 1.0) of weight-stable participants who did not intend to lose weight (p = 0.56).
There was a statistically significant interaction between intention to lose weight (yes/no) and the weight-change variable (loss, stable, gain) (p = 0.014).
The distribution of causes of death (Table 4) did not differ significantly between the six groups (global chi-square test p-value = 0.89), and the deaths in the two groups exhibiting excess mortality showed no distinct differences compared with the distribution overall. The distribution of the causes of death was in accordance with that expected from concurrent national statistics for the same sex and age groups (data not shown).
Table 4 Distribution of Underlying Causes of Death among the 268 Overweight or Obese Participants Who Died during the Follow-Up Period 1982 through 1999 by Intention to Lose Weight in 1975 and Weight Change between 1975 and 1981
The table gives the numbers of deaths (N) and the percentages within each group of those intending to lose weight and actual weight loss.
The ascertainment of causes of death was based on forensic autopsy in 40% of the deaths.
* In one case, cause of death was missing.
Analysis of Effects within Sex and Age Groups
The estimates of effects in the groups depicted in Table 3 showed generally the same pattern in men and women, in participants younger than 45 and participants 45 y or older, and in the three groups of younger and older men and older women (only 10 women younger than 45 y died, and none in the groups with no intention to lose weight and subsequent stable or increasing weight, so model-based estimates could not be produced). Table 5 shows that the estimated effects of weight change for those who intended to lose weight did exhibit the same pattern for the sex and age groups, and no statistically significant interactions by sex and/or age groups on mortality in the weight-loss group were found (all p-values > 0.23).
Table 5 HRs with 95% Confidence Intervals of Total Mortality between 1982 and 1999 within the Group of Overweight or Obese Participants Intending to Lose Weight by Sex and Age Group, for Those Who Did Lose Weight or Gained Weight between 1975 and 1981 Compared with Those Who Maintained Stable Weight (Reference Group)
The HRs were estimated by a fully adjusted Cox proportional hazards regression model.
The models were estimated on the basis of all participants who intended to lose weight. The fully adjusted model included sex, age (in 1981), and BMI (in 1975), hypertension, smoking, alcohol drinking, physical activity, life satisfaction, work status, and income.
Analysis of Effects of Weight Change within the Group Intending to Lose Weight
Among the participants intending to lose weight, those who lost weight showed excess mortality compared with those maintaining stable weight (HR = 1), the HR being 2.13 (CI 1.22–3.71). The corresponding HR for all cancer deaths as the outcome were 2.06 (CI 0.71–5.96), for all cardiovascular deaths, 2.04 (CI 0.64–6.49), and for all other natural causes, 1.31 (0.45–3.81). The participants who gained weight despite their intention to lose weight did not differ significantly from those who maintained weight, the HR being 1.06 (CI 0.56–2.01); also, none of the cause-specific outcomes were increased.
As seen in Figure 2, the excess mortality of the intentional-weight-loss group was numerically greater during the first 5 y of follow-up, during which period the HR was 6.26 (CI 0.33–118). After the first 5 y of follow-up, a statistically significant excess risk was still observed, the HR being 1.88 (CI 1.05–3.39).
Figure 2 Mortality by Weight Change in 1975–1981 among Those Reporting Trying to Lose Weight in 1975
Probability of survival from baseline in 1982 through 1999 among 1,058 participants who in 1975 reported intention to lose weight and who either lost weight, gained more than 1.0 kg/m2 in BMI, or remained stable, i.e., were unchanged or gained less than 1.0 kg/m2 in BMI, between 1975 and 1981.
The survival probability was adjusted using the Cox regression model for sex, median age, and median BMI. Note that the participants with weight loss had a lower survival rate throughout the 18 y of observation, whereas those with stable weight and weight gain did not differ.
When analysing weight loss and any gain as continuous variables among those intending to lose weight, the HR per BMI units were 1.27 (CI 1.07–1.49) and 1.03 (CI 0.87–1.23), respectively.
Analysis of Effects of Weight Change within the Group Not Intending to Lose Weight
Among the participants not intending to lose weight, those who gained weight showed excess mortality compared with those with stable weight (HR = 1.0), with the HR being 1.64 (CI 1.12–2.42). Those who lost weight did not differ significantly from those with stable weight, the corresponding HR being 1.19 (CI 0.83–1.72). This pattern of mortality was apparent throughout the follow-up period (Figure 3).
Figure 3 Mortality by Weight Change in 1975–1981 among Those with No Intention to Lose Weight in 1975
Probability of survival from baseline in 1982 through 1999 among 1,899 participants who in 1975 reported no intention to lose weight and who either lost weight, gained more than 1.0 kg/m2 in BMI, or remained stable, i.e., were unchanged or gained less than 1.0 kg/m2 in BMI, between 1975 and 1981. The survival probability was adjusted as in Figure 2. Note that the participants with weight loss had about the same survival rates throughout the 18 y of observation as those with stable weight, whereas those gaining weight showed a lower survival rate.
When analysing weight loss and any gain as continuous variables among those not intending to lose weight, the HR per BMI units was 1.02 (CI 0.87–1.19) and 1.17 (CI 1.02–1.34), respectively.
Analysis of Effects of Intention to Lose Weight within the Weight-Change Groups
Among the participants losing weight, those who intended to lose weight compared with those who did not intend to lose (HR = 1.0) showed a significantly increased mortality with a HR of 1.65 (CI 1.09–2.50) (in the basic model, the HR was 1.21 (CI 0.82–1.79), and this difference was due to the same confounding mentioned above and eliminated by the fully adjusted model).
Among participants who gained weight, those who had intended to lose weight had a lower mortality than those who did not intend to lose weight (HR = 1.0), the HR being 0.55 (CI 0.33–0.93). Participants who maintained stable weight had an insignificantly lower mortality if they had intended to lose weight, HR being 0.84 (0.49–1.48) (see Table 3).
Analysis of Effects of Methods for Intended Weight Loss and Actual Weight Loss
The eventual weight losses observed in each of the groups employing the various methods for losing weight at the beginning of the observation period were quite similar in magnitude (Table 6). When dieting was the intended method, those who lost weight compared with those who maintained stable weight showed significant excess mortality (Table 6). When exercise was used as the method, the HR for those who lost weight compared with those who maintained weight was increased but not statistically significant.
Table 6 HRs with 95% Confidence Intervals of Total Mortality between 1982 and 1999 within the Group of Overweight or Obese Participants Intending to Lose Weight by Reported Methods of Weight Loss, for Those Who Did Lose Weight between 1975 and 1981 Compared with Those Who Maintained Stable Weight
The HRs were estimated by a fully adjusted Cox proportional hazards regression model. Out of the 398 participants included in this analysis, 153 intended to use diet alone, 95 exercise alone, 140 both methods, five neither of the two (intended to use drugs only), and the intended method was unknown in five participants. 12 (of whom two died) intended to use some medication.
The models were estimated on the basis of all participants who intended to lose weight with the method indicated, irrespective of whether they lost or gained weight, and the group maintaining weight was used as the reference group (HR of 1.00).
The fully adjusted model included, in addition, variables for hypertension, smoking, alcohol drinking, physical activity, life satisfaction, work status, and income.
Discussion
In our study, both net weight loss and weight gain over a 6-y period among overweight or obese participants without known co-morbidities or high-risk conditions were associated with later increased long-term mortality, whereas attempts to lose weight at the beginning of this period in time, interpreted as intention to lose weight, by itself did not affect the long-term mortality. However, participants intending to lose weight and who experienced a net weight loss over the 6-y period had increased long-term mortality compared both with participants maintaining weight in spite of the intention to lose it and with participants with unintended weight loss. Those not intending to lose weight who later gained weight had increased mortality compared with participants maintaining stable weight and with participants gaining weight in spite of intending to lose weight.
Considering the arguments involved in the continuing controversy over the long-term health benefits of weight loss among overweight and obese individuals [14–18,22], the present study offers evidence of importance. Hitherto, excess long-term mortality after weight loss has been observed in several large-scale population-based prospective studies. Despite careful efforts to control confounding by risk factors and by underlying diseases associated with both weight loss and increased mortality, this excess long-term mortality has been attributed to inadequate control of such confounding [17,18]. The prevailing hypothesis has been that the obviously expected reduction in mortality after deliberate weight loss in the epidemiological observational studies has been masked by admixture of a large group of participants who, for various reasons, suffered from ill health and, as one sign thereof, had also lost weight.
A recent study, based on the National Health Interview Survey in the United States, addressed the question in a group of 6,391 overweight or obese participants at least 35 y old who were followed for 9 y after reporting on weight change and intention to lose weight during the past year [22]. Except for the finding that the lowest mortality was observed for those not trying to lose weight and who gained weight, the study yielded results in agreement with the prevailing expectation. It showed that intended weight loss compared with stable weight was associated with lower all-cause mortality, and that weight loss was associated with higher mortality only if it was unintentional. Furthermore, attempted weight loss as such was associated with lower mortality, independent of weight change; this suggests that confounding by medical indications was not a problem, but, on the other hand, that these participants may have been more healthy than those not intending to lose weight.
In this and most of the previous studies, the question about intention to lose weight was posed after the participants had achieved the weight loss, which implies a possible selection and recall bias. If overweight or obese participants who at the time of reporting are feeling healthy and have experienced a weight loss are more prone to consider that this was intentional, then a study design of this type may favour delineation of a particular group of participants with low morbidity and mortality [17].
Our study was based on a large sample of overweight and obese adult working-aged participants. We aimed to effectively minimize the effect of confounding due to underlying disease before and during the weight-loss period. Notably, those suffering from relevant illnesses, diagnosed by physicians and reported either by the participants themselves or in three national medical registers, were excluded or otherwise taken into account. The finding that those not intending to lose weight but who did lose weight did not experience excess mortality suggests that the exclusion of underlying diseases as a possible confounder was successful. In contrast to most previous studies on intentional weight loss [15,19–22], this study obtained the information about the intention to lose weight on the basis of a current attempt to lose weight before the actual subsequent weight changes were known and 6 y prior to the baseline of the follow-up. This timing eliminates the recall and selection bias that might hamper studies in which such information is obtained later. On the other hand, subsequent weight loss may not be a direct consequence of the intent to lose weight during the following 6 y, and similarly the weight loss among those not initially intending to lose weight may be due to a later intentional weight loss. These changes over time in possible causes of the weight loss imply that the observed associations in our study can be interpreted only as conservative predictions of the effects of intentional weight loss on long-term mortality. The finding that among those intending to lose weight mortality increased only if weight was actually lost, but not if it remained stable or increased after 6 y, suggests that the excess mortality following weight loss is not due to later regain in body weight.
The most likely behavioural and psychosocial risk factors that could have confounding effects—smoking habits, alcohol drinking, physical activity, life satisfaction, work status, and income—were included in the analysis. It is reassuring that the results (with a few exceptions) were essentially independent of the adjustment for these confounders in both the original versions and the simplified recoded versions, which minimizes the likelihood of residual confounding because of insufficient precision in their assessment. The fact that the study sample consisted of adult twin individuals is unlikely to have affected the results; twinship itself was taken into account in the statistical analysis. Moreover, the genetic influence on total mortality and its relation to body weight is small [33,34], and adult twins have the same overall mortality as singletons [35].
Several other aspects of the results qualify their interpretation. The findings that weight loss and weight gain, compared with stable weight, were both associated with increased mortality indicates that the present study sample of overweight individuals in this respect was similar to several others without overt disease in which the same relationships have been found [10–14]. The finding that intention to lose weight as such had no detectable effect on mortality suggests that this intention is not an indicator of the participants being especially concerned about health. In particular, it does not seem to be a marker of an overall healthier or, conversely, more hazardous lifestyle resulting in mortality different from that in participants not reporting attempts to lose weight. The finding that the excess mortality in the weight-loss group with initial intention to lose weight was present also after the first 5 y of observation and throughout the entire subsequent follow-up period also supports that the weight loss in the group intending to lose weight did not reflect effects of incident diseases or other health problem inducing the weight loss.
However, we cannot exclude the possibility that this group of participants was heterogeneous, with some being very healthy and health-conscious, and others for whom intention to lose weight was an indicator of insidious or imminent health problems. In view of the difficulties in maintaining deliberate weight loss during a 6-y period, the observation that this was in fact achieved could in itself be an indicator of the presence of underlying disease processes in some of the overweight or obese participants that could have motivated the intention to lose weight, facilitated its apparent success and maintenance, and led to later increase in mortality. If this is the explanation of the finding, then it must be symptomatic chronic diseases, ongoing for more than 6 y and escaping clinical diagnosis during this period in spite of the universal and practically free access to medical care in Finland. Moreover, the effect of such diseases on total mortality would have to be strong enough to completely eliminate any beneficial effects of intentional weight loss in the entire group and to be contributing to increased mortality throughout the observation period. Although this explanation would bring our results in line with the prevailing expectations, it seems implausible and possibly hazardous to rely upon.
The analysis of the causes of death did not indicate any single disease as responsible for all the excess mortality. Cause-of-death information has its well-known limitations, and we had relatively modest numbers by cause of death. It is likely that the effects of intentional weight loss is carried through to increased mortality by a more general deleterious effect on ability to cope with or resist the risks of incident diseases, which justifies that we focus on total mortality rather than cause-specific mortality.
One condition possibly contributing to the excess total mortality could be type 2 diabetes, which can remain undiagnosed for several years and may induce weight loss. However, analysis of supplementary information on later-onset diabetes suggests that occurrence of diabetes cannot explain the excess mortality among those intending to lose weight and who did lose weight (unpublished results, based on both a follow-up questionnaire in 1990, i.e., 8 y after the start of the follow-up, and on medical certificates for diabetes medication in the national drug prescription register for diagnoses covering 1983–1994). Another possibility is non-psychotic depression, but this was not supported by the analysis of causes of death, which showed no particular excess of suicides or of cardiovascular deaths. Moreover, the inclusion of the life satisfaction score, highly correlated to depression, in the analysis did not change the results.
Although we have no definite explanation of our finding of an excess mortality following intentional weight loss, we may consider some possibilities based on comparison with other studies [17]. Weight loss may release toxic substances from the fat tissue [36], but if this mechanism were important, we would expect excess mortality in the unintentional weight-loss group as well. We may assume that intentional weight loss as induced by deliberate calorie restriction has multiple biological effects—some potentially beneficial, some potentially harmful. The net effects on the health of participants may depend on their current condition. Thus, overweight and obese participants with clinically manifest or sub-clinical diseases such as type 2 diabetes may benefit from intentional weight loss, also in terms of mortality [37]. However, we may speculate that in overweight or obese participants without such co-morbidities or high-risk conditions, the harmful effects of intentional weight loss may predominate and lead to excess mortality. If so, the outcome of a study of the association of weight loss with mortality would then depend on the particular admixture of healthy and diseased overweight or obese participants. The privileged conditions for conducting this type of research in Finland may have secured the identification of a study sample with minimal co-morbidity, resulting in a more clear manifestation of the harmful effects of intentional weight loss and the lack of such effects of unintentional weight loss.
Intentional weight loss may induce loss of fat-free mass, which seems an unavoidable accompanying consequence [38–40]. In cohort studies with a single baseline measurement of fat mass and fat-free mass, mortality increases with the decrease in fat-free mass [41–44]. The interpretation is further supported by a detailed re-analysis of two large longitudinal population studies in the United States, addressing the relation between total mortality and weight change in comparison with the relation between total mortality and changes in skin-fold thickness as an indicator of changes in the amount of subcutaneous fat tissue [45]. This analysis showed that decline in skin-fold thickness (for a given weight loss) was associated with reduced mortality, whereas overall weight loss (for a given change in skin-folds) was associated with increased mortality.
Even if the method reported being used for the attempts to lose weight at the beginning of the observation period had not been sustained, the differences in mortality between them may provide useful suggestion for explanation of the overall findings. In the present study, the distribution of the excess mortality over many years of the follow-up period suggests that the reason is not the known hazardous effects of severely imbalanced dieting. Our finding that the excess mortality was worse in the group reporting using dieting than in the group reporting using exercise, is consistent with the possibly hazardous effects of loss of fat-free body mass. In large-scale population studies, the association of changes in lean body mass and physical activity is barely detectable [38]. It is likely that those reporting exercise as their weight-loss method had in fact been dieting to some extent, because it is very difficult to achieve weight loss by exercising alone [46,47]. On the other hand, using exercise as part of a controlled dieting-based weight-loss program may inhibit the tendency to lose fat-free body mass [39,48], which could explain why the deleterious effects were less in this group compared with those using diet. In this connection, it is important to note that in the present study sample, reported leisure exercise, as expected, had an overall strong beneficial effect on total mortality [27,28].
To understand the difference in mortality patterns associated with intentional and unintentional weight loss in the present study, we should also consider other mechanisms. On one hand, it seems plausible that unintentional weight loss may be associated with excess mortality in overweight or obese participants who suffer from some of the many diseases that may lead to both weight loss and increased mortality; these cases were excluded as effectively as possible. On the other hand, unintentional weight loss in overweight or obese participants without co-morbidities may be interpreted in a different way. We need to allow for the possibility that the size of the fat mass may change by mechanisms other than negative energy balance enforced by deliberate calorie restriction. Health-enhancing lifestyle changes such as increased physical activity or dietary modifications may lead to loss of body fat and increase in lean body mass. This may also be the explanation why weight gain in the group intending to lose weight was followed by a lower mortality than weight gain in the group not intending to lose weight. There appear to be still unknown factors contributing to weight fluctuations in healthy individuals, in whom weight changes in one direction were the strongest predictors of subsequent weight change in the opposite direction [49], i.e., prior weight loss best predicted future weight gain.
In conclusion, the long-term effects of weight loss are complex, and they may be composed of oppositely operating effects with net results reflecting the balance between these effects. We may cautiously suggest that future studies assess short-term advantages of planned weight loss against possible long-term risks, and assess if the optimal strategy among already overweight individuals could be to avoid further weight gain. If this is true, it puts a major emphasis on the need for prevention of development of overweight and obesity. This conclusion does not contradict the possible beneficial effects of planned weight loss in obese individuals who have already developed co-morbidities of their obesity, such as type 2 diabetes and symptoms of cardiovascular disease [50]. Obviously, more research will be needed to evaluate and explain our findings before they can be used as basis for advice about intentional weight loss in the large population of otherwise healthy overweight and obese individuals.
Patient Summary
Background
Although it seems obvious that when overweight people lose weight their health should improve, previous work has suggested that the relationship between weight loss and health may not be as simple as that. For example, it is difficult to control for all other possible things that might cause weight loss, such as other medical conditions that could then increase mortality.
What Did the Researchers Do?
They started with a population of 19,993 Finnish twins who were asked in 1975 about their weight and whether they intended to lose weight. In 1981, they were asked again about their weight, and then followed for up to 18 years to see if any died. The researchers took out of the analyses all the people who had illnesses, or those who had data missing. They analysed mortality against intention to lose weight in 1975 and actual change in weight. They found that those people who intended to lose weight and who actually did so had a slightly higher mortality than those who gained weight or whose weight remained the same. In people who did not intend to lose weight, gaining weight was associated with a slightly higher mortality.
What Do These Results Mean?
In the people studied here who were otherwise healthy and only moderately overweight, losing weight seemed to be associated with higher mortality. What makes these results quite difficult to interpret is that the actual number of people who died was not very high, but nonetheless intentional weight loss did not improve mortality. One reason for this result may be that when people diet to lose weight, they lose fat-free tissue as well as fat.
In people who have medical conditions related to obesity, losing weight is obviously desirable. But overall, preventing people, especially children, from becoming overweight in the first place seems crucial, since this work suggests that once weight is gained losing it again may not be good for health.
Where Can I Get More Information?
The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH) has several pages of information on weight control: http://www.niddk.nih.gov/health/nutrit/nutrit.htm
The International Association for the Study of Obesity has a Web site with information on obesity worldwide: http://www.iotf.org/
This study was supported by the Academy of Finland and the GenomEUtwin project (European Union Contract QLG2-CT-2002–01254). The Danish Epidemiology Science Centre is supported by the Danish National Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests. TIAS has been a member of the working group that prepared the governmental national action plan against the obesity epidemic. TIAS is currently president of the Danish Association for the Study of Obesity. TIAS has received research grants from the European Commission to conduct research in the aetiology, development, and treatment of obesity. TIAS has a contract with the research company Nestec, owned by Nestlé, to conduct metabolomic analysis aiming at identifying targets for obesity prevention and treatment. TIAS was recently included in the scientific advisory board of the local national branch of the drug company Sanofi-Aventis, which plans to market the new anti-obesity drug rimonabant. The other authors have declared that no competing interests exist.
Citation: Sørensen TIA, Rissanen A, Korkeila M, Kaprio J (2005) Intention to lose weight, weight changes, and 18-y mortality in overweight individuals without co-morbidities. PLoS Med 2(6): e171.
Abbreviations
BMIbody mass index
CIconfidence interval
HRhazard ratio
==== Refs
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| 15971946 | PMC1160579 | CC BY | 2021-01-05 10:39:54 | no | PLoS Med. 2005 Jun 28; 2(6):e171 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020171 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597194710.1371/journal.pmed.0020172Research ArticleObstetrics/GynecologyWomen's HealthObstetricsPregnancyWomen's HealthHigh Risk of Unexpected Late Fetal Death in Monochorionic Twins Despite Intensive Ultrasound Surveillance: A Cohort Study Intrauterine Death in Monochorionic TwinsBarigye Olivia
1
Pasquini Lucia
1
Galea Paula
1
Chambers Helen
2
Chappell Lucy
1
Fisk Nicholas M
1
*1Institute of Reproductive and Developmental Biology, Imperial College Londonand Centre for Fetal Care, Queen Charlotte's and Chelsea Hospital, LondonUnited Kingdom2Perinatal Pathology Unit, Department of HistopathologyHammersmith Hospital, LondonUnited KingdomBlickstein Isaac Academic EditorKaplan Medical CentreIsrael
Competing Interests: Nicholas Fisk is on the editorial board of PLoS Medicine.
Author Contributions: LC and NMF designed the study. OB, LP, PG, and NMF acquired data. OB, HC, LC, and NMF analyzed the data. OB, LP, PG, HC, LC, and NMF contributed to writing the paper.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e17222 12 2004 22 4 2005 Copyright: © 2005 Barigye et al.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Uncomplicated monochorionic diamniotic twins and the timing of delivery
Assessing the Risks of Twin Pregnancies
Background
The rationale for fetal surveillance in monochorionic twin pregnancies is timely intervention to prevent the increased fetal/perinatal morbidity and mortality attributed to twin–twin transfusion syndrome and intrauterine growth restriction. We investigated the residual risk of fetal death after viability in otherwise uncomplicated monochorionic diamniotic twin pregnancies.
Methods and Findings
We searched an electronic database of 480 completed monochorionic pregnancies that underwent fortnightly ultrasound surveillance in our tertiary referral fetal medicine service between 1992 and 2004. After excluding pregnancies with twin–twin transfusion syndrome, growth restriction, structural abnormalities, or twin reversed arterial perfusion sequence, and monoamniotic and high-order multiple pregnancies, we identified 151 uncomplicated monochorionic diamniotic twin pregnancies with normal growth, normal liquor volume, and normal Doppler studies on fortnightly ultrasound scans. Ten unexpected intrauterine deaths occurred in seven (4.6%) of 151 previously uncomplicated monochorionic diamniotic pregnancies, within 2 wk of a normal scan, at a median gestational age of 34+1 wk (weeks+days; range 28+0 to 36+3). Two of the five cases that underwent autopsy had features suggestive of acute late onset twin–twin transfusion syndrome, but no antenatal indicators of transfusional imbalance or growth restriction, either empirically or in a 1:3 gestation-matched case–control comparison. The prospective risk of unexpected antepartum stillbirth after 32 wk was 1/23 monochorionic diamniotic pregnancies (95% confidence interval 1/11 to 1/63).
Conclusion
Despite intensive fetal surveillance, structurally normal monochorionic diamniotic twin pregnancies without TTTS or IUGR are complicated by a high rate of unexpected intrauterine death. This prospective risk of fetal death in otherwise uncomplicated monochorionic diamniotic pregnancies after 32 wk of gestation might be obviated by a policy of elective preterm delivery, which now warrants evaluation.
Despite intensive fetal surveillance, structurally normal monochorionic diamniotic twin pregnancies were found to be complicated by a high rate of unexpected intrauterine death late in pregnancy.
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Introduction
Monochorionic diamniotic (MCDA) twin placentation occurs in one in every 400 pregnancies, and is characterised by placental vascular anastomoses and thus interfetal transfusion [1]. MCDA twins are considered high risk by virtue of their 3- to 5-fold increased perinatal morbidity and mortality compared to dichorionic (DC) twins. This is largely attributed to twin–twin transfusion syndrome (TTTS), which occurs in 15%–20% of MCDA twin pregnancies, and discordant intrauterine growth restriction (IUGR), which complicates an additional 25% [1–5]. Further, in the event of intrauterine death (IUD) of one twin, there is a 40%–50% risk of death or neurological damage in the co-twin from acute intertwin transfusion [6–8]. There is also a minor contribution to the risk of MCDA twin pregnancies from a 2- to 4-fold increased risk of structural anomalies, particularly congenital heart disease [9,10]. With the decline in perinatal mortality in singletons in recent decades, increasing attention is now being given to improving outcomes in high-risk multiple pregnancies.
The rationale for heightened fetal surveillance in monochorionic (MC) compared to DC twins is early detection of TTTS and IUGR, to allow timely treatment in an attempt to prevent adverse perinatal outcome, principally by amnioreduction, endoscopic laser ablation of anastomoses, bipolar cord occlusion, or early delivery. Although the frequency of monitoring has not been evaluated in randomised trials for any type of twin pregnancy, it is common practice to monitor MC twins more frequently than DC twins. Despite a vigilant policy in our centre of fortnightly ultrasound surveillance for MCDA pregnancies, we observed that unexpected late fetal deaths still occurred in seemingly uncomplicated MCDA twins.
We aimed to determine the prospective gestational age-specific risk of unexpected IUD in uncomplicated MCDA twins after viability (24 wk of gestation). We also investigated the cause of such deaths and whether they could be predicted antenatally.
Methods
We audited the perinatal outcome of all completed MC pregnancies seen over a 12-y period from 6 October 1992 to 31 August 2004, at the Centre for Fetal Care, Queen Charlotte's and Chelsea Hospital, a tertiary referral fetal medicine service in northwest London. Clinical details and ultrasound scan reports were retrieved from an electronic database (FileMaker Pro 5) of MC pregnancies, and augmented with case notes where necessary.
The term “uncomplicated” was used to denote pregnancies without features of TTTS on ultrasound that also had appropriate and concordant fetal growth, as well as normal growth velocity in each of two structurally normal twins. Appropriate growth was defined as an estimated fetal weight (EFW) greater than the fifth centile for gestational age [11]. The percentage EFW difference (ΔEFW) was calculated as the difference in weight, divided by the larger twin's weight, multiplied by 100, with growth defined as concordant if the ΔEFW was less than 25% [12]. Growth velocities were plotted against the charts of Chitty et al. [13–15]. The absence of TTTS in these pregnancies was denoted by normal and evenly distributed amniotic fluid volume (deepest vertical pool 2–8 cm in each sac) with concordant bladder dynamics [16]. These pregnancies also had normal umbilical artery (end diastolic frequencies present), umbilical vein (no pulsations), and/or ductus venosus (positive a wave) Doppler waveforms in each twin.
Uncomplicated MCDA pregnancies were monitored according to a standard protocol, which comprised routine first trimester nuchal translucency assessment and chorionicity determination, a detailed anomaly scan and fetal echocardiography at 20 wk, and then fortnightly scans for growth, amniotic fluid, and Doppler (umbilical artery, umbilical vein, and/or ductus venosus). Ductus venosus Doppler was added as a routine component in 1999 [16], and chorionic plate Doppler for compensatory artery-to-artery anastomosis (AAA) detection was added in 1995 [17]. Elective delivery was scheduled in otherwise uncomplicated pregnancies at between 36 and 37 wk of gestation.
We excluded pregnancies complicated by TTTS, IUGR, structural abnormalities, and twin reversed arterial perfusion, as well as high-order multiple, monoamniotic, and conjoined pregnancies. As this was a study of fetal death after viability, we also excluded pregnancies that delivered before 24 wk of gestation and those for which the pregnant woman returned to her local hospital for continued monitoring for geographical reasons. Six pregnancies with unavailable outcome data were also excluded.
The rate of fetal death in continuing uncomplicated MCDA pregnancies was derived for each 2-wk gestational block, beginning at 24 wk. It was calculated as the number of IUDs that occurred within the 2 wk following the beginning of week n divided by the number of continuing uncomplicated pregnancies at the beginning of week n. As we have previously applied to singleton pregnancies, the prospective risk of IUD was calculated as the total number of IUDs at or beyond the beginning of week n divided by the number of continuing pregnancies at or beyond the beginning of week n [18]. Prospective risk was not determined after 36 wk, as elective delivery resulted in too few continuing pregnancies beyond this time point.
To ensure that no features of IUGR or TTTS had been missed in pregnancies with an IUD, we compared antenatal indicators of these conditions at the last scan before the IUD in affected pregnancies with those in uncomplicated MCDA pregnancies unaffected by an IUD. The variables examined for IUGR were the abdominal circumference, head:abdominal circumference ratio, EFW, and ΔEFW. For TTTS, the amniotic fluid index (AFI) was used as the antenatal indicator; the deepest vertical pool of liquor in each twin's sac could not be compared, as they were not consistently recorded when the AFI was reported as normal and evenly distributed. The comparison used a 1:3 gestation-matched case versus control design (cases are pregnancies with an IUD; controls are those without). Variables in cases were from the last scan before the IUD; control scans were from the next three uncomplicated MCDA pregnancies appearing in the database for which the scan matched within 2 d of the case scan.
As the presence of an AAA reduces the risk of TTTS by a factor of four [17], and reduces severity in affected cases [19], we also compared the frequency of AAA detection by Doppler between cases and controls to determine whether functional AAAs were less common in pregnancies with an IUD; if so they would be more prone to develop TTTS than those unaffected by an IUD.
Finally, we reviewed the autopsy reports for any undetected pathology. In particular, we sought phenotypic features of TTTS such as organ hypertrophy, particularly cardiac hypertrophy in the larger twin.
For each variable in the 1:3 gestation-matched case–control comparison, we obtained three Δ values for every case by calculating the differences between the case and three control values. The mean and 95% confidence intervals (CIs) were then calculated for the Δ values (case variable minus control variable). Normally distributed variables were compared by one-sample t testing, and categoric variables by the Fisher exact test. Binomial CIs were calculated for proportions.
Results
The total number of MC pregnancies during the period of study was 480, from which we excluded 164 with TTTS, 62 with IUGR, 27 with structural abnormalities, 21 monoamniotic pregnancies, 14 high-order multiples, nine with twin reversed arterial perfusion sequence, and two with conjoined twins. From the remaining 181 uncomplicated MCDA pregnancies, we excluded four that delivered before 24 wk, 20 referred back to their local hospitals for continued monitoring, and six for which delivery data were unavailable. The final study population was 151 apparently uncomplicated, intensively monitored MCDA twin pregnancies. Monochorionicity was the indication for referral in each case, and median maternal age was 32 y (range 16–43).
From this cohort of 151, there were ten unexpected fetal deaths in seven uncomplicated MCDA pregnancies (three double deaths and four single deaths) after 24 wk, giving an overall incidence of 4.6% per pregnancy (CI 1.9%–9.9%). Deaths occurred at a median gestation of 34+1 wk (range 28+0 to 36+3).
Table 1 shows the rate by 2-wk blocks of fetal death per pregnancy, which, with the limits of small numbers, appeared to increase after 32 wk. The prospective risk of unexpected fetal death, also detailed in Table 1, remained relatively stable between 24 and 34 wk of gestation, 1/22 (4.5%) and 1/30 (3.3%) pregnancies, respectively.
Table 1 Rate and Prospective Risk of Unexpected Fetal Death in 151 Intensively Monitored, Uncomplicated MCDA Pregnancies after 24 wk of Gestation
DOI: 10.1371/journal.pmed.0020172.t001
Ultrasound findings on the last scan before IUD diagnosis and the clinical presentation at IUD are detailed in Table 2. Apart from one case that presented with reduced fetal movements, the finding of an IUD was incidental in six of the seven pregnancies at a routine scan. Growth was appropriate for gestational age and concordant, and the AFI was normal and evenly distributed in all seven affected pregnancies at the last scan prior to the diagnosis of IUD. All fetal deaths were diagnosed within 2 wk of a normal scan (median 1+4 wk, range 1+0 to 2+0).
Table 2 Antenatal Features of Pregnancies with an IUD (n = 7)
DOI: 10.1371/journal.pmed.0020172.t002
In the 1:3 gestation-matched case–control comparison, there was no significant difference between IUD-affected pregnancies and controls in any of the antenatal indicators of IUGR or TTTS. The mean Δ value for the abdominal circumference was 20 mm (CI −58 to +98), for the head:abdominal circumference ratio was −0.02 (CI −0.04 to 0.00), for EFW was −10 g (CI −142 to +122), for ΔEFW was −0.5% (CI −4.8 to +3.8), and for AFI was 1.2 cm (CI −1.4 to +3.8). AAA detection rates were similar in the two groups (54% in pregnancies with IUD and 60% in matched controls without an IUD). All the IUDs occurred in pregnancies monitored after the introduction of routine chorionic plate Doppler for AAA detection, as similarly did the selected 3:1 control pregnancies.
The post-mortem and placental findings are summarised in Table 3. After autopsy, deaths remained unexplained in three of five pregnancies (seven fetuses). However, two cases (A and F) had post-mortem evidence of TTTS, with the larger plethoric twins having cardiac hypertrophy, a feature that distinguishes TTTS from acute agonal intertwin transfusion, a common sequela of single IUD. In case F, a specific placental cause for sudden late onset TTTS was identified, both macroscopically and at histology, in the form of recent thrombosis in the recipient arterial component of a recipient–donor (compensatory) arteriovenous anastomosis. In case A, an AAA, which would otherwise decrease susceptibility to TTTS, was not detected antenatally nor on placental pathology, and hence the late onset of TTTS was unexplained. Both these double IUDs took place within 10–14 d of a normal scan, where the AFIs had been 16 and 17 cm and documented as evenly distributed, with fetal weight discordances of only 11% and 14%, respectively (Table 2). Thus, although these two double deaths could not be described as “unexplained” at autopsy, they were still unanticipated by serial fetal surveillance and thus “unexpected”.
Table 3 Autopsy and Placental Findings after IUD
DOI: 10.1371/journal.pmed.0020172.t003
Discussion
We investigated the prospective risk of unexpected IUD in MCDA pregnancies with structurally normal fetuses, uncomplicated by TTTS or IUGR, which has not to our knowledge been reported in the literature to date. Population-based twin studies provide little information of relevance to this question, as they fail to stratify for chorionicity, fail to distinguish complicated from uncomplicated twin pregnancies, and record gestational age not at fetal death but at delivery, often many weeks later [20,21]. Indeed the prospective risk of stillbirth in twins overall has yet to be addressed in the literature. Older studies in both singletons and twins have concentrated on overall risks of fetal death as a proportion of total births, or weekly rates, rather than deriving prospective risks by gestation as a function of continuing pregnancies [22–24]. We have previously applied the latter approach to singletons to illustrate how prospective risk declines with gestational age and is a more useful measure both for patient counselling and timing of delivery, as it expresses the risk of fetal death at any particular point in gestation for the remainder of the pregnancy [18].
Sebire et al. suggested that the main risk of fetal death in MC pregnancies was before 24 wk, as after this, the rate of perinatal loss was only slightly higher in MC than in DC pregnancies (4.9% versus 2.8% of pregnancies) [3]. This, however, was a referral population with a high incidence of TTTS and other pathologies, and no recent ultrasound in many cases, and so is not comparable with our study of otherwise uncomplicated MCDA pregnancies which suffered an unexpected death within 2 wk of a normal scan.
Our data suggest instead that even intensively monitored, apparently healthy MCDA pregnancies remain at substantial risk of IUD after 24 wk (4.6% of pregnancies and 3.3% of fetuses). IUDs after 24 wk occurred in the third trimester, and predominantly after 32 wk of gestation, at which time the prospective risk of subsequent IUD was 1/23 pregnancies. Our findings provide useful information for counselling parents with MCDA pregnancies, and notwithstanding the limitation of small numbers, may be used to inform decisions regarding the optimal timing of delivery.
Analysis of the antenatal findings on fortnightly scans shows that deaths after 24 wk occurred unassociated with any antenatal evidence of either TTTS or IUGR, and thus were unexpected. Only two of the seven pregnancies complicated by an IUD had a definite cause at autopsy. Late onset TTTS is rare but not unknown, and here seems to have been unpredictable. Recent case reports [25,26] suggest that thrombosis of a previously patent compensatory anastomosis, either AAA or reverse arteriovenous, can result in haemodynamic imbalance and thus acute onset TTTS, as suggested in one of the two cases in this study. The fetal deaths in the present study occurred despite strategies aimed at preventing them, specifically, fortnightly ultrasound and Doppler surveillance in a tertiary fetal medicine unit, and elective delivery at 36–37 wk).
We acknowledge a number of limitations of our study. First, there is the retrospective nature of the analysis. Notwithstanding this, clinical and ultrasound findings were entered prospectively into our database, and we reviewed all cases entered over the 12-y period since its inception. Second, caution is advised in view of small numbers. However, this is the first series to our knowledge to report this outcome in otherwise normal MCDA twins, and the deaths were spread relatively evenly over the study period.Third, there is a lack of comparative data in other twin pregnancies. Use of a DC control group was precluded by they fact that our protocol for DC pregnancies differs from that for MCDA pregnancies, as in most centres, with DC monitoring performed outside the fetal medicine unit and at less frequent intervals (every 4 wk rather than every 2 wk), and with elective delivery 1 to 2 wk later than for MCDA pregnancies. In terms of complicated MC twin pregnancies, few with TTTS or IUGR and two live fetuses remain undelivered after 32 wk. We chose not to use the brain:liver ratio as an indicator of IUGR at post-mortem because of its insensitivity [27]. Birth weights were also not used as an indicator of discordant IUGR in view of the variable interval from death to diagnosis, and the even greater interval between death and delivery (up to several weeks) following single IUD, in which varying degrees of post-mortem weight loss and continued growth of the surviving twin might confound measured birth weights.
Our findings have a number of clinical implications. The high rate of unexpected third trimester fetal death might be obviated by a range of preventative strategies. One would be to increase the frequency of monitoring. Although growth is only usefully measured every 2 wk, more frequent surveillance could include amniotic fluid volume and distribution, and fetal Doppler waveforms. This might allow earlier identification of the occasional case of late onset TTTS, and thus prevention of IUD through timely delivery. However, it is not clear how more frequent monitoring would prevent unexplained IUDs, just as this strategy does not appear to prevent them in singleton pregnancies [28,29].
Another preventative approach might be earlier delivery. Complicated MCDA twin pregnancies, such as those with TTTS or abnormal umbilical artery Doppler, are already delivered in many centres at 32 wk. At this stage neonatal survival is now comparable to that at term [30,31]. Minor neonatal morbidity is still likely at 32 wk, but the chance of major respiratory and neurological problems is reduced substantially by administration of antenatal steroids [32]. Elective vaginal delivery would increase the chance of failed induction, but there is an increasing view that elective caesarean section is preferable to preterm induction of labour in MCDA twin pregnancies, both to avoid a caesarean section being performed as an emergency and to obviate the risks of acute intertwin transfusion during labour [33]. Accepting that elective delivery of all MC twins at 32 wk would carry an attendant neonatal morbidity, the complications of iatrogenic prematurity could be lessened if an intermediate gestation were chosen, e.g., 34 wk. Although neonatal morbidity would be reduced, so would the number of fetal deaths prevented. Acknowledging the limitation of small numbers in this study, 80% of fetal deaths at 32 wk or greater and 60% at 34 wk or greater, respectively, would have been prevented if fetuses were delivered before these gestation time points. When expressed per pregnancy, one case of IUD would be prevented for every 23 MC pregnancies delivered at 32 wk and one for every 30 pregnancies at 34 wk. The above figures underestimate the potential gain of such strategy, because single IUD in MCDA pregnancy also exposes the surviving co-twin to a substantial risk of brain injury and thus long-term handicap from acute intertwin transfusion [6–8].
Major neurodevelopmental sequelae are now rare in otherwise well babies delivered after 32 wk. MC twins are at increased risk of cerebral palsy compared to DC twins, but this is mainly prior to 32 wk and in pregnancies complicated by TTTS and IUGR [34,35]. Prematurity per se is not considered responsible for the high risk of neurological morbidity in MC twins, but rather the haemodynamic imbalance unique to MC placentation [36,37]. Thus, uncomplicated MC twins should not be at substantially increased risk of neurological sequelae from elective premature delivery at or after 32 wk. Indeed, elective delivery may reduce their risk of neurodevelopmental injury, as single IUD in MC twins is a well-established risk factor for cerebral palsy [6,38].
In conclusion, apparently uncomplicated MCDA pregnancies remain at substantial risk of fetal loss as they approach 32 wk of gestation, whereafter one in 23 (4.3%) will suffer an unexpected IUD. This risk is exacerbated by the 40%–50% risk of co-twin death or neurological injury following a single IUD [6]. If our findings are confirmed in other observational series, our suggestion that earlier delivery might prevent this adverse outcome could be tested by randomised trial.
Patient Summary
Background
As mothers wait longer to have children and more of them make use of reproductive technologies (medications that stimulate egg production and ripening, and in vitro fertilization, for example), multiple pregnancies (mostly twins and triplets) have become a lot more common. Such pregnancies carry a higher risk for mothers and fetuses, and obstetricians have developed more involved regimens of check-ups and tests to minimize those risks.
Why Was This Study Done?
Guidelines for how to best manage multiple pregnancies are still under development. This study looked at how the risk of complications changed from 24 weeks of pregnancy (the earliest time point after which preterm babies can survive) on throughout the last trimester.
What Did the Researchers Do?
They looked at identical (monozygotic) twins that shared a common placenta but had their own amniotic sac (the sac that holds the protective liquid called amniotic fluid that surrounds the fetus inside the uterus). Twins with these characteristics make up about two-thirds of all monozygotic twins. The researchers studied the records of one United Kingdom hospital that specializes in fetal care over a 12-year period (from 1992 to 2004) and looked at the clinical details and ultrasound scan records.
What Did They Find?
Of a total of 480 completed identical twin pregnancies with shared placentas, 151 were found to be uncomplicated; that is, both fetuses seemed normal and healthy on all of their regular check-up scans (done every two weeks). In seven of those 151 pregnancies, however, either one or both of the fetuses died late during pregnancy, without any prior warning signs.
What Does This Mean?
Despite intensive surveillance of women during pregnancy, this particular group of pregnant women experienced a substantial rate of unexpected fetal death. The numbers in this study are small, however, and this is only one study (one needs to be careful about drawing conclusions based on one small study). In addition, the study was not designed to test a specific question but just looked at the records retrospectively. For all of these reasons, it is not clear whether these results are representative of what happens in general.
What Next?
Larger studies need to test whether this risk of fetal death late in pregnancy among identical twins sharing a placenta is common. If follow-up studies confirm the preliminary results here, it might be worth delivering such twins prematurely (that is, at around 32 weeks of pregnancy), when the risks associated with premature birth are relatively small compared to the risk of losing one or both of the fetuses after that date.
Additional Online Resources
Information about different types of multiple pregnancies, screening guidelines, and possible complications can be found at the following sources.
OMNI (Organising Medical Networked Information) Web page on multiple pregnancy: http://omni.ac.uk/browse/mesh/D011272.html
An editorial from the Medical Journal of Australia:
http://www.mja.com.au/public/issues/178_12_160603/ums10064_fm-2.html
Web page from the Health on the Net Foundation: http://www.hon.ch/Dossier/MotherChild/complications/complicate_multiple.html
Web Page from the University of Maryland Medical Center: http://www.umm.edu/pregnancy/specialcare/articles/multipreg.html
We acknowledge funding for our work on intertwin transfusion from the Richard and Jack Wiseman Trust, and the Institute of Obstetrics and Gynaecology Trust. These funding sources had no role in study design, collection or analysis of data, interpretation of results, or in writing of the report.
Citation: Barigye O, Pasquini L, Galea P, Chambers H, Chappell L, et al. (2005) High risk of unexpected late fetal death in monochorionic twins despite intensive ultrasound surveillance: A cohort study. PLoS Med 2(6): e172.
Abbreviations
ΔEFWpercentage estimated fetal weight difference
AAAartery-to-artery anastomosis
AFIamniotic fluid index
CI95% confidence interval
DCdichorionic
EFWestimated fetal weight
IUDintrauterine death
IUGRintrauterine growth restriction
MCmonochorionic
MCDAmonochorionic diamniotic
TTTStwin–twin transfusion syndrome
==== Refs
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Bejar R Vigliocco G Gramajo H Solana C Benirschke K Antenatal origin of neurologic damage in newborn infants. II. Multiple gestations Am J Obstet Gynecol 1990 162 1230 1236 2187353
Sebire NJ Snijders RJ Hughes K Sepulveda W Nicolaides KH The hidden mortality of monochorionic twin pregnancies Br J Obstet Gynaecol 1997 104 1203 1207 9333002
Lynch A McDuffie R Stephens J Murphy J Faber K The contribution of assisted conception, chorionicity and other risk factors to very low birthweight in a twin cohort BJOG 2003 110 405 410 12699803
Jain V Fisk NM The twin–twin transfusion syndrome Clin Obstet Gynecol 2004 47 181 202 15024284
Nicolini U Poblete A Single intrauterine death in monochorionic twin pregnancies Ultrasound Obstet Gynecol 1999 14 297 301 10623986
Fusi L McParland P Fisk N Nicolini U Wigglesworth J Acute twin–twin transfusion: A possible mechanism for brain-damaged survivors after intrauterine death of a monochorionic twin Obstet Gynecol 1991 78 517 520 1870813
van Heteren CF Nijhuis JG Semmekrot BA Mulders LG van den Berg PP Risk for surviving twin after fetal death of co-twin in twin–twin transfusion syndrome Obstet Gynecol 1998 92 215 219 9699754
Karatza AA Wolfenden JL Taylor MJ Wee L Fisk NM Influence of twin–twin transfusion syndrome on fetal cardiovascular structure and function: Prospective case-control study of 136 monochorionic twin pregnancies Heart 2002 88 271 277 12181221
Pasquini L Wimalasundera RC Fisk NM Management of other complications specific to monochorionic twin pregnancies Best Pract Res Clin Obstet Gynaecol 2004 18 577 599 15279818
Yudkin PL Aboualfa M Eyre JA Redman CW Wilkinson AR New birthweight and head circumference centiles for gestational ages 24 to 42 weeks Early Hum Dev 1987 15 45 52 3816638
Branum AM Schoendorf KC The effect of birth weight discordance on twin neonatal mortality Obstet Gynecol 2003 101 570 574 12636964
Chitty LS Altman DG Henderson A Campbell S Charts of fetal size: 4. Femur length Br J Obstet Gynaecol 1994 101 132 135 8305387
Chitty LS Altman DG Henderson A Campbell S Charts of fetal size: 3. Abdominal measurements Br J Obstet Gynaecol 1994 101 125 131 8305386
Chitty LS Altman DG Henderson A Campbell S Charts of fetal size: 2. Head measurements Br J Obstet Gynaecol 1994 101 35 43 8297866
Quintero RA Morales WJ Allen MH Bornick PW Johnson PK Staging of twin–twin transfusion syndrome J Perinatol 1999 19 550 555 10645517
Taylor MJ Denbow ML Tanawattanacharoen S Gannon C Cox PM Doppler detection of arterio-arterial anastomoses in monochorionic twins: Feasibility and clinical application Hum Reprod 2000 15 1632 1636 10875880
Cotzias CS Paterson-Brown S Fisk NM Prospective risk of unexplained stillbirth in singleton pregnancies at term: Population based analysis BMJ 1999 319 287 288 10426736
Tan TY Taylor MJ Wee LY Vanderheyden T Wimalasundera R Doppler for artery-artery anastomosis and stage-independent survival in twin–twin transfusion Obstet Gynecol 2004 103 1174 1180 15172849
Sairam S Costeloe K Thilaganathan B Prospective risk of stillbirth in multiple-gestation pregnancies: A population-based analysis Obstet Gynecol 2002 100 638 641 12383526
Kahn B Lumey LH Zybert PA Lorenz JM Cleary-Goldman J Prospective risk of fetal death in singleton, twin, and triplet gestations: Implications for practice Obstet Gynecol 2003 102 685 692 14550996
Luke B Reducing fetal deaths in multiple births: Optimal birthweights and gestational ages for infants of twin and triplet births Acta Genet Med Gemellol (Roma) 1996 45 333 348 9013999
Kilpatrick SJ Jackson R Croughan-Minihane MS Perinatal mortality in twins and singletons matched for gestational age at delivery at > or = 30 weeks Am J Obstet Gynecol 1996 174 66 71 8572036
Hilder L Costeloe K Thilaganathan B Prolonged pregnancy: Evaluating gestation-specific risks of fetal and infant mortality Br J Obstet Gynaecol 1998 105 169 173 9501781
Nikkels PG van Gemert MJ Sollie-Szarynska KM Molendijk H Timmer B Rapid onset of severe twin–twin transfusion syndrome caused by placental venous thrombosis Pediatr Dev Pathol 2002 5 310 314 12007025
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597194810.1371/journal.pmed.0020176Health in ActionBioethicsWomen's HealthInfectious DiseasesOtherEmergency MedicineEpidemiology/Public HealthHealth PolicyHIV/AIDSMental HealthSexual HealthPublic HealthEmergency MedicinePsychiatryHealth education (including prevention and promotion)International healthMedicine in Developing CountriesInfectious DiseasesConfidentialityEthicsMedical journalsPrivacyMeeting the Health Needs of Migrant Workers Affected by the Tsunami Health in ActionWilson David David Wilson is Medical Coordinator of Médecins Sans Frontières, Bangkok, Thailand. E-mail: [email protected]
Competing Interests: The author declares that he has no competing interests.
6 2005 28 6 2005 2 6 e176Copyright: © 2005 David Wilson.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Addressing Psychosocial Needs in the Aftermath of the Tsunami
How Women Were Affected by the Tsunami: A Perspective from Oxfam
Is the filming and photography of patients and dead bodies in hospitals during disasters ethically permissible?
Evidence Based Health-care Interventions after the Asian Tsunami: the response of the Cochrane Collaboration
Revisiting the South Asia Tsunami: Health Consequences of Flooding
After The Tsunami: Legal Implications of Mass Burials of Unidentified Victims in Sri Lanka
David Wilson of Médecins Sans Frontières, Thailand, discusses how the tsunami excacerbated the health status of Burmese migrants.
Burmese migrant workers in Thailand are a vulnerable group
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Following last December's tsunami, Thailand reported 10,385 deaths and missing persons among Thais and foreign tourists, 80% of them in Phang Nga province (Figure 1). These reported tsunami deaths do not include Burmese migrant workers, which one local organisation estimates as being as many as 1,000 individuals [1].
Figure 1 Four Provinces of Southern Thailand Most Severely Affected by the Tsunami
(Source: Thai Ministry of Interior Department of Disaster Prevention and Mitigation)
Medical relief to Thai nationals and Western victims was well organised [2], but Burmese migrants could not always benefit. This article briefly highlights how the tsunami affected this vulnerable group, exacerbating pre-existing difficulties in access to health care, and how Médecins Sans Frontières (MSF) is responding.
Migrant Workers in Thailand
Prior to the tsunami, 30,572 registered migrants, 98.6% of them Burmese, worked in fisheries, coastline construction, agriculture, and tourism in Phang Nga province [3].
Nongovernmental organisations estimate there are, in addition, an equal number of illegal migrants, who are in effect undocumented refugees. Migrant workers are vulnerable to exploitation, migration being largely unregulated and partially criminalized [4].
Access to Health Care
Registration requires participation in a health insurance scheme, giving registered migrants rights of access comparable to those of Thai citizens to Thailand's low-cost health-care system. Undocumented migrants cannot legally access health care. However MSF's observation is that sick Burmese, if they can speak Thai or are accompanied by a translator, do receive appropriate health care, whether registered or unregistered. Unregistered patients without money may be treated free for common complaints. Hospitals also subsidise more specialised treatment but approach the employer for a further contribution.
In practice, Burmese migrants rarely access even basic preventative health services such as vaccination. Very few married women we have met use any form of contraception, even if they do not want to become pregnant. Deliveries are at home, in poor conditions, often under the supervision of a traditional birth attendant.
Burmese to whom we spoke listed the following constraints to accessing adequate health care: (1) being unable to speak and read Thai, (2) not knowing what health services are available, (3) having fears about security, and (4) having no money for transport costs.
The Impact of the Tsunami
The tsunami killed employers and destroyed construction sites, leaving many migrants without work, and many returned to Burma or relocated to other areas of southern Thailand, either for fear of another tsunami or to find work. Police began arresting and deporting illegal migrants, but also registered migrants who may have lost their documentation [5]. MSF has met members of families who were split up in the deportation process.
Following a request to the Ministry of the Interior by international governmental and nongovernmental organisations on January 26th, these arrests and deportations have stopped, but a climate of fear remains—one month after the tsunami we met two Burmese with tuberculosis who had stopped treatment for fear of arrest whilst travelling to hospital.
Reconstruction in Phang Nga is proceeding slowly compared with other provinces [6], because of delay in distribution of government assistance [7], but as the reconstruction speeds up, further migrant workers are likely to arrive and problems to exacerbate. Community organisations already tell us of increasing numbers of migrant workers in Phang Nga, of whom more than half are unregistered.
Health-Care Priorities for Migrant Workers: High Levels of STIs
The need for post-tsunami assistance will be long term [8]. After supporting the Thai government's initial relief effort, MSF began to focus on strengthening the health-care system for migrant workers in Phang Nga. We had early contact with a community of 80 Burmese and Thai construction workers, at which point we referred two Burmese women with suspected AIDS (later confirmed) and one with gonorrhoea and pelvic inflammatory disease to hospital.
High levels of sexually transmitted infection (STIs) and HIV amongst Burmese migrants are unlikely to be confined to this site. Syphilis serology was positive (RPR+) in a high 6% of tested antenatal patients in MSF clinics in Yangon, Burma's capital, in 2004, having fallen from still higher rates in previous years following intensive STI prevention and treatment efforts.
STI rates are low in Thailand, with syphilis serology being positive (VDRL+) in only 0.18% of tested antenatal patients nationwide [9].
The low rate in Thailand is not a cause for complacency. Local nongovernmental organisations and migrant workers tell us that before the tsunami there were many sex workers along the coastal areas serving both migrant and host communities. Where destruction from the tsunami was less, we have seen that this behaviour continues. Lack of knowledge about STIs is a problem. Our first health education session for Burmese migrants revealed that none had ever used a condom; only four men knew anything about STIs: two of these had had an STI previously.
Conclusion
To improve knowledge and understanding of health by migrant workers, and their access to health care, we must first learn more about both the migrant and host communities and how they interact. Many migrants are undocumented, and some remain in hiding. We do not have a full appreciation of communities' own perspectives on health priorities. Working in this environment will require being sensitive to the concerns of other stakeholders, including employers who will want to know what a nongovernmental organisation is doing with their workforce.
For time given from busy schedules: Dr. Sombat Thanprasertsuk, Director, Division of AIDS, TB and STI, Thai Ministry of Public Health; Ass. Prof. Chanuantong Tanasugarn, Faculty of Public Health, Mahidol University, Bangkok; Mr. Wirat Phroplook, Director, Takau Pa District Health Office; and Mr. Sirin Srisuphan, Director, Thai Meuang District Health Office.
Citation: Wilson D (2005) Meeting the health needs of migrant workers affected by the tsunami. PLoS Med 2(6): e176.
Abbreviations
MSFMédecins Sans Frontières
STIsexually transmitted infection
==== Refs
References
Saydana Tsunami Situation report No. 1: Burmese migrant workers in Thailand 2005 1 6 Available: http://www.saydanatsunami.org/report.php?#report1 . Accessed 26 April 2005
Wacharong C Chukpaiwong B Mahaisavariya B After the tsunamis: Phang Nga, Thailand The Lancet 2005 365 203
International Organization for Migration United Nations High Commission for Refugees UNIFEM (United Nations Development Fund for Women) United Nations Office of the High Commission for Human Rights World Bank Technical Assistance Mission Report: Joint Tsunami Migrant Assistance Mission to the Provinces of Krabi, Phang Nga, Phuket and Ranong Thailand, 20–25 January 2005. (Available from author.) Accessed 01 May 2005
International Organization for Migration Migration in South East Asia 2003 Available: http://www.iom-seasia.org/index.php . Accessed 26 April 2005
Saydana Tsunami Situation report No. 2 2005 1 28 Available: http://www.saydanatsunami.org/report.php?#report2 . Accessed 26 April 2005
McGeown K Thailand's tsunami-hit tourism 2005 3 24 London BBC News Available: http://news.bbc.co.uk/1/hi/world/asia-pacific/4375815.stm . Accessed 29 April 2005
Srimalee S Tsunami: 100 days on, firms still await financial aid The Nation, Business p 2005 April 4 1B
Walker P Wisner B Leaning J Minear L Smoke and mirrors: Deficiencies in disaster funding BMJ 2005 330 247 250 15677661
Thai Ministry of Public Health Unpublished data, 29 April 2005, provided by Dr. Sombat Thanprasertsuk, Director, Division of AIDS, TB, and STI, Ministry of Public Health, Nonthaburi, Thailand
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597194910.1371/journal.pmed.0020177EssayBioethicsOtherEthicsPrivacyConfidentialityMedical journalsPublic HealthShould Health Professionals Allow Reporters Inside Hospitals and Clinics at Times of Natural Disasters? EssayBhan Anant Anant Bhan is at the Centre for Studies in Ethics and Rights (CSER), Mumbai, India, and is currently a Fogarty International Fellow at the University of Toronto Joint Centre for Bioethics (JCB), Canada. E-mail: [email protected]
Competing Interests: AB is a bioethics fellow studying in Canada and a public health physician from India, one of the countries most heavily affected by the tsunami. He is presently the recipient of a Fogarty International Fellowship.
6 2005 28 6 2005 2 6 e177Copyright: © 2005 Anant Bhan.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Addressing Psychosocial Needs in the Aftermath of the Tsunami
Meeting the Health Needs of Migrant Workers Affected by the Tsunami
Revisiting the South Asia Tsunami: Health Consequences of Flooding
How Women Were Affected by the Tsunami: A Perspective from Oxfam
Evidence Based Health-care Interventions after the Asian Tsunami: the response of the Cochrane Collaboration
After The Tsunami: Legal Implications of Mass Burials of Unidentified Victims in Sri Lanka
Journalists and health workers need to carefully consider whether it is ethical to show images of patients in medical settings, or of dead bodies in morgues.
Health professionals must protect their patients' dignity and privacy
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The tsunami that marked a solemn end to 2004 left behind unprecedented devastation. The world was shocked at the increasing casualty figures and the real-time images of the disaster brought by the news media. These included clips and photographs of dead bodies, grieving relatives, and suffering patients admitted to makeshift emergency wards.
The photographs did help in organizing a quick response from the rest of the world, as societal pressure led governments and relief agencies to respond with comprehensive relief measures. Graphic footage and newspaper headlines continue to dwell on this human tragedy. However, both health-care workers and journalists need to carefully consider whether it is ethical to show images of patients in obvious distress and undergoing medical attention in emergency camps, or of dead bodies in hospital morgues.
Ethical Guidelines
There are many published guidelines that journalists can turn to for guidance on the ethics of reporting. For example, the UNESCO International Principles of Professional Ethics in Journalism details the principle of respect for privacy and human dignity as an integral part of the professional standards of a journalist [1]. The Australian Journalists Code of Ethics calls upon journalists to respect private grief and personal privacy, and reinforces the right of journalists to resist their compulsion to intrude [2]. The Code of Ethics and Professional Conduct of the Radio-Television News Directors Association, the world's largest professional organization devoted exclusively to electronic journalism, expects professional electronic journalists to treat all subjects of news coverage with respect and dignity, showing particular compassion to victims of crime and tragedy [3].
During disasters, journalists and health professionals must respect patients' privacy (Illustration: Giovanni Maki)
However, there has been little consideration to date of the ethics of health-care staff allowing access to media inside medical institutions at times of natural disasters. In a 2003 editorial in the BMJ, which discussed “man-made” disasters such as war, Singh and DePellegrin questioned the use of footage of casualties from the Iraq war without the patients' consent [4]. An extensive debate followed publication of the commentary (see http://bmj.bmjjournals.com/cgi/eletters/326/7393/774); one of the views expressed was the need to show the world the extent of killing and maiming in the war (http://bmj.bmjjournals.com/cgi/eletters/326/7393/774#31147).
Media Coverage of the Tsunami: Benefits and Harms
In the post-tsunami scenario, the usefulness of the Internet and media was apparent. For example, a young Swedish child separated from his family was identified by his uncle on a hospital Web site and later reunited in an emotional moment with his father, who had been admitted to another hospital. The publication on government and hospital Web sites of the names of those admitted to hospitals, together with news releases, helped many identify their friends and relatives.
Furthermore, the aid response has been the largest of any disaster in history, which may have been due to the unprecedented media coverage. There has also been the advent of “disaster tourism”—the massive inflow of well-meaning, but often ill-organized, charitable organizations and aid volunteers to the tsunami-hit areas [5].
At the same time, the media coverage of wailing relatives and dead bodies lying in hospital morgues is deeply disturbing. The death of a loved one is a time for privacy and respect for the dead. As a South Asian, I am aware that in many communities the dead body is covered with a shroud that denotes purity. It is rare to photograph or film funerals. To infringe on the privacy of families when they are emotionally shattered is disrespectful to the living. Photographing and filming the deceased in various stages of undress and decomposition violates the dead and their dignitary rights, according to most cultures. In addition, the hordes of news media that descend on a hospital can hamper the efficiency of the medical staff providing emergency care, where even seconds are crucial.
The Role of Health Professionals in Protecting Privacy
Health professionals and administrators can and should control media access to hospitals and clinics. The public's right to information should not outweigh the right of victims of natural disasters to privacy, confidentiality, and dignity. Health professionals should be aware that the filming of patients under their care may be used not only for highlighting the extent of a disaster's human toll, but also for commercial purposes, such as selling programs and newspapers, and for raising funds. For these reasons, extreme caution should be used in giving permission to use images from inside hospitals in disaster-affected areas. Ideally, the consent of the patients or surrogate decision makers should be sought first.
It is now the ethical norm to seek consent of patients when photographs of them (or even of their body parts) are used in medical conferences or publications (see the guidelines on consent from the International Committee of Medical Journal Editors, at http://www.icmje.org/#privacy). A similar approach should be taken in the event of natural or man-made disasters. If photographs of the dead or those admitted to hospitals have to be publicized for identification purposes, this should be done keeping local sensibilities in mind.
It is difficult for health-care professionals to shoulder this social responsibility during a crisis when lifesaving measures come first. Community consent and monitoring through community leaders, tribal elders, or local authorities might be an option. Such community involvement would result in media coverage that would be socially and culturally acceptable. While the usefulness of documenting and transmitting such geographically and experientially diverse experiences around the world is undeniable, the terms of access for media have to be negotiated keeping the notion of consent central.
With the increasing focus in medicine and bioethics on individual rights, the right to privacy is pivotal. Doctors and other health professionals have a duty of care to their patients, which includes protecting their dignity and privacy. Ethical obligations of health professionals to monitor recording of images in health institutions need to be higher than those of society in other venues, such as the street or the beach.
It may be valuable for medical professionals to have a specific code, perhaps written by disaster-relief organizations (such as the Red Cross) together with the World Medical Association, that outlines how to deal with the media in disaster settings. Arguably, the universal obligation of health-care professionals and administrators to respect the privacy and confidentiality of their patients should suffice, but given the nature of realities on the ground in disasters and emergencies, a specific code would be useful.
Responsible Journalism
Responsible journalism in health-care settings at times of disaster, facilitated by guidelines that specifically address the ethical reporting of disasters and that are applicable universally across the world, will also help prevent exploitation of victims of a calamity. Such guidelines could be developed by a joint body comprised of international medical humanitarian agencies such as the Red Cross and Médecins sans Frontières (MSF), multinational agencies such as the United Nations, media representatives, and media watchdogs.
The guidelines need to be acceptable to the global media community and also need to be made binding. For example, sanctions could be imposed upon journalists (or their parent organizations) who ignore them, or perhaps only those journalists who have been accredited in “ethical reporting of disasters” should be given access to disaster sites.
The guidelines could also usefully be published together with a code for health professionals. An example of joint guidelines on ethical reporting on health issues for the media and health-care professionals are those adopted in Washington State (http://www.wsma.org/news/guide.html). These guidelines were jointly approved and prepared by media, publishers, broadcasters, and hospital and medical associations, and they could serve as a template for international guidelines on disaster reporting.
Conclusion
In disasters, the affected are often left with almost nothing and with negligible negotiating power. They might be left with only their pride and dignity, and they must not be robbed of that. Patients or affected families might not be in a condition to respond to encroachment on their rights. While health professionals want to facilitate recognition of their unidentified patients and also facilitate more aid to affected areas, they also have an enhanced responsibility to protect their patients' dignity and rights. We should not need to be voyeurs into the grief of vulnerable victims to launch an effective and humane response to any disaster.
AB would like to thank colleagues and faculty at CSER and JCB and the peer reviewer for helpful comments on this essay.
Citation: Bhan A (2005) Should health professionals allow reporters inside hospitals and clinics at times of natural disasters? PLoS Med 2(6): e177.
==== Refs
References
UNESCO International Principles of Professional Ethics in Journalism issued by the Fourth Consultative Meeting of International and Regional Organizations of Journalists in Paris (20 Nov 1983) Available: http://www.journalism-islam.de/konferenzen/codes.html . Accessed 10 April 2005
Media, Entertainment and Arts Alliance: Australian Journalists Association, Code of Ethics Available: http://www.nt.gov.au/pfes/media/aboutus/ethics . Accessed 15 April 2005
Code of Ethics and Professional Conduct of the Radio-Television News Directors Association Available: http://www.rtnda.org/ethics/coe.shtml . Accessed 15 April 2005
Singh JA DePellegrin TL Images of war and medical ethics BMJ 2003 326 774 775 12689950
IANS Tsunami spawns disaster tourism Times of India 2005 Jan 7 Available: http://timesofindia.indiatimes.com/articleshow/983383.cms . Accessed 10 Jan 2005
| 15971949 | PMC1160582 | CC BY | 2021-01-05 10:39:54 | no | PLoS Med. 2005 Jun 28; 2(6):e177 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020177 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597195010.1371/journal.pmed.0020178EssayBioethicsOtherEpidemiology/Public HealthWomen's HealthWomen's HealthMedicine in Developing CountriesPublic HealthConfidentialityEthicsMedical journalsPrivacyHow Women Were Affected by the Tsunami: A Perspective from Oxfam EssayMacDonald Rhona Rhona MacDonald is the editor of Oxfam.org.uk, the Web site of the development, relief, and campaigning organisation Oxfam, Oxford, United Kingdom. She is the lead of Oxdocs, a new initiative to enable doctors and medical students to get involved in the fight against poverty (http://www.oxfam.org.uk/doctors). E-mail: [email protected]
Competing Interests: RM is an employee of the charity Oxfam, but was not paid by Oxfam to write this article. She has financially supported Oxfam for 15 years and also financially supports other international humanitarian charities.
6 2005 28 6 2005 2 6 e178Copyright: © 2005 Rhona MacDonald.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Addressing Psychosocial Needs in the Aftermath of the Tsunami
Meeting the Health Needs of Migrant Workers Affected by the Tsunami
Is the filming and photography of patients and dead bodies in hospitals during disasters ethically permissible?
Evidence Based Health-care Interventions after the Asian Tsunami: the response of the Cochrane Collaboration
Revisiting the South Asia Tsunami: Health Consequences of Flooding
After The Tsunami: Legal Implications of Mass Burials of Unidentified Victims in Sri Lanka
When natural disasters hit poverty-stricken areas, women are more likely to be affected than men. MacDonald discusses the reasons for, and implications of, this gender disparity.
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In Oxfam's experience, disasters are profoundly discriminatory, even those that are “natural” rather than man-made. Factors that were present before a disaster, such as poor social conditions, mean that some people in the disaster zone will be more affected than others.
People living in poverty are much more vulnerable to the effects of natural disasters. As women account for 70% of the 1.3 billion people worldwide living in extreme poverty (less than $1 a day) (see http://www.oxfam.org.uk/what_we_do/issues/gender/introduction.htm), it follows that when natural disasters hit poverty-stricken areas, women are more likely to be affected than men.
Disproportionate Death Toll in Women
The tsunami decimated Southeast Asia on 26 December 2004, killing more than 220,000 people in 12 countries and leaving 1.6 million people homeless. According to a survey recently carried out by Oxfam, four times as many women than men were killed in the tsunami-affected areas of Indonesia, Sri Lanka, and India [1]. Some of the reasons for this are similar across these countries: women died because they stayed behind to look for their children and other relatives. Women in these areas often can't swim or climb trees, which meant that they couldn't escape.
Some cultural differences between men and women also contributed to the disproportionate death toll. Although traditionally many women go out to work in Aceh (Indonesia), the tsunami hit on a Sunday morning when they were at home and the men were out on errands and so away from the seafront. Women in India play a major role in fishing and were waiting on the shore for the fishermen to bring in the catch when the wave struck. In Batticaloa District of Sri Lanka, the tsunami hit at the time when women who lived on the east coast usually took their baths in the sea [1].
Gender-Specific Problems
The overcrowded camps (Figure 1) and resettlement sites for people who have been made homeless and the imbalanced male-to-female survival ratio have resulted in several gender-specific problems. For example, women in the camps are often verbally and physically harassed by men and are at risk of being sexually abused.
Figure 1 Camps and Resettlement Sites Are Overcrowded (Photo: Howard Davies, Oxfam)
It is also more likely that women will be pressured into marrying earlier than in the past and into having more children closer together, with significant implications for their education, livelihoods, and reproductive health. It is also more difficult for women to access money and emergency supplies because in some areas only men are recognised as head of the household, which has meant that women have been unable to collect entitled relief cash and goods [1].
All of the above problems are an escalation of the reality that women faced before the tsunami. For example, Oxfam has been working on gender issues in the Batticaloa District of Sri Lanka for four years. According to Shanthi Sivasanan, Oxfam programme assistant in the area: “Before the tsunami, there were some very serious issues regarding the level of violence against women in this area, and we need to continue considering this during our tsunami response. Our ongoing development programme was offering counselling to families and people affected by violence (whether through war or domestic violence, etc.), training for counsellors, paralegal training, and the protection of children.”
“From 2002 to 2004, we were aware of 400 cases of very critical domestic violence. We found that out of these 400, 150 women were physically injured in their own home every day. And 48 suffered mental harassment. Often, this was all due to stress and pressure in the home because of dowry payments, political issues, and poverty.”
The tight culture means that if women experience violence, they will never talk about it. Pathimalar, a survivor of the tsunami currently living in Vattavan Camp in Batticaloa District, said: “Many women, like me, are uneducated. We have a saying in Tamil which says, ‘Even if our husband is a stone, we worship him.’ We don't like to talk about harassments or what happens between man and wife. If something happens and we start to cry, or shout too much, we think it might create further problems and he starts to fight with us again.” Pathimalar explained how women deal with this situation: “Silently and patiently, we cry within us.”
Meeting Women's Needs in the Emergency and Recovery Phase
Oxfam's programme strategy is to assess the needs of women and men in all of its work. So in the relief phase, in addition to supplying shelter, mosquito nets, clean water, food, and cooking utensils, Oxfam distributed public-health packages that included toiletries and underclothes for women. Oxfam also consulted women in the camps about emergency water and sanitation interventions and also asked them about their specific needs. This has resulted in building well-lit toilets, where the women feel safe.
Immediately after the tsunami, it was very difficult for the survivors who had been made homeless to follow their domestic hygiene practices. Now that Oxfam has built clean latrines and distributed soap, health promoters have been reinforcing the basic rules of hygiene by using visual aids and drama.
According to Jastinahilari Ignatius and Jesudhas Yeogaraja, Oxfam health supervisors in Vattavan Camp, gender issues mean that they have to have separate groups for men and women when teaching health promotion: “There are some difficult gender issues here, and some of the women don't like to speak out in front of men, so we had separate groups for men and for women, with the result that all of the participants talked more than they would have done in a mixed group.”
Pregnant women living in the camps have a very uncomfortable time, according to V. Uijayaluxumi, a field health officer with Oxfam's partner, Sarvodaya, working in Vattavan Camp: “Many of the pregnant women have been really psychologically affected by the tsunami. They were scared for themselves and their families, but especially for their unborn children. Now they are [here] in camps and living in tents where it's hot, noisy, and away from familiar surroundings. There's just dust and tents here.”
“It is traditional here for women to use many herbs and remedies before and after giving birth, but they can't get these while they are in the camps. They use things like honey, tree bark, and leaves, etc., and there is one powder that is supposed to be good for the stomach and helps with feeding and sleeping.”
She also reported some good news: “One baby has already been born in this camp and is doing well, but there are nine more due before July.”
In the reconstruction work, Oxfam aims to ensure equal wages for equal work, regardless of gender. For example, in Culladore, India, Oxfam worked with the district administration to ensure that men and women were paid equally for the same work. Men, who were previously paid higher wages, resisted this initially, but Oxfam staff and partners talked to the community, and everyone eventually agreed that equal pay was the right thing to do [1].
There are some positive examples of governments recognizing the important role of women after the tsunami. For example, just after the tsunami, the governments of Tamil Nadu and Kerala (in India) implemented an initiative to post women fire officers, police officers, and doctors in the camps and affected villages. This helped to prevent violence against women and provided women survivors with a safer environment [1].
Conclusion: Gender Equality and Global Poverty
Oxfam bases its work on the common understanding that gender equality is the key to overcoming poverty and suffering (see http://www.oxfam.org.uk/what_we_do/issues/gender/policy.htm). The special needs of women—whether or not these needs are related to one of the biggest natural disasters the world has ever seen—must be urgently addressed by the international community. After all, world leaders agreed to a Millennium Development Goal to promote gender equality and empower women [2]. Those of us in the international health and development community must keep reminding these leaders of their commitment, and keep campaigning for some progress.
All interviews were conducted by Tori Ray at Oxfam.
Citation: MacDonald R (2005) How women were affected by the tsunami: A perspective from Oxfam. PLoS Med 2(6): e178.
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References
Oxfam The tsunami's impact on women 2005 Available: http://www.oxfam.org.uk/what_we_do/issues/conflict_disasters/downloads/bn_tsunami_women.pdf . Accessed 3 May 2005
United Nations UN millennium development goals 2000 Available: http://www.un.org/millenniumgoals . Accessed 3 May 2005
| 15971950 | PMC1160583 | CC BY | 2021-01-05 10:40:15 | no | PLoS Med. 2005 Jun 28; 2(6):e178 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020178 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597195110.1371/journal.pmed.0020179Health in ActionBioethicsOtherEmergency MedicineEpidemiology/Public HealthMental HealthWomen's HealthPsychiatryInternational healthPublic HealthConfidentialityEthicsMedical journalsPrivacyMedicine in Developing CountriesEmergency MedicineAddressing Psychosocial Needs in the Aftermath of the Tsunami de Jong Kaz *Prosser Sue Ford Nathan Kaz de Jong is a clinical psychologist and is mental health advisor of MSF Amsterdam. Sue Prosser is a psychotherapist and former field coordinator of MSF in Indonesia. Nathan Ford is the head of the medical unit of MSF in London.
Competing Interests: The authors declare that they have no competing interests.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e179Copyright: © 2005 de Jong et al.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
After The Tsunami: Legal Implications of Mass Burials of Unidentified Victims in Sri Lanka
How Women Were Affected by the Tsunami: A Perspective from Oxfam
Is the filming and photography of patients and dead bodies in hospitals during disasters ethically permissible?
Evidence Based Health-care Interventions after the Asian Tsunami: the response of the Cochrane Collaboration
Revisiting the South Asia Tsunami: Health Consequences of Flooding
Meeting the Health Needs of Migrant Workers Affected by the Tsunami
MSF discusses its response to tackling mental health problems in Aceh, Indonesia, and explores some of the main concerns in responding effectively to mental health problems in an emergency setting.
MSF shares its observations from its programme in Aceh, Indonesia
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On the morning of 26 December 2004, an earthquake of 9.2 on the Richter scale rocked the province of Aceh, Indonesia, followed soon after by a three-wave tsunami that smashed into the north, west, and topmost point of Aceh. The most heavily hit city, in terms of population, was Banda Aceh on the northernmost point of the Aceh peninsula, exposed between the north and west coast—two-thirds of the city was totally destroyed.
Médecins Sans Frontières (MSF) sent four teams consisting of medical, logistical, water and sanitation, and mental health workers (Figure 1), who undertook rapid assessments of the tsunami-affected areas in Aceh Province. This article outlines MSF's response to tackling mental health problems in the region, and discusses some of the main concerns in responding effectively to mental health problems in an emergency setting.
Figure 1 MSF Provides Emergency Medical Support in the Banda Aceh Province of Indonesia
In this province, MSF has five areas where teams are actively providing emergency medical support. Here in Meulaboh, 250 km west of Banda Aceh, MSF has started work in the local hospital as well as visiting the surrounding area with mobile teams. Half of the town was destroyed, and access to the area has been by helicopter or boat only. (Photo: © Francesco Zizola/MSF)
Assessing Mental Health Needs in an Emergency Setting
Aceh Province in Indonesia has been an area of conflict for more than 40 years. The GAM (Free Aceh Movement) was created in 1959 to pressure the government for independence, and there has been ongoing conflict between GAM and the Indonesian military. The general population is often caught in the middle. Civilians have suffered arrests and intimidation, and have been forced to flee their homes; the tsunami exacerbated what for many was already a fragile existence.
While aid organisations continue to build operational knowledge, there remains considerable debate over the extent to which general mental and psychosocial care programmes provided by these organisations are relevant to the local context [1]. These concerns also extend to the ability of aid organisations to assess needs.
Assessments rely on information gathered using quantitative and qualitative methods. Questionnaires such as the Impact of Events Scale [2], The General Health Questionnaire 28 [3], and the Hopkins Symptoms checklist [4] are used to gather objective data on mental and psychosocial needs. The use of these standard questionnaires has considerable limitations. First and foremost, we should be very modest in our expectations as to what extent it is possible to define the experience of living through a massive crisis in terms of a set of symptoms in a simple checklist. Furthermore, questionnaire tools often lack validation for local context, so they have to be interpreted with caution. Validation, while possible, can take several months and significant resources, and must therefore be done alongside the provision of assistance using approaches proven to be effective in other settings [5].
The limitations to gathering valid quantitative data in the short term requires additional qualitative information gathered through focus group discussions, key informant interviews, social mapping, and literature reviews. The aim is to obtain information that will help interpret individuals' subjective perceptions of their experiences, and to identify levels of resilience and past or present positive coping mechanisms. This information is also used to inform programme design. In the absence of valid and reliable quantitative data, agencies often have to use Western benchmarks.
Western research shows that after exposure to a traumatic event, about 20% of people need some kind of professional psychosocial support to deal with stress and related symptoms or problems, and 5% of people can be expected to have serious mental health disorders [6]. These disorders are mostly combinations of generalized anxiety disorders, post-traumatic stress disorder, and depression. Though the recovery environment in Aceh is less favourable due to the ongoing conflict, MSF uses this estimate in planning programme activities.
The Emergency and Post-Emergency Mental Health Response
Following initial assessments, and at the request of the Indonesian government, MSF established a community-based psychosocial care programme in January 2005. MSF staff work together with psychologists from the Ministry of Health, providing individual and group counselling.
The primary objective of this programme is to enable people to resume normal activities and to encourage active participation in the community. In Aceh, we use group and individual counselling to help those who are overwhelmed by the events or post-disaster circumstances. The reconnection of individuals with their environment runs parallel to community approaches focused on establishing a climate to facilitate this reconnection. Health education activities covering both physical and mental health issues are used to foster self-help and self-control mechanisms.
The programme promotes understanding and acknowledgement of people's problems, and actively involves formal and informal leaders, such as spiritual and village leaders, whose role in the re-establishment of the previously existing care system is crucial. These approaches are in line with the core strategies to respond to mental health in complex emergencies as defined through collaborative engagement of numerous implementing agencies in recent years (see Box 1).
Box 1. Refining the Response to Mental Health Needs in Humanitarian Crises
The Sphere Project (http://www.sphereproject.org) was launched in 1997 by a group of humanitarian NGOs and the Red Cross and Red Crescent movement in an effort to improve the quality of assistance provided to people affected by disaster and to enhance accountability. Chapter 5, “Minimum Standards of Health Service,” has a section on noncommunicable diseases, which includes the standard that “people have access to social and mental health services to reduce mental health morbidity, disability, and social problems.”
The Psychosocial Working Group (http://www.forcedmigration.org/psychosocial), established in 2000, is a collaboration between academic institutions and humanitarian agencies committed to the development of best practice in complex emergencies. Through extensive dialogue between organisations and practitioners representing a range of approaches and orientations to psychosocial intervention, the group has worked to develop a series of papers addressing different issues in psychosocial work.
The World Health Organization has been driving efforts toward a consensus on best public-health practice in respect of mental health. An update on the current state-of-play was given at a recent roundtable discussion, published in the WHO Bulletin (http://www.who.int/bulletin/volumes/83/1/en/71.pdf).
There are only about 500 clinical psychologists for all of Indonesia (about one per 420,000 people), and, in general, most Indonesian people do not know what a psychologist is or does. As a result, we found that mental health issues are often understood in terms of psychiatric disorder and are associated with shame. For this reason, it is important to avoid using psychiatric terms and diagnoses. Instead, in our assessments we frame discussions in terms of wanting to understand people's problems and how people are functioning in their environment.
Another important consideration is how people interpreted the tsunami through their local religions and cultural beliefs. In Banda Aceh, the Muslim religion is central to people's culture and traditions, but mixed with this is superstition and magic. Without exception, the people we spoke with during the assessment understood the tsunami as a punishment or a warning from Allah for being “immoral.” Many believe that the earth will continue to shake until all the dead are buried. Religion is an important tool that helps give people in Banda Aceh meaning and helps them accept what has happened. In our psychosocial activities, we incorporate the importance of religion as a coping mechanism, and we have active links with local religious representatives.
In Banda Aceh, we found that most people have a strong desire to move forward and to rebuild their lives. The men, especially, say that they are anxious about earning a living and supporting their family, and having nothing to do brings them to a state of “great sadness and memories.”
We have also found that it is important to link mental health support with practical support in our psychosocial programmes. Such a link may be a break from the traditional Western approach, where health (physical and mental) is usually considered separate from social and spiritual well being. MSF provides some practical support through health, water, and sanitation programmes. As part of the social activities of these programmes, we establish links on the ground with local and international development organisations, whose role it is to provide practical assistance such as micro-credit schemes, housing, and family-tracing services.
Despite our close collaboration with the existing health system, one problem that persists is the limited possibility for referral of severely traumatized patients to specialized services. There are only three psychiatrists in Aceh Province (which has a population of 4.2 million), and the only psychiatric hospital in Aceh was severely damaged and will not be fully functional for some months. While this hospital is being rehabilitated, mental health care falls to district hospitals and primary health-care clinics, which have little experience in dealing with severe mental health problems.
The Future
The enormous generosity of the public and governments has generated massive humanitarian assistance in tsunami-affected regions, raising concerns about poor coordination, fragmentation and duplication of relief efforts, and inappropriate assistance [7]. Collaboration between agencies and authorities on the ground is essential, but there is also a fine balancing act between working together and being told what to do. This balancing act is crucially important in areas of conflict where humanitarian agencies have to prevent collaboration from becoming co-optation. Many humanitarian emergencies aid agencies, while working together with governments, have also to insist upon their independence.
In the immediate short term community-based clinical services must be reinforced. Our experience clearly shows that people know how to support each other when distressed but have no referral mechanisms when that support is not enough. The self-help and healing capacity of communities needs to be mobilized in conjunction with culturally adapted psychological interventions in which religion is acknowledged as an important coping mechanism. Primary health-care structures are best placed to provide psychosocial services that are appropriate to the local community. Specialist psychiatric support, while not forming the bulk of the response, does need to be available to those who have more severe problems that cannot be managed at the primary-care level.
The evaluation of large humanitarian interventions in emergency settings is difficult as the quickly changing environment hinders appropriate comparisons, but there is a steadily growing body of research evaluating the effects of psychosocial interventions in emergency settings [8–12]. Culturally specific models and instruments to evaluate programs need to be further developed.
In Banda Aceh, many people expressed the hope that with the presence of Westerners the government will not forget them, and that the oppressive ongoing conflict will not resume as before. In other words, the mere presence of Westerners in the region is valued as important to their current sense of security. This underscores an important principle that holds in many other complex emergencies: not all benefits of humanitarian assistance are amenable to evidence-based assessments [13].
Citation: de Jong K, Prosser S, Ford N (2005) Addressing psychosocial needs in the aftermath of the tsunami. PLoS Med 2(6): e179.
Abbreviation
MSFMédecins Sans Frontières
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van Ommeren M Saxena S Saraceno B Mental and social health during and after acute emergencies: Emerging consensus? Bull World Health Organ 2005 83 71 75 Available: http://www.who.int/bulletin/volumes/83/1/en/71.pdf . Accessed 4 May 2005 15682252
Horowitz NJ Wlimer N Alvarez N Impact of events scale: A measure of subjective stress Psychosom Med 1979 41 209 218 472086
Goldberg DP Hillier VF A scaled version of the General Health Questionnaire Psychol Med 1979 24 18 26
Derogatis LR Lipman RS Rickets K Uhlenhuth EH Covi L The Hopkins Symptom Checklist (HSCL): A self-report symptom inventory Behav Sci 1974 19 1 15 4808738
Bolton P Tang AM An alternative to cross-section function assessment Soc Psychiatry Psychiatr Epidemiol 2002 37 537 543 12395144
Kleber RJ Brom D Coping with trauma 1992 Lisse (Netherlands) Swets & Zweitlinger 276
Sondorp E Bornemisza O Public health, emergencies and the humanitarian impulse Bull World Health Organ 2005 83 163 15798834
Mooren GTM de Jong KT Kleber RJ Kulenovic S Ruvic J The efficacy of a mental health program in Bosnia-Hercegovina: Impact on coping and general health J Clin Psychol 2003 59 57 69 12508331
Bolton P Bass J Neugebauer R Verdeli H Clougherty K Group interpersonal psychotherapy for depression in rural Uganda. A randomised controlled trial J Am Med Assoc 2003 289 3117 3124
Dybdahl R Children and mothers in war: An outcome study of a psychosocial intervention program Child Dev 2004 72 1214 1230
Neuner F Schauer M Klaschik C Karunakara U Elbert T A comparison of narrative exposure therapy, supportive counselling, and psychoeducation for treating post-traumatic stress disorder in an African refugee settlement J Consult Clin Psychol 2004 72 579 587 15301642
Igreja V Kleijn WC Schreuder BJN van Dijk JA Verschuur M Testimony method to ameliorate post-traumatic stress symptoms. Community-based intervention study with Mozambican civil war survivors Br J Psychiatry 2004 184 251 257 14990524
Robertson DW Bedell R Lavery JV Upshur R What kind of evidence do we need to justify humanitarian medical aid? Lancet 2002 360 330 333 12147390
| 15971951 | PMC1160584 | CC BY | 2021-01-05 11:13:39 | no | PLoS Med. 2005 Jun 28; 2(6):e179 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020179 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597195210.1371/journal.pmed.0020180PerspectivesObstetrics/GynecologyWomen's HealthObstetricsPregnancyUncomplicated Monochorionic Diamniotic Twins and the Timing of Delivery PerspectiveCleary-Goldman Jane *D'Alton Mary E Jane Cleary-Goldman and Mary E. D'Alton are at the Division of Maternal Fetal Medicine, Columbia University Medical Center, New York, New York, United States.
Competing Interests: The authors declare that they have no competing interests.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e180Copyright: © 2005 Cleary-Goldman and D'Alton.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
High Risk of Unexpected Late Fetal Death in Monochorionic Twins Despite Intensive Ultrasound Surveillance: A Cohort Study
Assessing the Risks of Twin Pregnancies
Cleary-Goldman and d'Alton discuss the implications of a new study in PLoS Medicine examining the risk of fetal death in uncomplicated monochorionic diamnotic twin pregnancies.
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In the past 20 years, advancing maternal age and greater reliance on assisted-conception techniques have dramatically increased the incidence of multiple births, including monochorionic twins (see Glossary) [1,2]. Concomitantly, there have been advances in our understanding of these high-risk pregnancies.
Glossary
Diamniotic: Twins with two amniotic sacs.
Monochorionic: Twins that share one placenta.
Monochorionic diamniotic: Twins that share one placenta with two amniotic sacs.
Monoamnionicity: Twins that share one placenta and one amniotic sac.
Twin-to-twin transfusion syndrome (TTTS): This occurs in monochorionic twins. Twins with TTTS have vascular connections that result in unequal sharing of blood. The smaller twin (donor) does not get enough blood, while the larger twin (recipient) becomes volume overloaded.
Intrauterine fetal demise: This occurs when a fetus dies in utero (before birth). This is also known as stillbirth.
Multicystic encephalomalacia: This refers to the formation of multiple cystic lesions in the cerebral cortex of fetuses, neonates, and infants following injury usually secondary to a hypoxic event.
Twin reversed arterial perfusion sequence: This occurs in monochorionic twins. In this disease, there is a normally developing twin (pump twin) and a twin that does not develop a normal heart (acardiac twin). The pump twin pumps blood for the acardiac twin and for itself by reversing the blood through the umbilical artery. The pump twin is at risk for heart failure due to the increased demands on its heart.
Nuchal translucency: This describes the area in the back of the fetal neck during ultrasound between ten and 14 weeks' gestation. Nuchal translucency screening can be used as a method of first-trimester screening for fetal aneuploidy.
Nonstress test: A test assessing fetal well-being. It involves attaching fetal heart rate and uterine contraction monitors around the patient's abdomen for approximately 20 to 30 minutes and recording the fetal heart-rate patterns. A reactive nonstress test suggests fetal well-being.
Risks of Multiple Pregnancy
The offspring of multiple pregnancies are at greater risk for adverse perinatal outcomes compared to their singleton counterparts, predominantly due to increased risks for preterm delivery and due to monochorionicity [3]. The link between monochorionicity and adverse perinatal outcomes has become increasingly strong [4]. Monochorionic pregnancies have an approximately 15% risk of developing the twin-to-twin transfusion syndrome (TTTS), which can be associated with perinatal mortality and morbidity despite treatment [5]. In addition, single intrauterine fetal demise (IUFD) in a monochorionic pregnancy may be associated with a more-than 20% risk for multicystic encephalomalacia in the surviving co-twin [6].
Managing High-Risk Pregnancies
Despite these advances in knowledge about risks, there is very little consensus about the management of these high-risk pregnancies. In addition, there is the temptation to be reassured by increasing gestational age as the potential complications of prematurity recede. Recent studies suggest, however, that the offspring of a multiple gestation may benefit from delivering prior to their expected date of delivery [7,8].
Several studies have focused on the “prospective risk of fetal death” to help determine by which gestational age a multiple pregnancy should be delivered [7,8]. For twins, the prospective risk of fetal death appears to be equivalent to that of post-term singletons at approximately 37 to 38 weeks' gestation [7,8]. The prospective risk of fetal death for twins intersects with neonatal death at approximately 39 weeks' gestation, indicating that it may be reasonable to consider delivery of uncomplicated twins prior to 40 weeks' gestation [8]. These studies, however, did not address the impact of chorionicity on the decision to deliver a multiple pregnancy.
A New Study of Monochorionic Twins
In this issue of PLoS Medicine, Barigye et al. report the prospective risk of fetal death in uncomplicated monochorionic diamniotic twin pregnancies derived from a cohort of pregnancies that were managed at a single tertiary care referral center and that delivered after 24 weeks' gestation [9]. Patients were excluded from the study if the pregnancy was complicated by TTTS, monoamnionicity, intrauterine growth restriction, growth discordance, structural anomalies, or twin reversed arterial perfusion sequence. Conjoined twins and high-order multiples were also excluded. All patients with uncomplicated monochorionic twins were managed according to a standard protocol. They received first-trimester nuchal translucency assessment and chorionicity determination, a midtrimester anatomical survey, and fetal echo followed by growth scans, amniotic fluid assessment, and Doppler studies every two weeks. The rate of fetal death was derived for two-week periods starting at 24 weeks' gestation. The prospective risk of fetal death was calculated by determining the number of IUFDs that occurred within the two-week block divided by the number of continuing uncomplicated monochorionic twin pregnancies during that same time period.
There were ten unexpected deaths (three double IUFDs and four single IUFDs) in a total of seven (4.6%) of the 151 seemingly uncomplicated monochorionic diamniotic pregnancies. These IUFDs occurred at a median gestational age of 34 weeks 1 day (range 28 weeks 0 days to 36 weeks 3 days). Between 24 and 34 weeks' gestation, the prospective risk of fetal death varied between 1/22 and 1/30 pregnancies. After 32 weeks' gestation, the prospective risk of unexpected antepartum stillbirth was 1/23. In six out of the seven pregnancies, the fetal death was incidental and found on routine ultrasound. (One case presented with decreased fetal movement.) There were no significant differences between the IUFD-affected pregnancies and the unaffected pregnancies with regards to antenatal indicators of intrauterine growth restriction and TTTS. In three of the five pregnancies (autopsy was declined in two cases), deaths remained unexplained after autopsy. Two cases, both double IUFDs, exhibited signs of TTTS. The authors concluded that despite intensive fetal surveillance, uncomplicated monochorionic diamniotic twin pregnancies are at risk for unexpected intrauterine death. The deaths occurred in the third trimester and predominantly after 32 weeks' gestation. As a result, the authors suggested that after 32 weeks' gestation, the prospective risk for fetal death in these pregnancies might be eliminated by elective preterm delivery.
Implications of the Study
Despite the limitations of the study, which Barigye et al. elucidate well (small numbers, retrospective nature, lack of non-monochorionic twin comparative data), this study highlights an important question that many practicing obstetricians are confronting increasingly often: when is the ideal gestational age to deliver apparently uncomplicated monochorionic twins? The authors suggest that 32 weeks' gestation may be reasonable. At that gestational age, many of the risks associated with prematurity, such as intraventricular hemorrhage, necrotizing enterocolitis, and respiratory distress syndrome, have abated.
Nonetheless, the risks of prematurity are not negligible at 32 weeks' gestation. Balancing the risk of iatrogenic preterm birth in an apparently uncomplicated monochorionic twin pregnancy with the risk of double IUFD or single IUFD with the concomitant risk of multicystic encephalomalacia for the surviving co-twin is challenging. In our practice, we have been conducting antenatal surveillance more frequently than once every two weeks, and we have been using the nonstress test in addition to ultrasound and Doppler studies. Although in our preliminary experience we have not had any unexplained IUFDs, we are uncertain if the frequency of testing could account for the prospective risk of fetal death found in the study by Barigye et al. In addition, we have been offering delivery of these apparently uncomplicated monochorionic twins at approximately 34 to 35 weeks' gestation following antenatal corticosteroid administration and thorough counseling regarding the risks of expectant management versus elective preterm delivery. While we acknowledge that our practice pattern is by no means a standard-of-care requirement, we feel it is a reasonable approach to this dilemma until larger, prospective observational studies have been conducted to better elucidate the natural history of these high-risk pregnancies and to better answer the question of when the ideal gestational age is to deliver apparently uncomplicated monochorionic twins.
Citation: Cleary-Goldman J, D Alton ME (2005) Uncomplicated monochorionic diamniotic twins and the timing of delivery. PLoS Med 2(6) e180.
Abbreviations
IUFDintrauterine fetal demise
TTTStwin-to-twin transfusion syndrome.
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Martin JA Hamilton BE Sutton PD Ventura SJ Menacher F Births: Final data for 2002 Natl Vital Stat Rep 2003 52 1 113
Blickstein I Estimation of iatrogenic monozygotic twinning rate following assisted reproduction: Pitfalls and caveats Am J Obstet Gynecol 2005 192 365 368 15695973
Bajoria R Kingdom J The case for routine determination of chorionicity and zygosity in multiple pregnancy Prenat Diagn 1997 17 1207 1225 9509540
Dube J Dodds L Armson BA Does chorionicity or zygosity predict adverse perinatal outcomes in twins? Am J Obstet Gynecol 2002 186 579 583 11904627
Fisk NM Galea P Twin-twin transfusion—As good as it gets? N Engl J Med 2004 351 182 184 15238623
Weiss JJ Cleary-Goldman J Budorick N Tanji K D Alton ME Multicystic encephalomalacia after first trimester intrauterine fetal demise in monochorionic twins Am J Obstet Gynecol 2004 190 563 565 14981409
Sairam S Costeloe K Thilaganathan B Prospective risk of stillbirth in multiple-gestation pregnancies: A population based analysis Obstet Gynecol 2002 100 638 641 12383526
Kahn B Lumey LH Zybert PA Prospective risk of fetal death in singleton, twin, and triplet gestations: Implications for practice Obstet Gynecol 2003 102 685 692 14550996
Barigye O Pasquini L Galea P Chambers H Chappell L High risk of unexpected late fetal death in monochorionic twins despite intensive ultrasound surveillance: A cohort study PLoS Med 2005 2 e172 15971947
| 15971952 | PMC1160585 | CC BY | 2021-01-05 11:13:39 | no | PLoS Med. 2005 Jun 28; 2(6):e180 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020180 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597195310.1371/journal.pmed.0020181PerspectivesDiabetes/Endocrinology/MetabolismEpidemiology/Public HealthGeneral MedicineObesityEpidemiologyChronic Disease ManagementWeight Loss and Mortality: What Does the Evidence Show? perspectiveStampfer Meir Meir Stampfer is a professor of epidemiology and nutrition at the Harvard School of Public Health, Massachusetts, United States. E-mail: [email protected]
Competing Interests: The author declares that he has no competing interests.
6 2005 28 6 2005 2 6 e181Copyright: © 2005 Meir Stampfer.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Intention to Lose Weight, Weight Changes, and 18-y Mortality in Overweight Individuals without Co-Morbidities
Weight Loss and Mortality
A study in this month's PLoS Medicine concludes that, "deliberate weight loss in overweight subjects, without known co-morbidities, may be hazardous in the long term." Stampfer questions whether the data really support such a conclusion.
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Randomized trials and observational studies have conclusively shown a marked improvement in several cardiovascular risk factors when obese individuals lose weight, including decreases in blood pressure, and improvements in the lipid profile and glucose metabolism. These results are closely consistent with observational data linking weight gain to worsening of these cardiovascular risk factors, and to their natural sequelae, clinical cardiovascular events. Obesity is also strongly associated with increased risk of cancer at various sites. With such compelling evidence of the adverse effects of obesity and overweight [1–4], why has there been a continuing controversy about its association with overall mortality?
In their paper in this month's PLoS Medicine, Jaakio Kaprio and colleagues conclude that “deliberate weight loss in overweight subjects without known co-morbidities may be hazardous in the long term” [5]. Do the data really support such a conclusion? An examination of this paper illustrates several of the complexities in approaching this seemingly simple question.
Evidence from Epidemiological Studies
Epidemiologic studies of the relation between overweight and mortality typically must address three principal concerns [6]. First, in many populations, cigarette smokers tend to be leaner than nonsmokers. Because cigarette smoking is such a strong risk factor for mortality, failure to adjust for this adequately can lead to confounding, with the erroneous conclusion that leanness carries increased risk of death. Statistical adjustment for smoking is often insufficient to account for this difficulty. Smoking can lead to medical conditions, sometimes sub-clinical, that are associated with decreased body weight, such as chronic obstructive pulmonary disease. The presence of symptoms or diagnosed conditions may induce smokers to quit. Moreover, the intensity of smoking is related to both risk of death and body mass index. For these reasons, the best way to assess the impact of overweight on risk of mortality is simply to exclude current and past smokers. Kaprio and colleagues' study differentiated only current smoker or nonsmoker. Thus, never-smokers were included in the same category as past smokers, regardless of how much the past smokers had smoked or their reasons for quitting.
A second difficulty in some epidemiologic studies is the inclusion of intermediary factors as co-variates. Weight loss improves hypertension and diabetes, so including these as co-variates would tend to attenuate the apparent benefit of weight loss. In the present study, the authors appropriately excluded people with diabetes, and adjustment for hypertension appeared to have little impact.
The third and most difficult issue in studies of overweight and mortality is reverse causation, the impact of disease on body weight. This can occur either through the biological impact of a condition (diagnosed or preclinical) or as an inducement to attempt to lose weight as a means to improve health. The authors' keen recognition of this problem provides a significant strength to the present study. The authors appropriately excluded individuals with a wide range of conditions to identify an apparently healthy cohort. This critical step is often ignored. Sometimes, investigators also exclude deaths that occur in the first few years after follow-up, to reduce the impact of reverse causation. Such lagged analyses can be helpful, but some chronic conditions of long duration, such as depression, chronic lung disease, and heart failure (conditions that often may not reach the level of clinical diagnosis) can lead to lower body weight and higher mortality risk. Hence, that strategy (not employed by Kaprio and colleagues) may not fully avoid the problem.
The Difficulties of Assessing the Effects of Intention to Lose Weight
As a further step toward reducing the potential bias of the impact of disease on body weight, the authors identified individuals with intent to lose weight. Unintended weight loss is well known as an ominous clinical sign, usually signifying a serious illness. Just over a third of the overweight subjects in the Kaprio cohort had expressed intent to lose weight at baseline in 1975. It is interesting to note that despite this intention, as a group, the median weight change in the ensuing six years was a gain of 0.33 kg/m2, almost identical to the weight change in the group that had not expressed intent to lose weight (gain of 0.31 kg/m2). Thus, this study cannot fairly be characterized as an assessment of intentional weight loss and its subsequent effect on mortality. Because the changes in weight are so similar in these two groups, it is implausible to attribute the weight loss in the intent-to-lose group to that intention. These findings render the results particularly difficult to interpret.
Other important limitations include the very small number of endpoints—only 268 total deaths in the cohort. When further subdivided according to intention to lose weight, and categories of weight change, the numbers are far from sufficient for reliable estimates. For example, the main conclusions are based on the subgroup of those with intent to lose weight who actually lost weight; this group had only 42 deaths, of which ten were violent. Another related difficulty is that this cohort, though overweight, is not drastically obese. The median body mass index (BMI) was 26.7 kg/m2, and fewer than 10% of people had a BMI greater than 30. With such a narrow range of BMI, coupled with the very small changes in weight during the six years of initial follow-up, even a very large study would not have sufficient power to detect plausible relative risks associated with weight change.
What Advice Should Be Given?
Given the well-known adverse metabolic consequences of overweight and obesity, the strong links with cardiovascular disease and several other serious illnesses, and the absence of any plausible adverse biologic consequences of maintaining normal weight (BMI 19 to 25), it seems prudent to continue advising adults to seek to maintain a weight within that range. The results from Kaprio et al, although they raise questions about the effects of weight loss, do not cast serious doubt on that advice.
Citation: Stampfer M (2005) Weight loss and mortality: What does the evidence show? PLoS Med 2(6): e181.
Abbreviation
BMIbody mass index
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Tuomilehto J Lindstrom J Eriksson JG Valle TT Hamalainen H Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance N Engl J Med 2001 344 1343 1350 11333990
National Task Force on the Prevention and Treatment of Obesity Overweight, obesity, and health risk Arch Intern Med 2000 160 898 904 10761953
Willett WC Manson JE Stampfer MJ Colditz GA Rosner B Weight, weight change, and coronary heart disease in women: Risk within the “normal” weight range JAMA 1995 273 461 465 7654270
Hu FB Willett WC Li T Stampfer MJ Colditz GA Adiposity as compared with physical activity in predicting mortality among women N Engl J Med 2004 351 2694 2703 15616204
Sørensen TIA Rissanen A Korkeila M Kaprio J Intention to lose weight, weight changes, and 18-year mortality in overweight subjects without co-morbidities PLoS Med 2005 2 e171 15971946
Manson JE Stampfer MJ Hennekens CH Willett WC Body weight and longevity: A reassessment JAMA 1987 257 353 358 3795418
| 15971953 | PMC1160586 | CC BY | 2021-01-05 11:13:39 | no | PLoS Med. 2005 Jun 28; 2(6):e181 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020181 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597195410.1371/journal.pmed.0020183Research ArticleInfectious DiseasesVirologyEpidemiology/Public HealthWomen's HealthVaccinesInfectious DiseasesGlobal healthImmunology and allergyTravel MedicineDevelopment of a New Vaccine for the Prevention of Lassa Fever Vaccine against Lassa FeverGeisbert Thomas W
1
2
*Jones Steven
3
4
Fritz Elizabeth A
1
Shurtleff Amy C
1
Geisbert Joan B
1
Liebscher Ryan
3
Grolla Allen
3
Ströher Ute
3
5
Fernando Lisa
3
Daddario Kathleen M
2
Guttieri Mary C
1
Mothé Bianca R
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Larsen Tom
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Hensley Lisa E
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Jahrling Peter B
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Feldmann Heinz
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5
1Virology Division, United States Army Medical Research Institute of Infectious DiseasesFort Detrick, MarylandUnited States of America2Department of Pathology, Uniformed Services University of the Health SciencesBethesda, MarylandUnited States of America3Special Pathogens Program, National Microbiology LaboratoryPublic Health Agency of Canada, Winnipeg, ManitobaCanada4Department of Immunology, University of ManitobaWinnipeg, ManitobaCanada5Department of Medical Microbiology, University of ManitobaWinnipeg, ManitobaCanada6Department of Biological Sciences, California State UniversitySan Marcos, CaliforniaUnited States of America7Pathology Division, United States Army Medical Research Institute of Infectious DiseasesFort Detrick, MarylandUnited States of America8Headquarters, United States Army Medical Research Institute of Infectious DiseasesFort Detrick, MarylandUnited States of AmericaHoerauf Achim Academic EditorUniversity of BonnGermany
Competing Interests: The authors have declared that no competing interests exist.
Author Contributions: Please see acknowledgements section
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e18323 2 2005 2 5 2005 Copyright: © 2005 Geisbert et al.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
A Promising Candidate for a Lassa Fever Vaccine
Some Tolerance for Fur-Animal Studies in PLoS Medicine
Background
Recent importation of Lassa fever into Germany, the Netherlands, the United Kingdom, and the United States by travelers on commercial airlines from Africa underscores the public health challenge of emerging viruses. Currently, there are no licensed vaccines for Lassa fever, and no experimental vaccine has completely protected nonhuman primates against a lethal challenge.
Methods and Findings
We developed a replication-competent vaccine against Lassa virus based on attenuated recombinant vesicular stomatitis virus vectors expressing the Lassa viral glycoprotein. A single intramuscular vaccination of the Lassa vaccine elicited a protective immune response in nonhuman primates against a lethal Lassa virus challenge. Vaccine shedding was not detected in the monkeys, and none of the animals developed fever or other symptoms of illness associated with vaccination. The Lassa vaccine induced strong humoral and cellular immune responses in the four vaccinated and challenged monkeys. Despite a transient Lassa viremia in vaccinated animals 7 d after challenge, the vaccinated animals showed no evidence of clinical disease. In contrast, the two control animals developed severe symptoms including rashes, facial edema, and elevated liver enzymes, and ultimately succumbed to the Lassa infection.
Conclusion
Our data suggest that the Lassa vaccine candidate based on recombinant vesicular stomatitis virus is safe and highly efficacious in a relevant animal model that faithfully reproduces human disease.
A replication-competent vaccine based on attenuated recombinant vesicular stomatitis virus vector protects non- human primates.
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Introduction
Among the viruses causing severe hemorrhagic fever in Africa, Lassa fever is a significant public health problem in Nigeria, Liberia, Sierra Leone, and the Republic of Guinea. It is estimated that Lassa virus infects over 200,000 individuals per year across this region, causing over 3,000 deaths [1]. The case fatality rate for Lassa fever is around 15% in hospitalized patients and has been greater than 50% in several outbreaks [2,3]. Human infection is associated with contact with a widely distributed and highly commensal rodent, Mastomys natalensis, or by contact with infected patients. Recent importation of Lassa fever into Germany, the Netherlands, the United Kingdom, and the United States by travelers on commercial airlines [4–8] illustrates the potential for the spread of this highly dangerous and contagious pathogen. In addition, Lassa virus has gained notoriety because it is classified as a Category A bioweapon agent [9].
Lassa virus is an enveloped, bisegmented RNA virus belonging to the Old World group within the family Arenaviridae [10]. Arenavirus particles contain a genome consisting of two ambisense single-stranded RNA molecules, designated small (S) and large (L), of a length about 3.4 kb and 7.2 kb, respectively. The S segment contains two genes that encode three structural proteins, the nucleoprotein (NP) and the envelope glycoproteins GP1 and GP2. The L segment contains two genes that encode two proteins, the viral polymerase (L protein) and the Z protein. NP and L protein associate with the genomic RNA in a ribonucleoprotein complex or nucleocapsid structure. It is thought that the Z protein functions as a matrix protein and is responsible for the formation of virus particles [11]. GP1 and GP2 are initially synthesized as a precursor molecule, glycoprotein C (GPC), which is posttranslationally cleaved by the protease SKI-1/S1P [12]. GP1 is the portion of the surface glycoprotein spike that is thought to be the effector for receptor binding, while GP2 is structurally consistent with viral transmembrane fusion proteins of other enveloped viruses [13].
Currently, there are no vaccines or antiviral drugs approved for Lassa fever. Treatment with intravenous ribavirin was shown to reduce mortality from Lassa fever in high-risk patients and presumably decreases morbidity in all patients with Lassa fever [14]. However, the availability of ribavirin is very limited in endemic areas, and treatment is most effective if initiated within the first week of disease onset [14]. Preventing contact with the reservoir host in endemic areas is currently unachievable; therefore, a preventive vaccine is a critical public health need, especially to protect health care providers, who are often the most at risk. Indeed, the recent untimely death of a well-known doctor in Sierra Leone who treated thousands of cases of Lassa fever and ultimately contracted the disease as a result of these laudable efforts [15] underscores the need for a preventive vaccine.
Early attempts to develop a vaccine against Lassa fever focused on classical approaches such as killed vaccines. A whole-virion vaccine inactivated by gamma irradiation provided a good humoral response to Lassa viral proteins, NP, and GP, but failed to protect nonhuman primates from a lethal Lassa challenge [16]. As these animals were unprotected despite a strong humoral response, it was suggested that protection would depend on a robust cellular response. Subsequent efforts to develop an efficacious vaccine against Lassa fever focused on genetically engineered vaccines. Specifically, studies focused on recombinant vaccinia viruses expressing the NP, the full-length GPC, and combinations of GP1 and GP2. In a series of studies by Fisher-Hoch and colleagues, approximately 90% of nonhuman primates that received a vaccine containing both GP1 and GP2 survived a lethal Lassa challenge [1,17,18]. Protection did not correlate with humoral immunity, as none of these animals had demonstrable Lassa virus-specific antibody before challenge. Consequently, cell-mediated immunity was implicated in protecting these animals, although T-cell responses were not measured in these studies. In comparison, vaccination of macaques with NP alone resulted in the development of relatively high antibody titers before Lassa challenge, but only 20% survival.
While the recombinant vaccinia vaccines expressing the full-length Lassa GPC protected nearly 90% of nonhuman primates from a lethal challenge, it was noted that the vaccinia platform is not suitable for human use because of potential side effects, which is a particular concern in areas where HIV prevalence is high [1]. However, it appears from all previous studies that a strong cellular response may be required for protection. Vaccination with a live virus characteristically elicits strong cellular immune responses, and live vaccines are particularly attractive because they can confer protection in a single injection. Therefore, the ideal Lassa vaccine would be based on a replication-competent format engineered for enhanced safety and with the ability to overcome technical obstacles such as prior immunity to the vector. Recently, we described the generation of a live attenuated, recombinant vesicular stomatitis virus (rVSV) expressing the GPC of Lassa virus, strain Josiah [19]. Vaccines based on live attenuated rVSV have been highly effective in animal models and are particularly attractive because they can be administered by the mucosal route [20–23]. Furthermore, vesicular stomatitis virus (VSV) infections in humans occur fairly rarely worldwide, mainly in the enzootic regions of the Americas, and consequently, global pre-existing immunity is negligible [24]. Because the guinea pig model of Lassa fever does not faithfully mimic human disease [25] and is not generally predictive for efficacy of vaccines and antivirals [1], we evaluated the protective efficacy of the replicating rVSV vector in nonhuman primates.
Methods
Vaccine Vectors and Viruses
The recombinant VSV expressing the glycoprotein of Lassa virus, strain Josiah, and Zaire ebolavirus (ZEBOV), strain Mayinga, were generated as described recently using the infectious clone for the VSV, Indiana serotype (kindly provided by John Rose, Yale University, New Haven, Connecticut, United States) [19]. Briefly, the appropriate open reading frames for the glycoproteins were generated by PCR, cloned into the VSV genomic vectors lacking the VSV glycoprotein gene, sequenced-confirmed, and rescued using the method described earlier [26]. The recombinant viruses expressing Lassa virus glycoprotein and ZEBOV glycoprotein were designated VSVΔG/LVGPC (Figure 1A) and VSVΔG/ZEBOVGP, respectively. Cynomolgus macaques were challenged with Lassa virus isolated from a human case in 1976 in Sierra Leone [27].
Figure 1 Lassa Virus Vaccine Design and Vaccination Regimen
(A) Left, schematic diagram of the recombinant VSV expressing the glycoprotein of Lassa virus. The VSV glycoprotein (G) was replaced with the Lassa virus glycoprotein (LV GPC). Shown are the nucleocapsid (N), phosphoprotein (P), matrix (M), and RNA-dependent RNA polymerase (L). Right, gold sphere labeling (10-nm spheres) of VSVΔG/LVGPC particles by immunoelectron microscopy using a pool of two murine monoclonal antibodies directed against Lassa virus GP1 and GP2; particles were negatively contrasted with 1% uranyl acetate.
(B) Time line for vaccination and Lassa viral challenge study. Vaccination of cynomolgus monkeys was done with a single intramuscular dose of 10 7 PFU of either VSVΔG/LVGPC (four animals) or VSVΔG/ZEBOVGP (two animals). Challenge was performed with a single intramuscular dose of 104 PFU of Lassa virus, strain Josiah. Arrows indicate day of sampling (blood and swabs).
Animal Studies
Six cynomolgus macaques (Macaca fascicularis), 4–6 y old and weighing between 3 kg and 8 kg, were obtained from approved suppliers. Four of the animals were vaccinated intramuscularly with approximately 2 × 107 PFU of VSVΔG/LVGPC, and two with an equivalent dose of VSVΔG/ZEBOGP (controls). The six cynomolgus macaques were challenged intramuscularly 28 d after the single-dose immunization with 1 × 104 plaque-forming units (PFU) of Lassa virus, Josiah strain. Animals were closely observed twice daily after the immunization and the challenge for clinical symptoms. Swab samples (oral, nasal, rectal, vaginal) and blood were collected as shown in Figure 1B. Studies were performed in biosafety level (BSL)-4 biocontainment. Research was conducted in compliance with the Animal Welfare Act and other federal statues and regulations relating to animals and experiments involving animals, and adheres to the National Research Council laboratory animal care principles [28]. The facility used is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Hematology and Serum Biochemistry
Total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, hematocrit values, total hemoglobin, mean cell volume, mean corpuscular volume, and mean corpuscular hemoglobin concentration were determined from blood samples collected in tubes containing EDTA, by using a laser-based hematologic analyzer (Coulter Electronics, Hialeah, Florida, United States). The white blood cell differentials were performed manually on Wright-stained blood smears. Serum samples were tested for concentrations of albumin (ALB), amylase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), glucose, cholesterol, total protein, total bilirubin (TBIL), urea nitrogen (BUN), and creatinine (CRE) by using a Piccolo Point-Of-Care Blood Analyzer (Abaxis, Sunnyvale, California, United States).
Virus Detection
RNA was isolated from blood and swabs using the appropriate RNA isolation kits from QIAGEN (Mississauga, Ontario, Canada). To detect VSV we used an RT-PCR assay targeting the matrix gene. Lassa viral RNA was detected using a primer pair targeting the gene encoding L protein. Virus was titrated by plaque assay on Vero E6 cells from all blood and selected and swab samples and from tissue samples collected at necropsy (liver, spleen, lung, kidney, adrenal gland, pancreas, lymph nodes, bone marrow, ovary, testis, and brain) [29].
Humoral Immune Responses
IgG antibodies against Lassa virus were detected with an ELISA using purified viral particles as an antigen source [30]. Neutralization assays were performed by measuring plaque reduction in a constant virus:serum dilution format as previously described [29]. Briefly, a standard amount of Lassa virus (approximately 100 PFU) was incubated with serial 2-fold dilutions of the serum sample for 60 min. The mixture was used to inoculate Vero E6 cells for 60 min. Cells were overlaid with an agar medium and incubated for 8 d. Plaques were counted 48 h after neutral red staining. Endpoint titers were determined by the dilution of serum which neutralized 50% of the plaques.
Cellular Immune Responses
Peripheral blood mononuclear cells (PBMCs) were isolated by histopaque gradient (Sigma, St. Louis, Missouri, United States) from each animal before vaccination and transformed with Herpesvirus papio to serve as antigen-presenting cells. Each day on which assays were performed, 5 × 105 transformed cells from each animal were dispensed and infected with VSVΔG/LVGPC at a multiplicity of infection of 1.0. At 12 h postinfection, transformed cells from each animal were individually mixed with PBMCs collected from the respective animals. For example, transformed cells from subject 1 were mixed with PBMCs collected from subject 1. Anti-CD28 and anti-CD49d antibodies (1.0 μg of each antibody; BD Pharmingen, San Diego, California, United States) and GolgiPlug (BD Pharmingen) were added to each coculture and incubated for 6 h at 37 °C. Staphylococcal enterotoxin B was added as a positive control to one set of cultures. Cultures were placed at 4 °C overnight. Cocultured cells were stained for CD8, CD3, and CD4 in Pharmingen stain buffer (BD Pharmingen) for 20 min at 4 °C in the dark. Cells were washed with Pharmingen stain buffer and fixed/permeabilized with Cytofix/Cytoperm buffer (BD Pharmingen) for 1–1.5 h in the dark at 4 °C. Cells were then washed in 1× Perm/Wash buffer (BD Pharmingen) and stained with interferon gamma (IFN-γ)-allophycocyanin or tumor necrosis factor alpha (TNF-α)-allophycocyanin in 1× Perm/Wash buffer for 45 min at 4 °C in the dark. Cells were next washed in 1× Perm/Wash buffer and resuspended in Cytofix/Cytoperm buffer for analysis. Acquisition was performed on a FACS Calibur flow cytometer collecting 100,000 to 200,000 lymphocyte-gated events per sample, and analyzed using FloJo software (Tree Star, Ashland, Oregon, United States). Cytokine-positive cells were defined as a percentage of double-positive cells for CD8 and CD4 populations within the lymphocyte gate.
Statistics
Vaccine efficacy was calculated using Fisher's exact test. A p-value of 0.05 or lower was considered significant.
Results
Protection against Lassa Fever
We tested the Lassa fever rVSV vaccine candidate in nonhuman primates. Four cynomolgus macaques (M. fascicularis) were immunized by intramuscular injection with a single dose of approximately 2 × 107 PFU of VSVΔG/LVGPC, and two control animals received VSVΔG/ZEBOVGP (same route and dose). The animals were monitored closely for clinical symptoms and shedding of rVSVs. Figure 1B shows the inoculation and sampling protocol that was followed for this study. After vaccination, none of the nonhuman primates displayed any signs of clinical symptoms, indicating that the rVSVs were apathogenic for these animals. All six animals were subsequently challenged intramuscularly on day 28 postvaccination with a high dose (1 × 104 PFU) of Lassa virus. The two control animals (vaccinated with irrelevant VSVΔG/ZEBOVGP) started to show clinical signs of illness on day 3, when one of the animals had a fever (defined as a temperature over 104 °F). By day 10, both control animals developed macular rashes and anorexia, and one animal had severe facial edema, which is prognostic for a poor outcome in humans [31]. These control animals succumbed to the Lassa virus challenge and were euthanized on day 11 and day 13, respectively. At necropsy, both controls showed lesions and pathological changes consistent with Lassa fever in nonhuman primates [25,32]. In contrast, none of the VSVΔG/LVGPC-vaccinated animals became sick, and all four animals were fully protected against the high Lassa challenge dose. Survival was statistically significant (p = 0.0079 by Fisher's exact test) when the four VSVΔG/LVGPC-vaccinated macaques were compared to the two nonspecifically vaccinated animals and three recent controls that succumbed to a Lassa challenge using the same dose of the same virus isolate delivered by the same route (T. W. G., unpublished data).
Analysis of blood chemistry and hematology was performed on the indicated days (Figure 1B; Table 1); values in the VSVΔG/LVGPC-vaccinated animals did not differ significantly from pre-challenge values. There were slight perturbations in platelet counts at day 7 after Lassa challenge in three of the four VSVΔG/LVGPC-vaccinated animals, with a small decrease in platelet numbers. This modest drop in platelets was also noted in one of the two VSVΔG/ZEBOVGP-vaccinated control animals at day 7. In addition, a marked lymphopenia developed in both control animals, but was not observed in any of the four VSVΔG/LVGPC-vaccinated macaques. Significant changes in clinical chemistry (greater than 3-fold changes from baseline values) were seen in both control animals in ALT and AST (Figure 2). In addition, increases were also seen in serum levels of ALP, GGT, TBIL, and BUN, while decreased levels of ALB and CRE were evident in one of these animals. The only perturbation in clinical chemistry values among the four VSVΔG/LVGPC-vaccinated animals was very slight increases in the ALT levels in two of the animals at day 7 and a third monkey at day 10.
Figure 2 Hepatic Enzyme Levels in Sera of Cynomolgus Monkeys After Challenge with Lassa Virus
Results of ALT (A) and AST (B) assays are shown for the four VSVΔG/LVGPC-vaccinated macaques (closed symbols) and the two control monkeys (open symbols).
Table 1 Blood Chemistry and Hematology Results for Cynomolgus Monkeys before and after Challenge with Lassa Virus
VSVΔG/LVGPC-vaccinated, four cynomolgus macaques vaccinated with VSV expressing the Lassa virus glycoprotein; controls, two cynomolgus monkeys vaccinated with VSV expressing an irrelevant antigen; LV, Lassa virus; NC, no change detected; ↑, increase in parameter; ↓, decrease in parameter.
DOI: 10.1371/journal.pmed.0020183.t001
No Viremia or Shedding of Vaccine Vector
To determine if viremia or shedding of the rVSVs occurs after vaccination, whole blood and swab samples (oral, nasal, vaginal, and rectal) from all six vaccinated animals were analyzed by virus isolation and RT-PCR assay. A moderate viremia was detected on day 2 postimmunization by virus isolation (103–104 PFU/ml) and RT-PCR in the two VSVΔG/ZEBOVGP-vaccinated control animals, but viremia was not detected at any time point in any of the four VSVΔG/LVGPC-vaccinated macaques (Figure 3A). No evidence of shedding was detected by virus isolation or RT-PCR in any of the swab samples from any of the six animals evaluated. Thus, there is no evidence that virus could be shed under the conditions tested.
Figure 3 Viremia in Nonhuman Primates after Vaccination and Lassa Virus Challenge
Viremia levels after vaccination (A) and Lassa virus challenge (B) were determined in plasma taken at the indicated times after vaccination and Lassa virus challenge (see Figure 1B). RNA was isolated from plasma, and PCR specific for VSV and Lassa virus were performed as described in Methods. Note: Subjects 1–4 were vaccinated with VSVΔG/LVGPC, while controls 1 and 2 were vaccinated with VSVΔG/ZEBOVGP.
Vaccine Protects against Lassa Fever but Immunity Is Not Sterile
Lassa viral replication and shedding was analyzed by virus isolation and RT-PCR from the blood and swab samples taken after Lassa challenge (see Figure 1B). Organ samples from the two control animals (vaccinated with irrelevant VSVΔG/ZEBOVGP) were also available. At day 3 after Lassa challenge, viremia was detected in one of the two control animals (Figure 3B), but not in the second control animal or in any of the four VSVΔG/LVGPC-vaccinated monkeys. By day 7, all six monkeys were viremic (viremia up to approximately 104 PFU/ml) (Figure 3B). However by day 10, all four of the VSVΔG/LVGPC-vaccinated animals had cleared the viremia, while both control animals had high viremias (more than 106 PFU/ml), which was maintained until euthanasia. High levels of Lassa virus were also detected in organ samples of these two control animals (104–109 PFU/g) (unpublished data).
Strong Humoral and Cellular Immune Response after Lassa Virus Challenge
By the day of Lassa challenge, all four VSVΔG/LVGPC-vaccinated monkeys had developed moderate- to high-level IgG antibody titers (Figure 4A), with three of four animals having titers over 1 in 800. In addition, all four VSVΔG/LVGPC-vaccinated monkeys developed low-level neutralizing antibody titers against Lassa virus (Figure 4B). After Lassa virus challenge, ELISA and neutralizing antibody titers markedly increased in all four VSVΔG/LVGPC-vaccinated monkeys (Figure 4). The cellular immune response was measured by intracellular cytokine staining for IFN-γ and TNF-α in CD4- and CD8-positive lymphocytes. Before Lassa virus challenge, the production of IFN-γ and TNF-α was detectable in CD8-positive lymphocytes in one of four VSVΔG/LVGPC-vaccinated monkeys (subject 2). After Lassa virus challenge, three of the four VSVΔG/LVGPC-vaccinated animals responded positively, with values between 0.15% to 0.93% positive IFN-γ- or TNF-α-positive CD8 cells (Figures 5 and 6). Two of these three animals also ranged between 0.19% and 0.36% positive IFN-γ- or TNF-α-positive CD4 cells. These results indicate that the VSVΔG/LVGPC was a potent stimulator of humoral and cellular immunity.
Figure 4 Humoral Immune Response in Nonhuman Primates to Lassa Virus before and after Lassa Challenge
(A) IgG responses were measured using an established ELISA (see Methods). The titers are presented as endpoint dilutions.
(B) Neutralizing antibodies were detected by a plaque reduction neutralization assay as described in Methods. Titers are presented as endpoint dilutions. Note: Subjects 1–4 were vaccinated with VSVΔG/LVGPC, while controls 1 and 2 were vaccinated with VSVΔG/ZEBOVGP.
Figure 5 Cellular Immune Response (IFN-γ) in Nonhuman Primates before Vaccination, after Vaccination, and after Lassa Virus Challenge
Intracellular levels of IFN-γ were determined in CD4- and CD8-positive T-cell populations before vaccination (−28), after vaccination (−14 and 0), and after challenge with Lassa virus (7, 14, and 30); challenge was administered on day 0. Strong cellular responses following restimulation with transformed cells expressing Lassa GPC were seen in three of the four animals after challenge. Note: Subjects 1–4 were vaccinated with VSVΔG/LVGPC, while controls 1 and 2 were vaccinated with VSVΔG/ZEBOVGP.
Figure 6 Cellular Immune Response (TNF-α) in Nonhuman Primates before Vaccination, after Vaccination, and after Lassa Virus Challenge
Intracellular levels of TNF-α were determined in CD4- and CD8-positive T-cell populations before vaccination (−28), after vaccination (−14 and 0), and after challenge with Lassa virus (7, 14, and 30); challenge was administered on day 0. Strong cellular responses following restimulation with transformed cells expressing Lassa GPC were seen in three of the four animals after challenge. Note: Subjects 1–4 were vaccinated with VSVΔG/LVGPC, while controls 1 and 2 were vaccinated with VSVΔG/ZEBOVGP.
Discussion
The VSV-based vector expressing the Lassa virus GPC mediated complete protection of four of four cynomolgus monkeys from a high-dose lethal challenge of Lassa virus. Protection was associated with the generation of Lassa-specific CD8+ T cell and antibody responses. Although the presence of preexisting ELISA and neutralizing anti-GP antibodies were associated with protection, previous studies in nonhuman primates showed little to no anti-glycoprotein antibodies after vaccination with vaccinia recombinants expressing the full-length Lassa viral glycoprotein [18]. Notably, significant increases in the levels of antibodies were detected after the vaccinia-immunized macaques were challenged with infectious Lassa virus, but levels of neutralizing antibodies were not observed before or after Lassa virus challenge in these studies. Indeed, in the current study, we also observed increased levels of antibodies in the VSVΔG/LVGPC-vaccinated animals after Lassa virus challenge, including substantial increases in the levels of neutralizing antibodies. While previous studies have implied that cellular immunity is necessary for protection against Lassa fever, no previous study to our knowledge has evaluated the cellular immune response in nonhuman primates. Here, we show that cytotoxic T lymphocytes appear to play an important role in protection, as production of IFN-γ and TNF-α was detected in CD8-positive lymphocytes in three of the four VSVΔG/LVGPC-vaccinated macaques after Lassa challenge. However, we cannot discount the importance of neutralizing antibodies as another potential mechanism of protection.
In this study, protection against Lassa fever was dependent on vaccination with an attenuated, replication-competent virus, which may raise questions regarding the safety of live-attenuated vectors. Most importantly, we demonstrated here that, despite a short-term viremia in two animals immunized with the control VSVΔG/ZEBOVGP vaccine, rVSV replication and shedding were not detectable in nonhuman primates, and the animals did not develop fever or other symptoms, nor were there changes in blood chemistry or hematology.
Increased concern about natural or artificial introductions of emerging viruses such as Lassa virus has driven increased investment in basic research and construction of a network of biocontainment laboratories. In the past, a relatively small global market and a lack of biocontainment facilities generated little commercial interest for developing a Lassa fever vaccine. Therefore, there have been relatively few attempts to evaluate vaccines against Lassa fever in nonhuman primate models. It is difficult to compare results across these few studies because of a number of variables, including differences among animal species and dose, route, and strain of challenge virus employed. It is important to note that two different species of nonhuman primates (M. fascicularis and M. mulatta) were employed in the previous studies evaluating vaccinia virus as a platform for a Lassa fever vaccine [1,17,18]. Those studies, which used seven cynomolgus monkeys, offer a good comparison with the current study, as these animals were immunized with a vaccinia construct expressing the Lassa viral GPC and were challenged with 104 PFU of Lassa virus (Josiah strain) [18]. Five of these seven vaccinia-Lassa GPC-vaccinated cynomolgus monkeys were protected from a lethal Lassa viral challenge, compared to the result of protection of four of four cynomolgus monkeys from 104 PFU of Lassa virus (Josiah strain) in the current study. Differences between these two vaccine platforms were also noted in circulating Lassa viral loads and clinical illness. While Lassa viremia was detected in both studies in all vaccinated animals at postinfection day 7, we failed to detect viremia in any VSVΔG/LVGPC-vaccinated animal at day 10. In comparison, in the previous study, which employed the vaccinia virus platform, viremia was detected in four of the seven vaccinated animals at day 10 [18]. Consistent with differences in the duration of viremia, we failed to detect any evidence of clinical illness in any of the four VSVΔG/LVGPC-vaccinated macaques after Lassa virus challenge, while a transient febrile illness with moderate physiological changes was reported in the vaccinia-Lassa GPC-vaccinated monkeys after Lassa virus challenge [1]. All experimental controls died in the current study and the previous studies [1,18]. The Lassa viral challenge seed used in our study was uniformly lethal in 12 of 12 cynomolgus monkeys challenged by the same route with a comparable dose [33] and three of three cynomolgus monkeys with the exact same dose (T.W.G., unpublished data); thus, protection of four of four VSVΔG/LVGPC-vaccinated monkeys is highly significant (p = 0.0079).
It will be important to determine whether this rVSV vaccine can protect against other strains of Lassa virus or whether successful vaccination will require additional genes to confer heterologous protection. Others have demonstrated that vaccination of nonhuman primates with the nonpathogenic and closely related Mopeia virus protects animals from a lethal Lassa virus (Josiah strain) challenge [17,18] suggesting that broad protection is an attainable goal.
The potency of the immune responses induced in this study led us to speculate that complete protection could be achievable in less than the 28 d between immunization and challenge, but this notion, of course, must be experimentally tested. A shortened regimen would be a significant advance in the event of either a natural outbreak or bioterrorism, as both occur rapidly with little warning and require swift and effective measures to reduce their impact on risk groups such as health-care and military personnel. An immediate response is needed to contain an outbreak and prevent the spread of disease to other geographic regions. So far, quarantine practices appear to be effective in limiting Lassa outbreaks, which have been most severe in rural hospitals [2,3]. However, infection and mortality have been devastating in the quarantined community [2,3], and modern advances in global travel cannot ensure that future outbreaks will be as easily contained. While rodent control would be an effective way to greatly reduce the number of cases of Lassa fever in West Africa, current economic barriers make such control unlikely. Thus, the availability of a vaccine that could be rapidly employed to create a ring of vaccination around an epidemic zone would be critical to controlling the subsequent spread of Lassa virus. Here, our one-dose, 4-wk, rVSV-based Lassa immunization regimen demonstrates the plausibility of developing a candidate vaccine to meet this need.
The recent focus on development of vaccines against highly pathogenic viruses has been concentrated on various recombinant vectors (e.g., adenoviruses, alphavirus replicons, vaccinia, DNA vaccination) for expression of virus-encoded proteins in various combinations to induce protective immunity [29,34,35]. While many of these approaches show promise, an important technical obstacle that is inherent with most of these platforms is the potential for prior immunity to the vector either through natural exposure or prior immunization [36–38]. This is an area in which the rVSV platform has a competitive advantage over many of the other formats. Preexisting immunity in the human population against VSV is minimal and may not be important at all, because neutralizing antibodies are directed against the VSV glycoprotein [21], which is not expressed using the recombinant vector described here.
The primary concern regarding use of the rVSV vaccine platform in humans is related to the fact that this is a replication-competent vaccine, and thus demonstration of safety is of paramount importance. Pathogenicity associated with VSV and rVSVs is a concern that will require further investigation. However, the VSV glycoprotein, which is thought to be the main determinant of pathogenicity for VSV, has been completely deleted in the VSV platform that we describe for Lassa virus. In fact, it has been shown that mutations truncating the VSV glycoprotein cytoplasmic domain from 29 to nine or one amino acid are nonpathogenic in mice [21,39], and, not unexpectedly, there is an absence of vector-associated pathogenesis in mice receiving vectors with complete deletion of the VSV glycoprotein [39]. Reassortment and recombination are other concerns that have been associated with the use of replication-competent vaccine platforms. Importantly, the VSV single-stranded RNA genome does not undergo reassortment and therefore lacks the potential of reassorting with wild-type viruses in vivo. Furthermore, VSV replicates within the cytoplasm of infected cells and does not undergo genetic recombination.
Durability is an important concern and a desirable trait of any vaccine platform. Indeed, this is an even more important consideration when developing vaccines against exotic pathogens such as Lassa virus that exist in remote geographic locales where boosting is often problematic or not feasible at all. In these settings, the ideal vaccine would be a single-shot vaccine capable of providing protection for life or for a very long period of time. Replication-defective vaccines typically do not provide such durability. Live viral vaccines have traditionally offered the most effective long-term protection against viral infections. Such vaccines tend to induce strong cellular and humoral host immune responses as a result of the intracellular synthesis of specific antigens at high levels over a prolonged period. Thus, in addition to low seroprevalence in humans, durability is another important advantage of exploiting VSV as a vaccine platform. While safety remains a hurdle that must be overcome before the potential of this platform can be fully realized, a number of groups are currently working toward developing the rVSV vaccine platform for eventual use in humans. Research with rVSV-based vaccines is underway for HIV, cancer therapy, and other applications. Notably, the National Institute of Allergy and Infectious Disease recently awarded a 5-y, 22.8-million dollar contract to Wyeth to expand research on a candidate rVSV-based HIV vaccine that was shown to prevent AIDS-like disease in monkeys [40]. Indeed, the use of replicating rVSV-based vectors has proven to be a potent and promising concept for future vaccine development against Lassa fever and perhaps other lethal viral hemorrhagic fever agents.
Patient Summary
Background
Lassa fever is a disease caused by a virus that is often spread by rodents. The disease is common in parts of West Africa where it causes a significant amount of death and disability among the population. Recently, Lassa fever has been imported by travelers to the United States and Europe. The Lassa virus that causes the disease is also on the list of potential bioweapons agents.
Why Was This Study Done?
A vaccine against the Lassa virus could help to control the disease in Africa, protect health workers, and help contain viral outbreaks. Several groups had developed different vaccines, some of which could protect monkeys from getting sick. However, none of the vaccines developed so far have shown all the characteristics one needs to have before testing them in humans.
What Did the Researchers Do?
Many vaccines combine specific parts of the harmful (pathogenic) virus with other components from harmless viruses, resulting in an effective but safe vaccine. The researchers developed a vaccine using a virus called vesicular stomatitis virus as the harmless component (known as the carrier). They inserted some genetic material from the harmful Lassa virus. In their study, they gave four macaque monkeys one shot of this combination vaccine and two others shots of the “empty” carrier (i.e., just the harmless component).
What Did They Find?
When four weeks later they injected all six monkeys with a lethal dose of Lassa virus, the four monkeys injected with the combination vaccine stayed healthy. The two monkeys who had been given just the “empty” carrier died.
What Does This Mean?
These are promising early results that justify further testing of this particular vaccine.
What Next?
Issues that need to be resolved before the vaccine can be tested in humans include the safety of the vesicular stomatitis carrier virus, how long the vaccine protects after the shot, and whether it is active against different strains of the Lassa virus (there are at least four strains, each with slightly different genetic material).
More Information Online
Information on Lassa fever can be found at the following Web sites.
The US CDC Special Pathogens Branch Web site: http://www.cdc.gov/ncidod/dvrd/spb/
The WHO fact sheet on Lassa Fever (from 2000): http://www.who.int/mediacentre/factsheets/fs179/en/
The authors thank Denise Braun, Daryl Dick, Friederike Feldmann, and Carlton Rice for technical assistance and assistance with animal care. The study was supported in part by a grant from the Canadian Institute of Health Research (CIHR-MOP-43921) awarded to HF and by the Medical Chemical/Biological Defense Research Program and Military Infectious Diseases Research Program, US Army Medical Research and Material Command (project numbers 04–4-7J-012 and 05–1-TT-001). Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author Contributions
TWG, SJ, US, LEH, PBJ, and HF designed the study. SJ, RL, US, and HF designed and rescued the vaccine vectors. TWG, JBG, and KMD carried out infection experiments. EAF and LEH performed intracellular cytokine staining to measure cellular immune responses. AG, US, and LF performed RT-PCR assays. JBG performed viremia and virus neutralization assays. ACS performed ELISAs to measure humoral immune responses. JBG and KMD performed hematology and clinical chemistry assays. TWG, SJ, EAF, ACS, JBG, AG, US, MCG, BRM, TL, LEH, PBJ, and HF analyzed the data and interpreted results of the study. TWG, SJ, and HF contributed to writing the paper.
Citation: Geisbert TW, Jones S, Fritz EA, Shurtleff AC, Geisbert JB, et al. (2005) Development of a new vaccine for the prevention of Lassa fever. PLoS Med 2(6): e183.
Abbreviations
ALBalbumin
ALPalkaline phosphatase
ALTalanine aminotransferase
ASTaspartate aminotransferase
BUNurea nitrogen
CREcreatinine
GPglycoprotein
GPCglycoprotein C
IFN-γinterferon gamma
NPnucleoprotein
PBMCperipheral blood mononuclear cell
PFUplaque-forming unit(s)
TBILtotal bilirubin
TNF-αtumor necrosis factor alpha
rVSVrecombinant vesicular stomatitis virus
VSVvesicular stomatitis virus
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| 15971954 | PMC1160587 | CC BY | 2021-01-05 10:40:11 | no | PLoS Med. 2005 Jun 28; 2(6):e183 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020183 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597195510.1371/journal.pmed.0020184Policy ForumBioethicsToxicology/Environmental HealthWomen's HealthInfectious DiseasesOtherEmergency MedicineEpidemiology/Public HealthHealth PolicyHIV/AIDSMental HealthSexual HealthEmergency MedicineMedicine in Developing CountriesInternational healthHealth education (including prevention and promotion)Infectious DiseasesPsychiatryPublic HealthEpidemiologyConfidentialityEthicsMedical journalsPrivacyRevisiting the Tsunami: Health Consequences of Flooding Policy ForumMorgan Oliver *Ahern Mike Cairncross Sandy Oliver Morgan is a visiting research fellow, Mike Ahern is a research fellow, and Sandy Cairncross is a professor in Environmental Health at the London School of Hygiene and Tropical Medicine, London, United Kingdom.
Competing Interests: The authors declare that they have no competing interests.
*To whom correspondence should be addressed. E-mail: [email protected] 2005 28 6 2005 2 6 e184Copyright: © 2005 Morgan, Ahern, and Cairncross.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
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Morgan and colleagues critically review the evidence on the health consequences of flooding disasters, and consider what interventions are appropriate.
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The South Asian tsunami on 26 December 2004 was one of the largest flooding disasters in recent history (Figure 1), causing about 280,000 fatalities in eight countries stretching from Asia to Africa [1]. Shortly after the disaster, the World Health Organization warned that disease could claim as many lives as the tsunami itself [2].
Fig 1 Flooding on the East Coast of Sri Lanka after the Tsunami
(Photo: Copyright, Aur lie Gr maud/MSF)
Early-warning communicable disease surveillance systems were established in the affected areas. However, in the following weeks, no large disease outbreaks were reported. In this article, we review the evidence for the health consequences of flooding disasters and consider what interventions are appropriate.
The Evidence on Flooding and Health
A recent systematic review of published literature found limited epidemiological evidence about the health effects from flooding disasters [3]. Notably, there were fewer studies from developing countries, where the disease burden is likely to be higher.
Diseases transmitted by the faecal–oral route.
The review found that diseases transmitted by the faecal–oral route were the main flood-related health impact. Such diseases include nonspecific diarrhoea, cholera, dysentery, and typhoid (Figure 2) [4]. For example, diarrhoea increased by a factor of two to four after flooding in Mozambique during 2000 [5]. Although increased incidence of diarrhoeal disease in affected populations is not usually associated with increased mortality, there have been some exceptions. Flooding in West Bengal in 1998 was followed by an outbreak of diarrhoea, suspected to be cholera, which resulted in 16,590 cases and 276 deaths (case–fatality ratio 1.7%) [6].
Fig 2 Conditions inside the Water Closet of the Index Case Residence during a Typhoid Outbreak in Cite Roche Bois, Mauritius
Salmonella typhi–contaminated sewage was aspirated into the main water distribution system after Hurricane Claudette's flooding, evidenced by high water marks on the walls. Note the leaking water pipe on the rear wall, and general unsanitary conditions.
(Photo: CDC)
Mosquito-borne diseases and other infections.
Flooding may also create a large number of breeding sites for mosquito-borne diseases such as malaria, and there have been numerous reports of increased incidence in previously endemic countries throughout Africa, Asia, and Latin America. This increase can be particularly important when populations are displaced. After the Mozambique floods of 2000, the number of malaria cases within the displaced population increased by a factor of 1.5 to two times previous levels [5].
Outbreaks of leptospirosis, a zoonotic bacterial disease associated predominantly with rats, have occurred when mud and water are contaminated by the urine of infected rodents. Flooding in Guyana in February 2005 led to more than 40 cases of leptospirosis. Other nonspecific infections such as conjunctivitis and ear, nose, and throat infections also increase [7].
After natural disasters, the media, health professionals, and relief workers often say that dead bodies of victims can cause epidemics of diseases such as cholera [8]. The fear caused by these claims encourages communities, local authorities, and governments to rapidly dispose of victims without identification. This contributes to psychological distress among surviving relatives and creates legal problems where there are property, inheritance, or insurance claims. However, victims of natural disasters die from trauma, burns, or drowning and are unlikely to harbour pathogenic organisms such as cholera, which can cause epidemics [9]. For the public, the risk of infectious disease from dead bodies after natural disasters is negligible. Individuals handling cadavers may have a small risk of exposure to tuberculosis, blood-borne viruses (such as Hepatitis B or C and HIV), and gastrointestinal infections. However, the risk of infection can be greatly reduced by following basic hygiene precautions [9].
Injuries.
The idea that flooding disasters cause large numbers of injuries is a common disaster myth that has regularly been proven to be wrong [10,11]. Even after violent flooding events such as the recent tsunami, the number of serious injuries is much lower than many medical emergency teams expect [11,12]. However, information about the number and type of injuries is often lacking, and improved data collection would improve our understanding of injury risk due to floods. Nevertheless, even in the absence of serious injury, wound infections of cuts and abrasions are common [7,12]. For example, in the Indonesian Province of Aceh, 106 cases of tetanus and 20 deaths were reported (case–fatality ratio 18.9%) after the tsunami at the end of 2004 (unpublished data).
Mental health problems.
Mental health impacts, which include common mental disorders, post-traumatic stress syndrome, and suicide, have not been well documented [13]. Again, much of the existing evidence comes from Western countries [3], where coping mechanisms and cultural contexts are likely to be different than in many lower-income countries [14]. However, several studies have reported increased symptoms, such as anxiety, depression, and sleeplessness, among flood victims [3,15]. Behaviour change in children has also been observed, with Durkin et al. reporting increased bed-wetting and aggression [16], and other studies reporting post-traumatic stress disorder and dissatisfaction with life [3]. Only two studies have studied suicide among flood victims, and the evidence of an effect is unclear [3].
Addressing the Health Consequences of Flooding
Appropriate and timely intervention can significantly reduce the risk of mortality and morbidity from infectious diseases after flooding disasters. In the short term, preventing the diseases spread via the faecal–oral route is the most important public health intervention. This relies on three measures: provision of clean water, suitable sanitation, and hygiene promotion (Figure 2) [17].
Providing clean water.
Each person requires a minimum of 15 litres per day for drinking, cooking, and washing [17]. Re-establishing a basic supply of clean water in urban areas may be complicated by damage to water treatment works or distribution pipelines. Electricity distribution grids may also need repair to run water pumps. In rural areas, open wells and hand pumps are often easier to rehabilitate. However, lack of access to affected areas usually hampers response efforts; flooding makes roads impassable, and access to all affected areas can often take several days or weeks.
Sanitation.
Sanitation is particularly important when affected populations seek shelter in communal settings such as schools. Additional latrines may have to be constructed in the short term. Household facilities such as pit latrines may be flooded or destroyed, often leaving returning communities without sanitation. Similar to urban water supply, urban sanitation systems are more complex and more costly to repair. Temporary low-tech solutions may be able to bridge the gap while longer-term repairs are made. It is much easier to promote such solutions among people who have owned toilets but lost them in a flood, than among communities unaccustomed to sanitation.
Personal hygiene.
Personal hygiene is especially important when individuals have reduced access to clean water and sanitation or are living in crowded or temporary accommodation. Floodwaters often carry substantial faecal contamination, and people may need to be alerted to the need to clean all household possessions that they have touched. Hygiene promotion messages highlighting the importance of hand washing should be considered as necessary as provision of clean water [18]. Such activities should also be supported by provision of basic materials such as buckets and soap.
Planning for the Future: Disaster Preparedness and Mitigation
It is often believed that casualties from natural disasters are unavoidable, but this belief is false [8]. There are many measures that can reduce morbidity and mortality following large and potentially catastrophic flooding events [19].
Early warning of impending floods and natural events such as hurricanes allows sufficient time for communities to be evacuated to safe areas. Although Florida experienced one of the worst hurricane seasons on record in 2004, the number of fatalities was lower than expected because of early warning and evacuation. Early warning also provides sufficient time to prepare when evacuation is not possible. Hurricane George in 1998 caused widespread damage and several fatalities in the Dominican Republic, where residents were not warned. In contrast, Cuba and Puerto Rico experienced relatively limited damage and loss of life, because preparations were made in the hours before the storm.
Disaster preparedness can also be developed for communities that are regularly exposed to flooding disasters. Cyclone shelters built by the Red Cross in Orissa, India, saved many thousands of lives in 1999 when two cyclones struck. In the region of the Americas, the Pan American Health Organization has spent many years promoting and integrating disaster preparedness into building health facilities to ensure that medical services needed to treat victims and maintain ongoing care for patients with chronic conditions will not be disrupted by disasters.
A recent global-scale review of health risks from flooding highlights that in flood-prone areas, disaster preparedness within the health system as a whole is particularly important [20]. In addition to infrastructure and early-warning systems, another key element in disaster preparedness is education and raising awareness about disaster risks and response plans. Had there been greater awareness about the risk of tsunamis, perhaps many lives could have been saved in the South Asian disaster in December 2004.
Conclusion
Disasters such as the tsunami in South Asia underline the vulnerability of many communities in developing countries. Although the importance of clean water and sanitation get top billing during natural disasters, their absence during normal conditions carries a still greater price: 2.5 million children in developing countries die each year of diarrhoeal illnesses [21].
Scientists project that climate change may increase the frequency and severity of flooding [22]. Despite this and the potentially large-scale impacts caused by flooding, our understanding of the health impacts is limited, especially of longer-term effects on mental health and the health effects of lost livelihoods. The latter may prove to be even greater than the short-term impacts. For example, it has been estimated (Cairncross, unpublished data) that the total mortality directly attributable to the Mozambique floods of 2000 was less than the increase in child mortality in Mozambique expected as a result of the reduction in GDP caused by the floods. Further work is needed to develop our understanding of the health impacts, especially on mental health, and to develop better disaster preparedness and response measures.
Citation: Morgan O, Ahern M, Cairncross S (2005) Revisiting the tsunami: Health consequences of flooding. PLoS Med 2(6): e184.
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Ahern M Kovats R Wilkinson P Few R Matthies F Global health impacts of floods: Epidemiological evidence Epidemiol Rev 2005 (In press)
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| 15971955 | PMC1160588 | CC BY | 2021-01-05 10:40:11 | no | PLoS Med. 2005 Jun 28; 2(6):e184 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020184 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597195610.1371/journal.pmed.0020185Policy ForumBioethicsSexual HealthToxicology/Environmental HealthWomen's HealthInfectious DiseasesOtherEmergency MedicineEpidemiology/Public HealthHealth PolicyHIV/AIDSMedical LawMental HealthForensic MedicineInternational healthPsychiatryHealth education (including prevention and promotion)Infectious DiseasesEpidemiologyPublic HealthHealth PolicyConfidentialityEthicsMedical journalsPrivacyMedicine in Developing CountriesEmergency MedicineAfter the Tsunami: Legal Implications of Mass Burials of Unidentified Victims in Sri Lanka Perera Clifford Clifford Perera is a lecturer at the Department of Forensic Medicine, Galle, Sri Lanka. E-mail: [email protected]
Competing Interests: The author declares that he has no competing interests.
6 2005 28 6 2005 2 6 e185Copyright: © 2005 Clifford Perera.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Addressing Psychosocial Needs in the Aftermath of the Tsunami
How Women Were Affected by the Tsunami: A Perspective from Oxfam
Is the filming and photography of patients and dead bodies in hospitals during disasters ethically permissible?
Evidence Based Health-care Interventions after the Asian Tsunami: the response of the Cochrane Collaboration
Revisiting the South Asia Tsunami: Health Consequences of Flooding
Meeting the Health Needs of Migrant Workers Affected by the Tsunami
Perera discusses how the tsunami highlighted gaps in the laws in Sri Lanka on death investigation and mass burials.
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On 26 December 2004, Sri Lanka was affected, along with many other countries in the region, by deadly tsunami waves. The estimated death toll in Sri Lanka was more than 40,000; many more thousands of people were missing and displaced. Sri Lanka had not experienced a disaster of such magnitude in its 2,000 years of recorded history. The administrative, health, and judicial services were simply not able to respond rapidly to the workload demands created by the disaster.
The disaster itself also highlighted gaps in the laws on death investigation and mass burials, which required the rapid introduction of many emergency regulations and modification of existing laws. This article discusses these gaps, and categorises the post-tsunami events into two phases: acute and secondary. The acute phase concerns the events that occurred during the first week with regard to the management of the deceased; the secondary phase represents the events that took place subsequently.
Legal Provision for a Death Investigation System
Death investigation is an essential process to maintain law and order in any civilized society. Sri Lanka adopted the Coroner system from Britain in the 18th century, along with other South Asian countries, and still uses it, with some modifications. The legal provisions for death investigation are elaborated in Sections 369–373 in the Code of Criminal Procedure, Act 15, of 1979 of Sri Lanka (available at http://www.lawnet.lk). These sections do not, however, provide any instructions with regard to a mass disaster situation. Under the present system, death investigation is principally carried out by an Investigator into Sudden Death appointed by the Minister of Justice for a particular jurisdiction. Unlike in European countries, most of these investigators have neither medical nor legal professional skills. On certain occasions, such as homicides and custodial deaths, the magistrates may act as investigators of death.
When emergency laws are activated, the usual death investigation procedures may deviate from accepted norms, and the authority for disposal of bodies—even without post-mortem examinations—has been given in the past to prescribed police authorities. Many such emergency regulations were enacted during the secondary phase of the tsunami in order to meet socio-political requirements. However, there were no specific regulations that related to death investigation or disposal of the deceased.
Death Investigation System after the Tsunami
In a disaster situation, it is very difficult to follow usual death investigation procedures, especially if the numbers of deceased are very high. Hence, it was not possible to follow usual procedures of death investigation in any of the countries affected by the tsunami in December 2004.
Investigators into Sudden Death and magistrates in the tsunami-affected areas were joined by their counterparts in non-affected areas in performing inquests into the deaths of the deceased. However, many deficiencies were observed in the formal procedure of death investigation and certification, mainly due to lack of descriptive guidelines to follow in a mass disaster situation.
Identification of the Deceased: Acute Phase
The most important aspect of mass disaster investigation, from the medico-legal perspective, is the identification of the deceased. The cause of death of the deceased is of secondary importance in these circumstances. The importance of the issue of identification of the deceased after the tsunami was brought to light by human rights groups in the early stages. Radhika Coomaraswamy, chairperson of the Human Rights Commission of Sri Lanka, said: “The identification of dead bodies is one of the most basic of all human rights. Due to the situation, many bodies were buried without identification. It is absolutely essential that forensic expertise is marshalled to identify all dead bodies so that their next of kin may be informed. The government must make this an important matter of priority” [1].
However, medico-legal services were not given priority during the acute phase management of the tsunami disaster in Sri Lanka, despite Sri Lanka having the best medico-legal services of the countries affected. The number of qualified full-time forensic pathologists in Sri Lanka is almost equal to that of Australia, although infrastructure facilities are underdeveloped in most centres.
After the tsunami, the deceased were sent to the nearest hospital morgues during the initial stages, and within hours all available space was occupied. After the second day of the disaster, the deceased were sent to mass burial sites, bypassing the hospitals. As a result, thousands of deceased were neither imaged nor documented in appropriate registries before being sent to mass burial grounds. The reasons for this administrative decision are shown in Box 1.
Box 1. Reasons Why the Deceased Were Neither Imaged nor Documented before Being Sent to Mass Burial Grounds
Minimal recognition given to the medico-legal services at the initial stages
Rapid recovery of dead during first few days
Lack of storage facilities for the deceased in hospitals
The advanced state of decomposition observed in the deceased
Lack of rapid documentation facilities (e.g., digital cameras, computers)
Minimal use and construction of temporary mortuary spaces
Lack of transport facilities to bring deceased into hospitals
Poor coordination amongst forensic pathologists
Lack of support staff to continue medico-legal work
As most of the statistics on the mass burials were not reliable nor available on demand, the total number of people who died in each region in the acute phase was calculated from hospital data, which were not representative of the true figure. If during the acute phase the deceased had at least been imaged and documented by an organised team of forensic experts in the few hours before mass burials, the overall picture would have been very different.
Legal Consequences of Mass Burials
The rapid disposal of the deceased into mass burial sites without any sort of documentation had serious effects on issuing death certificates subsequently. Many mass burial sites were not planned and not well documented. The police figures about the deceased in many burial sites were contradictory. In some instances, the identified and unidentified deceased were buried together. In none of the sites were the buried bodies given any permanent identification marks or tags, which would have been helpful for future possible exhumations. In addition, many burial sites were used for reburials.
The system established to collect information on missing persons functioned poorly. The police and courts maintained separate offices for this purpose, and much of their information was duplicated. Therefore, the credibility of the official statistics on missing persons is questionable. Some victims were recorded missing and deceased at two different centres; in other cases, coroners were reluctant to certify death due to lack of vital information.
The provisions of the Legal Aid Commission Act of 1978 should be adopted to ease the problem of lack of death certificates. A special mechanism will need to be derived that can authorise the issuing of death certificates on missing people after an agreed-upon brief period of search by the authorities. During the aftermath of the tsunami, the police, coroners, and courts were acting without coordination. Because of irregularities affecting identification of the deceased and the issuing of death certificates, there remains the possibility of the public raising the issue of their fundamental rights being denied. Such public protest may be driven by the many exhumations performed in January–March 2005 to identify missing foreigners in various parts of southern Sri Lanka.
Identification of the Deceased: Secondary Phase
The secondary phase of the disaster was dominated by two events: recovery of human remains from lowlands and seashore, and exhumation of suspected mass burial sites to search for missing foreigners (Figures 1 and 2).
Figure 1 Exhumations Performed in the Southern Province in Search of Missing Foreigners (Photo: Clifford Perera)
Figure 2 More Exhumations Performed in the Southern Province in Search of Missing Foreigners
(Photo: Clifford Perera)
Although the acute phase was carried out with much enthusiasm and with the participation of many segments of civil society, the secondary phase lacked momentum due to the lack of interest among law enforcement authorities and some forensic practitioners. The strain of the increased workload during the first few weeks, lack of coordination amongst the senior law enforcement and medico-legal officials, and lack of infrastructure facilities led to slow continuation of the secondary phase since January 2005. Some of the human remains recovered in this period, even within eight weeks of the disaster, are almost skeletalised—for example, those from marshy lands in the southern province collected within eight weeks. The high temperatures of 30–35 °C prevailing for weeks after the disaster may have had a direct effect on the speed of decomposition. Most of the deceased recovered during the second phase were females and children without any specific identification details.
An international commission was formed in mid-February to identify missing foreigners in Sri Lanka, with the participation of British, German, and French investigators, the officers of the Criminal Investigation Department, judicial medical officers from Colombo and Galle, and a coroner in Colombo. This commission undertook the task of continuing all the previous investigations started in the search for missing foreigners, including exhumations already performed in many parts of the country. The distinguishing feature of the commission's involvement was performing complete autopsy examinations and identification procedures on all suspected bodies of missing foreigners. This commission functioned until April, and many foreigners were positively identified following secondary (specific) investigations such as DNA profiling. The formation of such a commission led local experts to re-evaluate their strategies in disaster management, and many voices were raised demanding urgent attention to establish proper investigative mechanisms in the state sector, including DNA-profiling facilities to identify the deceased in disasters.
Local experts are realising that the secondary phase of identification may continue for months, despite having minimal resources and innovation for such an exercise. The seabed surrounding the affected coastal regions contains innumerable deceased, most of whom may never be seen or touched. However, the human remains recovered from the beaches of these affected regions must be treated as potential tsunami deceased for many months to come.
Implications for the Future
Sri Lanka has experienced many man-made disasters, such as bomb explosions, and natural disasters, such as regional floods, in the past three decades. Currently, many countries in Asia have their own disaster management and emergency plans; a few countries such as Sri Lanka lag far behind in modern disaster management. Therefore, it is crucial that Sri Lanka formulate a national plan for disaster management, including national guidelines for disaster victim identification, to overcome the problems discussed in this article.
Previously, Sri Lanka had enacted an act on a reconstruction and rehabilitation fund (Reconstruction and Rehabilitation Fund Act, No. 58, in 1993). A bill called “Sri Lanka Disaster Counter-Measures” was published in the government gazette issued in November 2002 and placed on the order paper of the parliament. The Supreme Court made a determination of the bill in January 2003 after its legality was challenged, but it was not taken for debate in the parliament afterwards. However, this time the Sri Lankan government introduced more or less the same bill under the title “Sri Lanka Disaster Management” in February 2005, two months after the disaster. The long title of the bill states that it is “to provide for the establishment of the National Council for Disaster Management, the National and Human Disaster Management Centre; the appointment of Technical Advisory Committees; the preparation of Disaster Management Plans; the declaration of a state of disaster, the award of compensation, and for matters connected thereto.”
Further, this bill contains the first legal definition of a disaster in Sri Lanka and also includes a separate definition for human disaster (man-made disaster). Section 10 of the bill states that every ministry, government department, and public cooperation prepare a disaster management plan—a long-awaited request. The framework suggested by this bill for disaster management in Sri Lanka is commendable and is an effort to uplift the local standards to the existing regional and international standards through a strong legal framework. Although the bill does not specify a time frame to establish disaster management structures, the legal implications of it will drastically affect the management of future disasters.
In its determination in 2005, the Supreme Court was critical of the failures of the previous parliament and reiterated the necessity of implementing relevant legal infrastructure: “We have to note that if the Bill in respect of which the determination was made by this court in January 2003 was proceeded with in parliament, a National Disaster Management plan would have been in place to cover natural disasters including a ‘tsunami.’ For some reason the Bill was not proceeded with by Parliament, and the court has once again to make a determination on substantially the same bill, two years after a devastating ‘tsunami’ has affected Sri Lanka, at a time when the country was totally unprepared to meet that situation” [2].
Conclusion
It is clearly a priority for Sri Lanka to have an effective medico-legal scheme to deal with problems of identification of deceased in future disasters. It is a collective responsibility of the attorney general's department, health department, police department, administrative authorities, and professional organisations of forensic pathologists, scientists, legal practitioners, and many others concerned to ensure that this happens.
Citation: Perera C (2005) After the tsunami: Legal implications of mass burials of unidentified victims in Sri Lanka. PLoS Med 2(6): e185.
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References
Human Rights Commission of Sri Lanka Protecting the rights of tsunami victims Daily News 2005 Jan 12 6
Supreme Court of Sri Lanka Supreme Court Special Determination 2005 4 3
| 15971956 | PMC1160589 | CC BY | 2021-01-05 10:40:15 | no | PLoS Med. 2005 Jun 28; 2(6):e185 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020185 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0020186Correspondence and Other CommunicationsEpidemiology/Public HealthHealth PolicyHIV/AIDSMedical EthicsEthicsHealth PolicyHIV Infection/AIDSMedicine in Developing CountriesResource allocation and rationingAuthors' Reply CorrespondenceCapron Alexander M Reis Andreas World Health OrganizationGenevaSwitzerlandE-mail: [email protected]
Competing Interests: The authors have declared that no competing interests exist.
6 2005 28 6 2005 2 6 e186Copyright: © 2005 Capron and Reis.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Designing Equitable Antiretroviral Allocation Strategies in Resource- Constrained Countries
Allocating Antiretrovirals in South Africa: Using Modeling to Determine Treatment Equity
Designing an Equitable Strategy for Allocating Antiretroviral Treatments
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In response to our commentary [1] on their paper, “Designing Equitable Antiretroviral Allocation Strategies in Resource-Constrained Countries” [2], Wilson and Blower assert that we misunderstood both their analysis and the importance of their results [3]. Rather than “setting the record straight,” what may be needed is more effort to bridge the differences in disciplinary approach that create a greater appearance of disagreement than is actually the case.
On a factual level, we believe Wilson and Blower's results were appropriately described for the purposes of our commentary. As their letter points out, they applied the operations research methods to model the allocation of antiretrovirals (ARVs) among 17 health-care centers in KwaZulu–Natal, based on a hypothetical distribution of HIV/AIDS among the communities in that province. Their article characterizes this as “an elegant and simple theoretical framework,” but they object to our concluding that it could “inform policy-makers' decisions regarding the location of HIV services,” since they took the treatment sites as given. Yet their article compared the alternatives of using all 54 centers in the province, at one extreme, and of using only a single treatment site (in Durban), at the other extreme; in each case, the possibility of allocating to a larger number of centers is equivalent to the creation of additional centers closer to remote groups of patients.
Wilson and Blower write that geographic accessibility is improved if the number of health-care facilities is increased, and they calculated that it would be optimal if all 54 facilities in the province of KwaZulu–Natal distributed the medicines, instead of just 17. We took this result to confirm the need to reach out and build capacity. We are sorry if we were mistaken in assuming that Wilson and Blower would want to see their stated objective of ensuring fair distribution applied in the real-world context of many poor countries with a high HIV burden and where fairness in ARV care cannot be achieved solely by allocating resources among the existing sites.
A wider gap in perception can be seen in Wilson and Blower's repeated conflation of “optimal,” “equal,” and “equitable,” combined with their suggestion that decision makers who fail to apply their model must be following an “ad hoc approach.” The central point of our commentary was that various ethical theories reach very different conclusions about what result would be optimal, and that even among those aiming to achieve the greatest equity (rather than some other optimum), many would not take equality as the measure of equity. Wilson and Blower themselves recognize that apparent equality of access (in terms of distance to treatment) needs further study to determine whether patients can in fact access treatment. We need to know whether some distances are simply too far for patients to travel for chronic care, and when distances of equal length affect access very differently because of the characteristics of particular patient populations, transportation systems, and so forth.
Wilson and Blower seem unwilling to accept the notion that, in the furtherance of a rational strategy to achieve equity, some health authorities might decide, for example, to allocate a disproportionate share of ARVs to traditionally disadvantaged populations. Wilson and Blower's model could still be useful in allocating resources among the centers chosen (or established) to reach the target population, but the calculation would have to take account of more information about the centers and the population, lest assumptions about catchment areas produce a formal equality that does not translate into actual equality in access, much less into equitable access in light of all relevant factors.
Plainly, we share Wilson and Blower's aim of optimizing countries' responses to the tragedy of treatable, but untreated, HIV/AIDS. Any tools that are useful to that end are welcome. But besides using models to distribute ARVs in a way that optimizes spatial equality, governments that want to achieve equity will need also to overcome nongeographic barriers to accessing treatment. These include ignorance, stigma, discrimination, and outright criminalization of vulnerable groups, as well as fees at point of service that are prohibitive for the poor. All of these are given attention within the context of the “3 by 5” program of the World Health Organization and the United Nations Joint Programme on HIV/AIDS, including in the guidance document on equitable access to ARV treatment cited in our commentary [4].
Citation: Capron AM, Reis A (2005) Authors' reply. PLoS Med 2(6): e186.
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References
Capron AM Reis A Designing an equitable strategy for allocating antiretroviral treatments PLoS Med 2005 2 e69 10.1371/journal.pmed.0020069 15737013
Wilson DP Blower SM Designing equitable antiretroviral allocation strategies in resource-constrained countries PLoS Med 2005 2 e50 10.1371/journal.pmed.0020050 15737005
Wilson DP Blower S Allocating antiretrovirals in South Africa: Using modeling to determine treatment equity PLoS Med 2005 2 e155 10.1371/journal.pmed.0020155 15971938
World Health Organization/UNAIDS Guidance on ethics and equitable access to HIV treatment and care 2004 Geneva WHO/UNAIDS Available at http://www.who.int/ethics/en/ethics_equity_HIV_e.pdf . Accessed 12 May 2005
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0020187Correspondence and Other CommunicationsOtherMedical EthicsClinical TrialsEthicsAuthors' Reply CorrespondenceTurner Erick
1
Tramèr Martin
2
1Portland VA Medical Center and Oregon Health and Science UniversityPortland, OregonUnited States of America2Geneva University HospitalsGenevaSwitzerlandE-mail: [email protected]
Competing Interests: EHT is on the speaker's bureaus of Eli Lilly, AstraZeneca, and Bristol-Myers Squibb. He has provided outside consulting to Bristol-Myers Squibb, Eli Lilly, GlaxoSmithKline, and Sepracor. He has also received funding for clinical drug trials, which can be spent only for research purposes and which has no effect on his income, from Abbott Laboratories, AstraZeneca, Bristol-Myers Squibb, and DOV Pharmaceuticals. MRT has been a scientific consultant to Pfizer, Merck, Janssen-Cilag, and Sintetica. He has also received lecture fees from various pharmaceutical companies.
6 2005 28 6 2005 2 6 e187Copyright: © 2005 Turner and Tramèr.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Is It Always Unethical to Use a Placebo in a Clinical Trial?
The Debate over Placebo-Controlled Trials
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We appreciate Dr. Miller's contribution to this debate [1]. Whether one uses his phrase “established treatment” or “proven therapy,” we urge caution in using such a litmus test to decide whether the use of placebo is or is not acceptable. Such terms beg to be defined carefully. Should nonsteroidal anti-inflammatory drugs be considered “proven” for arthritis, despite their problems with assay sensitivity [2,3]? Dr. Miller states that there is no established treatment for adenomatous polyps. However, there is evidence from epidemiological studies and clinical trials supporting the use of aspirin and other nonsteroidal anti-inflammatory drugs for this condition [4–10]. Armed with such evidence—whether it qualifies aspirin as “proven therapy” is open to subjective interpretation—many placebo opponents, we maintain, would argue for an active-controlled design as a more ethical alternative to the placebo-controlled design actually used in APPROVe [11].
Dr. Miller focuses on the question of defending the use of placebo in the APPROVe study. This focus reflects the prevailing bias, which is to choose when it is acceptable to use placebo rather than to choose when it is acceptable to omit placebo. This bias is evident in the current version of the Declaration of Helsinki, with wording such as, “Extreme care must be taken in making use of placebo-controlled trials.” Thus, the use of placebo is typically presumed “guilty until proven innocent,” while active-controlled designs are presumed “innocent until proven guilty.” The declaration is silent on the possibility that omitting placebo can lead to problems, too, as we have now witnessed with Vioxx.
So perhaps the more important question should be whether it was defensible to exclude placebo in the VIGOR study. Dr. Miller acknowledges that placebo would have provided a better assessment of the safety signal in the VIGOR study. Indeed, because placebo was not used, the authors were able to plausibly conclude that the difference between the two groups was due to naproxen causing benefit rather than to Vioxx causing harm [12]. (The plausibility of this conclusion has since been questioned [13,14].) This misleading safety signal only delayed the withdrawal of Vioxx. Its design was superficially ethical, but science was not advanced, and the public health was ill-served.
One might protest that these comments are made with the benefit of hindsight. It is true that the medical community at large did not become aware of this safety issue until September 2004, when the results of the placebo-controlled APPROVe study were made public and Vioxx was withdrawn. However, according to David Graham's testimony to the United States Senate [15] and a report on internal Merck documents [16], there was good reason for concern about a possible safety signal before 1999, when Vioxx was approved and recruitment for VIGOR began [15].
If the VIGOR study had included a placebo arm, the truth about Vioxx could have been learned in February 2001 instead of September 2004 [14]. That is over 180 weeks during which the now infamous “two to four jumbo jetliners” were allowed to continue “dropping from the sky every week” [15]. Using the midpoints of Graham's range estimates, this works out to 94,500 excess heart attacks and strokes, including 33,000 deaths, in the US alone.
The authors of the VIGOR study said, “We could not include a placebo group” [12]. Was the idea of including a placebo arm suggested but rejected as “unethical,” even though rescue medication could have been used? Or did they take the path of least resistance in the interest of rapid institutional review board approval and ease of patient recruiting? Whatever the reason, the decision to omit placebo led to ambiguity and inaction.
In clinical trials, whether one looks at efficacy (please see our opening argument in this debate [17]) or safety, omitting placebo often muddies the scientific waters and places the public health at increased risk. Good science and good ethics cannot be divorced from one another. We believe these considerations should factor into discussions on the ethics of clinical trial design. Before we experience another Vioxx, we hope that a future version of the Declaration of Helsinki will add, “Extreme care must be taken when omitting placebo.”
Citation: Turner E, Tramèr M (2005) Authors' reply. PLoS Med 2(6): e187.
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0020191Correspondence and Other CommunicationsMedical EthicsMedical HistoryHistoryEthicsAuthor's Reply CorrespondenceHayden Deborah San Anselmo, CaliforniaUnited States of AmericaE-mail: [email protected]
Competing Interests: The author has declared that no competing interests exist.
6 2005 28 6 2005 2 6 e191Copyright: © 2005 Deborah Hayden.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Alas, Poor Yorick Alas, Poor Yorick: Digging Up the Dead to Make Medical Diagnoses
Call for Biohistory Guidelines
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The excellent article by Jordan Paradise, Lori B. Andrews, and colleagues, “Ethics. Constructing Ethical Guidelines for Biohistory” [1], neither advocates nor argues against biohistorical research; instead, it points out that such investigations are currently taking place without guidelines—ethical, scientific, moral, or religious. The question remains: if such guidelines were to be established, what individuals, institutions, governments, medical examiners, family members, or intrepid biographers are to be given permission? Who is to decide what is “historically significant”? Not to mention the meta-question: who is to decide who is to decide? I apologize to the authors if my brief comments [2] implied that they took a position on this issue.
Citation: Hayden D (2005) Author's reply. PLoS Med 2(6): e191.
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References
Andrews LB Buenger N Bridge J Rosenow L Stoney D Ethics. Constructing ethical guidelines for biohistory Science 2004 304 215 216 15073360
Hayden D Alas, Poor Yorick: Digging Up the Dead to Make Medical Diagnoses PLoS Med 2005 2 e60 15783251
| 0 | PMC1160592 | CC BY | 2021-01-05 10:40:17 | no | PLoS Med. 2005 Jun 28; 2(6):e191 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020191 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597196010.1371/journal.pmed.0020192Correspondence and Other CommunicationsMedical EthicsMedical HistoryHistoryEthicsCall for Biohistory Guidelines CorrespondenceParadise Jordan Chicago-Kent College of LawChicago, IllinoisUnited States of AmericaE-mail: [email protected]
Competing Interests: The author has declared that no competing interests exist.
6 2005 28 6 2005 2 6 e192Copyright: © 2005 Jordan Paradise.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Alas, Poor Yorick Alas, Poor Yorick: Digging Up the Dead to Make Medical Diagnoses
Author's Reply
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I am writing in response to an essay published in the most recent issue of PLoS Medicine by Deborah Hayden, entitled “Alas, Poor Yorick: Digging Up the Dead to Make Medical Diagnoses” [1]. As a co-author of the Science piece with Lori B. Andrews that Hayden references, I am troubled by her comment on our article. Nowhere in that article, “Ethics. Constructing Ethical Guidelines for Biohistory” [2], do we suggest that genetic testing be done on deceased individuals for historically significant questions. In fact, we specifically highlight some of the ethical, legal, social, and scientific issues that such testing raises and recommend that guidelines be developed in order to monitor current research that is being undertaken in this area. The article does not advocate biohistorical research. This distinction is very important and one that is quite evident upon a careful reading of our article.
Citation: Paradise J (2005) Call for biohistory guidelines. PLoS Med 2(6): e192.
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References
Hayden D Alas, Poor Yorick: Digging Up the Dead to Make Medical Diagnoses PLoS Med 2005 2 e60 15783251
Andrews LB Buenger N Bridge J Rosenow L Stoney D Ethics. Constructing ethical guidelines for biohistory Science 2004 304 215 216 15073360
| 15971960 | PMC1160593 | CC BY | 2021-01-05 11:13:39 | no | PLoS Med. 2005 Jun 28; 2(6):e192 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020192 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0020194SynopsisNeurosciencePsychologyOtolaryngologyNeurologyOtolaryngology/ENTBrain Activity and Tinnitus Synopsis6 2005 28 6 2005 2 6 e194Copyright: © Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Tinnitus Perception and Distress Is Related to Abnormal Spontaneous Brain Activity as Measured by Magnetoencephalography
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Exposure to short periods of very loud noise can cause tinnitus—a persistent ringing or buzzing in the ears that cannot be blocked out. Tinnitus may affect around 10%–15% of the population; severe tinnitus is very debilitating (1%–2% of the population). Previous work has shown that tinnitus has a neurophysiological basis, but precisely which parts of the brain and the auditory circuits are involved is not yet understood.
The human ear is essentially a very sensitive vibration sensor, one that is able to receive the minute longitudinal vibrations in air that make up sound waves. It can detect sounds from 20 Hertz (Hz) (very low pitch) to 20,000 Hz (very high pitch) but is particularly sensitive to sounds in the range of 500–5,000 Hz—the so-called speech frequencies. However, the ear, and in particular the cochlea, or inner ear, can be damaged by exposure to excess noise, leading to permanent damage to the ear, i.e., deafness.
Some studies in both animals and humans have suggested that tinnitus and hearing loss may be related. These studies have found that neurons in regions of the auditory cortex that have been deprived of stimuli because of hearing loss change their receptive field and may develop enhanced spontaneous activity. Other studies, such as some involving neuroimaging using positron emission tomography, have suggested that parts of the brain involved in attention and emotional regulation might be involved in the production of tinnitus.
One of the key research targets in tinnitus has been investigation of cortical activity, especially in animal models of tinnitus, but studies in humans have been rare. Previous studies have identified temporal and frontal temporal changes in individuals whose tinnitus is severely disabling; however, there have been no group studies comparing abnormalities of ongoing, spontaneous neuronal activity in people with and without tinnitus.
In this month's PLoS Medicine, Nathan Weisz and colleagues studied 17 patients with chronic tinnitus and hearing loss and 16 control individuals with normal hearing. Patients were asked to fill in a questionnaire about the impact of tinnitus on their lives and had their levels of tinnitus assessed.
The team's methods differed from previous work in that the team chose to examine the power spectrum of neuromagnetic oscillatory activity during rest, whereas previous studies had focused on measuring neurophysiological responses following sounds.
Normally in awake and healthy subjects a certain rhythm of brain activity at 8–12 Hz—the so-called alpha rhythm—is dominant. Finding enhanced slow-wave, or delta, activity (<4 Hz) in awake subjects is usually a sign of a dysfunctional neuronal network, as these waves can be observed in various neurological and psychiatric disorders. Weisz and colleagues' analysis of the frequency spectrum of recorded magnetic fields revealed that the energy in the alpha band was strongly reduced and that of the delta band enhanced in the group with tinnitus compared with the individuals with normal hearing. This pattern was particularly pronounced in the temporal regions, and overall the effects were stronger for the alpha than for the delta frequency band.
This is the first study to show these changes in delta and alpha spontaneous cortical activity, say the authors. But they concede it is still unclear whether the enhancement of delta activity compared with alpha is the abnormal activity perceived as tinnitus. However, the fact that regions that show slow-wave activity during slow-wave sleep are also regions of low alpha activity supports the idea that changes in cortical activity might be mediated by sensory deprivation, in this case that partial hearing loss might be involved in producing tinnitus.
Tinnitus-related distress as assessed by the questionnaire was strongly associated with this abnormal spontaneous activity, especially in the right temporal and left frontal areas, thus pinpointing a possible tinnitus-related cortical network.
A limitation of this study was that the tinnitus group also had high-frequency hearing loss, whereas the control group did not; the ideal control group would have been patients with the same sort of hearing loss but no tinnitus.
In discussing their findings, the authors suggest that their study supports previous work indicating that the prefrontal cortex is a candidate region for integration of the sensory and emotional aspects of tinnitus. Further studies should focus on frontal areas, which could allow identification of interactions and modulating influences that higher-order psychological processes (e.g., emotions and thoughts) may have on the generation of tinnitus in the auditory cortex.
| 0 | PMC1160594 | CC BY | 2021-01-05 11:13:39 | no | PLoS Med. 2005 Jun 28; 2(6):e194 | utf-8 | PLoS Med | 2,005 | 10.1371/journal.pmed.0020194 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0020195SynopsisObstetrics/GynecologyWomen's HealthObstetricsPregnancyWomen's HealthAssessing the Risks of Twin Pregnancies Synopses6 2005 28 6 2005 2 6 e195Copyright: © Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
High Risk of Unexpected Late Fetal Death in Monochorionic Twins Despite Intensive Ultrasound Surveillance: A Cohort Study
Uncomplicated monochorionic diamniotic twins and the timing of delivery
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As mothers get older and assisted conception becomes more common in developed countries, the incidence of multiple births—primarily of nonidentical siblings, but also of identical ones—has dramatically increased. Multiple pregnancies are high-risk pregnancies, with preterm delivery and monochorionicity (shared placenta) the major problems. Consequently, efforts are underway to optimize the management of these pregnancies.
While identical (monozygotic) twins are much less common than dizygotic ones, monozygotic twinning events are increased after induced ovulation and in vitro fertilization. Monozygotic twins can be diamniotic dichorionic (two amniotic sacs, two placentas), monoamniotic monochorionic (one amniotic sac, one placenta), or diamniotic monochorionic (two amniotic sacs, one placenta). The last type accounts for approximately two-thirds of all monozygotic twins.
Monochorionic twins are at higher risk because they share a common placenta; they are primarily at risk from circulation abnormalities like twin–twin transfusion syndrome (the smaller twin [donor] does not get enough blood while the larger twin [recipient] becomes volume overloaded) and intrauterine growth restriction. However, the majority of diamniotic monochorionic twin pregnancies do not develop such complications.
Nicholas Fisk and colleagues have studied records of 151 seemingly uncomplicated diamniotic monochorionic pregnancies and found a surprisingly high rate of fetal death: ten unexpected intrauterine deaths occurred in seven of the 151 pregnancies with no prior signs of complications. All deaths occurred within two weeks of a normal scan, at a median gestational age of 34 weeks and 1 day.
The authors conclude that “despite intensive fetal surveillance, structurally normal monochorionic diamniotic twin pregnancies without twin–twin transfusion syndrome and intrauterine growth restriction are complicated by a high rate of intrauterine death.” As the deaths occurred predominantly after 32 weeks' gestation, the authors suggest that the prospective risk for fetal death in these pregnancies might be eliminated by elective preterm delivery after 32 weeks.
In an accompanying Perspective (DOI: 10.1371/journal.pmed.0020180), Jane Cleary-Goldman and Mary D'Alton agree that, despite the limitations of the study (its retrospective nature, small numbers, and lack of dichorionic controls), it highlights a critical question for obstetricians, namely, “when is the ideal gestational age to deliver apparently uncomplicated monochorionic twins?” As Cleary-Goldman and D'Alton discuss, at 32 weeks of gestation many of the risks associated with prematurity have abated, but the remaining ones are not negligible. Until larger prospective observational studies have been conducted, balancing these risks remains challenging.
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0020196SynopsisInfectious DiseasesVirologyEpidemiology/Public HealthWomen's HealthVaccinesInfectious DiseasesGlobal healthImmunology and allergyTravel MedicineA Promising Candidate for a Lassa Fever Vaccine Synopsis6 2005 28 6 2005 2 6 e196Copyright: © Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Development of a New Vaccine for the Prevention of Lassa Fever
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Lassa fever, a viral hemorrhagic fever caused by the Lassa virus and commonly transmitted by its rodent host, is endemic in certain areas of West Africa, where several hundred thousand people are estimated to be infected each year. The disease is asymptomatic or mild in approximately 80% of infected patients, but the remaining 20% have severe multisystem disease. Estimated overall mortality is 1%–2%.
Death rates are particularly high for women in the third trimester of pregnancy, and for fetuses, about 95% of which die in the uterus of infected pregnant mothers. The most common complication of Lassa fever is deafness. Various degrees of deafness occur in approximately one-third of cases, and in many cases hearing loss is permanent. Disease severity does not seem to affect this complication: deafness may develop in mild as well as in severe cases.
Lassa fever remains a serious challenge to public health in West Africa, threatening both local residents in rural areas and those who serve them, particularly medical care providers. Ribavirin, an antiviral drug, has been used successfully in Lassa fever patients, but it needs to be given early and is not readily available in the infected areas. Given the ecology of the rodent host and conditions in the endemic area, a vaccine is mandatory for control. Lassa vaccine initiatives have suffered from a lack of funding in the past, but bioterrorism and recent importation of the disease to the United States and Europe have brought new resources to Lassa virus science.
Early attempts to develop a Lassa fever vaccine in the 1980s focused on killed pathogens, which caused a strong humoral response but failed to protect nonhuman primate test animals. Subsequently, recombinant vaccines used vaccinia vectors carrying different combinations of structural Lassa proteins. Some of these protected 90% of nonhuman primates from a lethal challenge in the absence of a strong humoral response, suggesting that cellular responses are important for protection.
Use of vaccinia vectors in humans is problematic, especially in areas where HIV infection is common—immune-suppressed individuals can develop serious skin lesions—and several alternative vaccines based on other vectors as well as harmless vaccinia ones are under development. Thomas Geisbert and colleagues now report promising results with a replication-competent vaccine based on attenuated recombinant vesicular stomatitis virus vectors expressing the Lassa viral glycoprotein. A single intramuscular vaccination protected all four vaccinated cynomolgus macaques against a lethal challenge of a particular Lassa strain, while two control monkeys that had received empty vector died after injection with the same dose of virus.
These are encouraging results, but future larger studies will need to assess the duration of protection and demonstrate the safety of this replication-competent vaccine. Another crucial question is how quickly vaccinated individuals acquire protection, and thus whether the vaccine would be suitable for creating a ring of vaccination around an outbreak zone, the most likely early application of a promising candidate vaccine. In addition, there are at least four different strains of the Lassa virus, and an ideal vaccine should provide protection across all strains. Finally, conducting trials in endemic areas, many of which lack political stability, remains a serious challenge.
Color-enhanced transmission electron micrograph of Lassa virus particles
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0020198SynopsisImmunologyEpidemiology/Public HealthGeneral MedicinePublic HealthSmokingImmunology and allergySmoking and Inflammation Synopsis6 2005 28 6 2005 2 6 e198Copyright: © Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Smoking Cessation and Cardiovascular Disease Risk Factors: Results from the Third National Health and Nutrition Examination Survey
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Smoking is the single largest preventable cause of disease and premature death, according to the World Health Organization. Smoking-related diseases kill one in ten adults globally, i.e., 4 million deaths annually; by 2030, if current trends continue, smoking will kill one in six people. Smoking is a prime factor in heart disease, stroke, and chronic lung disease, which cost the United States more than $150 billion a year. The relationship between smoking and cardiovascular disease is well documented, as is the association of smoking with increased levels of inflammatory markers and accelerated atherosclerosis. It is also well known that when smokers quit, their risk of mortality and future cardiac events declines, but there is little data quantifying the rate of this risk reduction.
Smoking triggers an immunologic response to vascular injury, which is associated with increased levels of inflammatory markers, such as C-reactive protein and white blood cell count. Several studies have shown that such markers predict future cardiovascular events. Markers such as C-reactive protein are also increasingly implicated in the pathogenesis of atherosclerosis. There are, however, still some gaps in our knowledge of cardiovascular disease, smoking, and the predictive use of such markers. For example, few studies have examined the impact of smoking cessation on levels of inflammatory markers or on cardiovascular risk reduction; the level and rate at which the inflammatory response subsides following smoking cessation is also uncertain. Furthermore, whether traditional risk factors can explain the decline in cardiovascular risk following smoking cessation is also unclear.
Short- and long-term health-care savings may be realized if smoking cessation is made a priority
In this month's PLoS Medicine, Arvind Bakhru and Thomas Erlinger investigate the association between smoking and smoking cessation and levels of inflammatory markers and cardiovascular risk factors. Data were gathered on 15,489 US adults between 1988 and 1994 in the Third National Health and Nutrition Examination Survey. Of these, 7,665 were classified as never smokers, 3,459 were former smokers, and 4,365 were current smokers.
The investigators focused on changes in C-reactive protein, white blood cell count, albumin, and fibrinogen, and the traditional risk factors—total cholesterol, high-density lipoprotein cholesterol, triglycerides, systolic blood pressure, and diabetes—that occurred with decreased smoking intensity and increased time since smoking cessation. They found that inflammatory markers had a dose-dependent and temporal relationship to smoking and smoking cessation. They noted that both inflammatory and traditional risk factors improved with less smoking, but as the time since smokers quit increased, inflammatory markers resolved more slowly than traditional cardiovascular risk factors. Still, the smoking-associated inflammatory response returned to normal within five years after smokers quit, suggesting that the vascular effects were reversible and that cardiovascular risk subsides gradually with reduced exposure.
The authors conclude that these findings support the hypothesis that cardiovascular risk falls as inflammatory response falls, and that inflammatory markers are good indicators of this risk reduction. Despite limitations of the study, including possible errors from self-reporting and lack of data on second-hand smoke and newer measures such as interleukin-6 and high-sensitivity C-reactive protein, the inflammatory markers studied here demonstrated a much clearer trend and longer-lasting effect after smoking cessation than traditional risk factors, and hence were more useful and accurate markers of disease.
As with related studies, these results suggest that smoking cessation should be a more prominent goal of public policy, and the authors conclude that policymakers must pursue smoking cessation plans as an opportunity to make savings on health care through cardiovascular risk reduction. Further research should explore the acute phase response in the months after smoking cessation, which this and other studies have not been able to study adequately.
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0020200SynopsisNutritionNutrition and MetabolismObesityWeight Loss and Mortality Synopsis6 2005 28 6 2005 2 6 e200Copyright: © Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Intention to Lose Weight, Weight Changes, and 18-y Mortality in Overweight Individuals without Co-Morbidities
Weight Loss and Mortality: What Does the Evidence Show?
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If you are overweight, then losing weight is good for your health, surely? Unfortunately, the evidence on which an answer to this seemingly simple question might be based is at best equivocal, and at worst very controversial. Previous work has shown that weight loss in obese people improves risk factors associated with cardiovascular diseases and diabetes, but studies are conflicting on the long-term effects of weight loss on mortality. A study in this month's PLoS Medicine by Jaakko Kaprio and colleagues on a Finnish dataset adds more evidence to this debate, but experts are divided on what can be concluded from it.
The major difficulty in getting clear results on this question is that it is virtually impossible to do a controlled trial to answer it. Hence, the evidence accumulated has come mostly from epidemiological studies, but it is notoriously difficult to remove all confounding factors from these studies. Kaprio and colleagues' study is another epidemiological study, but we should not simply dismiss the data as unreliable just because of the problems inherent to such a study design. Instead, we should consider their study in the light of all the other evidence available.
Starting from a group of 19,993 twins from Finland who have been studied since 1975, the authors gathered data from the 2,957 overweight participants who remained after they had excluded people with pre-existing disease, and those with missing data. These twins had been asked in 1975 if they intended to lose weight, and then had information on weight collected in 1981. Information on mortality was then collected over the next 18 years; the authors then analyzed mortality in relation to intention to lose weight and actual weight change.
In total, 268 people died. When the results were analyzed, the surprising finding was that people who intended to lose weight, and who did so, had a somewhat higher mortality than those who intended to lose weight but whose weight remained stable, or went up. People who intended to lose weight, and who did so, also had a slightly higher mortality than those who did not intend to lose weight and whose weight was stable.
The problems with such a study are outlined in an accompanying Perspective (DOI: 10.1371/journal.pmed.002018) by Meir Stampfer from Harvard School of Public Health, and there is no doubt that these results seem counterintuitive. Some readers may take away the idea from this paper that overweight people should not be advised to lose weight, but Stampfer cautions against that interpretation. Perhaps the safest interpretation of these results is that by the time adults are overweight, the health benefits of losing weight are not clear-cut. If there is one message therefore that should be taken from the paper it is this: in order to prevent the associated health effects of obesity, preventing obesity, especially in childhood, should be an overriding public health priority.
The study leaves us with the question of how intentional weight loss could lead to excess mortality. The authors suggest that this could be due to the unavoidable loss of lean body mass, which according to several other studies may increase mortality, and which may outweigh the beneficial effects of losing fat mass in healthy individuals. The authors therefore conclude that “the long-term effects of weight loss are complex, and they may be composed of oppositely operating effects with net results reflecting the balance between these effects.”
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0020201SynopsisBiotechnologyDevelopmentPathologyTurning Stem Cells into Mesenchymal Tissues Synopsis6 2005 28 6 2005 2 6 e201Copyright: © Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Derivation of Multipotent Mesenchymal Precursors from Human Embryonic Stem Cells
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As cells specialize during development they pass through different levels of differentiation, from the earliest stem cells through to the highly specialized types that make up the body's organs. Hence, a number of different tissues may derive from common precursors. For example, muscle, fat, cartilage, and bone are all derived from a group of mesenchymal precursor cells that originate in the paraxial mesoderm. So pluripotent (i.e., able to differentiate into any cell type) human embryonic stems cells are potentially a starting point for the regeneration of all types of diseased or damaged organs (and already researchers have shown that it is possible to stimulate human embryonic stem cells to differentiate into specific cell types such as neural or hematopoietic cells). The isolation of intermediate multipotent stem cells (which can differentiate into a limited number of cell types) may also be valuable. For example, the production of an unlimited supply of mesenchymal precursors would be very useful, not only for the understanding of how cells differentiate, but also for eventual practical application.
In this month's PLoS Medicine, Lorenz Studer and colleagues from the Sloan-Kettering Institute in New York describe a protocol for deriving mesenchymal precursors, which they then show are capable of differentiating into specialized cell types.
They used two undifferentiated stem cell lines—from the 22 lines that were approved in 2001 by President Bush for use in federally funded research in the United States. The specifications for approval for these lines are clear—see the guidelines at http://stemcells.nih.gov/research/registry/eligibilityCriteria.asp. The number of human embryonic stem cell lines available for researchers are strictly limited, making it necessary to develop protocols that expand these cells along various lineages.
In order to differentiate the cells into mesenchymal precursors, the stem cell lines were cocultured with mouse feeder cells to produce five different polyclonal lines. The authors then cultured these polyclonal precursors with appropriate tissue-specific stimulation in attempt to produce fat, bone, cartilage, or muscle cells. The evidence that the authors provide for these cells being differentiated includes analysis of gene expression, surface antigens, and immunocytochemistry typical of the mature tissues. For example, the authors were able to show the presence of fat granules in adipocytes, calcium in the matrix of osteogenic cells, and collagen in chondrocytes. It was harder to produce muscle cells, but even these types of cells could eventually be induced by specific culture conditions.
Myotubes formed in vitro from human ES–derived mesenchymal precursors upon cocultures with C2C12 myoblasts
What are the possible concerns about these types of studies? One obvious one is the potential for residual undifferentiated cells to turn into tumors, but the authors tested the differentiated cell cultures for cell surface markers characteristic of undifferentiated cells and found no evidence of them. Another worry for the use of these cells directly in humans is the need, at least at the beginning, to culture the cells with mouse feeder cells—obviously no human treatment could contain cells contaminated with mouse cells. Further development of protocols will be needed to address this issue. However, as the authors comment, “the high purity, unlimited availability, and multipotentiality of hESMPCs [human embryonic stem cell–derived mesenchymal precursor cells] will provide the basis for future therapeutic efforts using these cells in preclinical animal models of disease.” In addition, the techniques described here will provide a very useful resource for studying mesenchymal cell development.
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1597196110.1371/journal.pmed.0020203EditorialInfectious DiseasesMicrobiologyVirologyVaccinesSome Tolerance for Fur—Animal Studies in PLoS Medicine
EditorialThe PLoS Medicine Editors 6 2005 28 6 2005 2 6 e203Copyright: © Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Development of a New Vaccine for the Prevention of Lassa Fever
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PLoS Medicine is a sufficiently new journal that we are often doing something for the first time. This issue's “first” is the publication of a research article that reports data exclusively from animals, more precisely from six cynomolgus macaques used to test the efficacy of a new Lassa fever vaccine. There is no question that animal studies are an important part of medical research, but which ones, if any, belong in a medical journal? More to the point, which ones belong in PLoS Medicine? Although our journal's focus is on human studies, we have decided, on occasion, to publish animal studies that have important and proximal implications for clinical research, and maybe even practice.
Lassa fever causes serious morbidity and mortality in West Africa. The virus's natural hosts are rodents, and as there is little chance for effective rodent control in the endemic areas, a vaccine is the most feasible way to gain control of the disease. Several research groups around the world have worked on vaccine development—and their efforts have been boosted by the classification of Lassa virus as a Category A bioweapons agent—but to date no vaccine is available for either general or high-risk application in humans.
Thomas Geisbert and colleagues have developed and now report tests of a recombinant vaccine based on a replication-competent vesicular stomatitis virus (DOI: 10.1371/journal.pmed.0020183). One shot of this vaccine protected four out of four vaccinated monkeys against a lethal virus challenge, whereas the two control animals died, making the vaccine a serious candidate for future application in humans. Clearly many issues about this vaccine still need to be resolved, such as vector safety, duration of protection, and breadth of protection (there are at least four distinct Lassa virus strains). Nevertheless, we accepted this paper because we and our advisers felt that the research was at a stage where clinical questions, such as patient safety and design of early human trials, should inform any additional studies in animals. The proper place for such a study is, we believe, a clinical journal.
Brueghel's The Entry of the Animals into Noah's Ark
There are several other types of animal studies we consider appropriate for publication in PLoS Medicine. These include studies that explore off-label uses of approved medical interventions in validated animal disease models, again based on the studies' direct relevance to potential treatment of human patients. More often, we would publish human studies that also include experimental animal data, which typically explore molecular mechanisms suggested by the human data.
Animal studies that are submitted to medical journals can be broadly divided into two groups—“animal clinical trials” and exploratory studies. We will assess the former in a similar way to how we look at human trials. This assessment will include not only the ethical conduct of the study and approval by the respective regulatory authority, but also the rigor of the methodology. Too often, animal clinical trials, i.e., prospective, hypothesis-testing studies that evaluate the effects of a health-related intervention in animals, are not performed with the same rigor that has been developed over past decades and widely adopted by the clinical research community. In particular, animal studies often have inappropriate controls, are underpowered, involve researchers monitoring outcomes who are not blinded to treatment allocation, or lack proper statistical analysis.
Exploratory studies, designed to yield insight into disease etiology, pathology, or the mechanisms by which a particular treatment affects a disease state, are a crucial early part of the translation of basic research findings into clinical practice. However, by and large, they will not be appropriate for PLoS Medicine. Instead, we encourage submission of important advances from this early translational stage to PLoS Biology, our flagship open-access biology journal (www.plosbiology.org).
It would be foolish to deny the existence of a sizeable “grey area” between these types of study. Between our journals, we will try to provide open-access publication for any important study at this interface between biology and medicine, and will be happy to talk with authors on a case-by-case basis. In addition, we currently cross-reference studies between the two journals—and will do so even more when our new journals come on line. For example, PLoS Medicine has published a number of Perspectives on research articles published in PLoS Biology, and PLoS Biology regularly highlights papers from PLoS Medicine on its home page. In this way, because all our journals are open access, the difference to the reader between a paper published in PLoS Medicine and any other PLoS journal, is just a rodent click.
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Ann Gen PsychiatryAnnals of General Psychiatry1744-859XBioMed Central London 1744-859X-4-121595525610.1186/1744-859X-4-12Case ReportPregnancy-induced obsessive compulsive disorder: a case report Kalra Harish [email protected] Rajul [email protected] Jitendra kumar [email protected] Aleksandar [email protected] Department of Psychiatry, Royal Perth Hospital, Perth WA 6000, Australia2 Grampians Psychiatric Services, Ballarat Health Services, Ballarat VIC 3350, Australia3 Department of Psychiatry, King George Medical University, Lucknow – 226003, U.P., India4 University of Western Australia, School of Psychiatry and Clinical Neurosciences, Perth WA 6000, Australia2005 15 6 2005 4 12 12 17 5 2005 15 6 2005 Copyright © 2005 Kalra et al; licensee BioMed Central Ltd.2005Kalra et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Pregnancy is a well-recognised risk factor in precipitating obsessive-compulsive disorder. We present and discuss a case with the onset of obsessive-compulsive disorder in the fourth month of gestation, which fully recovered two weeks after delivery. The phenomenology of the observed disorder was similar to earlier reports of obsessive-compulsive disorder in pregnancy, i.e. the obsessions and compulsions were predominantly related to the concern of contaminating the foetus resulting in washing compulsions. Despite the initial success with anti-obsessional drugs, the patient stopped the medication in the last month of gestation. Nevertheless, she fully recovered two weeks after the delivery without any psychiatric intervention. There were no obsessive-compulsive symptoms at one-year follow up. The possible mechanisms involved in the aetiology of this case, and future research directions in understanding the role of pregnancy in OCD are discussed.
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Introduction
Pregnancy and the postpartum period are known to influence the onset and course of various psychiatric disorders such as mood disorders, psychotic disorders, and anxiety disorders. [1-3] There is considerable evidence that suggests the role of stressful events, including pregnancy and childbirth, in precipitating or exacerbating obsessive-compulsive disorder (OCD). [4] Various studies have evaluated the role of pregnancy in OCD and have reported onset and exacerbation of OCD in a significant percentage of their study groups. [5-7]
Postpartum OCD has been described as having onset within the first three weeks of delivery. [8,9] Here we report a patient whose OCD had its onset during pregnancy and remitted following delivery. To the best of our knowledge, this is the first report describing onset of OCD during pregnancy with spontaneous complete recovery following delivery.
Case presentation
A 30-year old primigravida woman presented to the outpatient department in the fourth month of gestation. She had no past history of psychiatric illness. Her chief complaints were contamination obsessions and washing compulsions in the preceding one month. Preoccupied with thoughts of contamination, she had started spending the majority of time washing herself or cleaning various household items. She described these thoughts as being her own and recognised them to be "irrational", but she could not resist them. She was distressed and unable to maintain her employment. Washing compulsions relieved her anxiety. However, she could not offer an explanation as to what she feared about contamination. No depressive or psychotic symptoms were elicited. Biochemical investigations, including metabolic and thyroid function studies, were in the normal range. She was diagnosed with OCD according to ICD-10 criteria [10].
Pharmacotherapy, offered at the first consultation, was refused by the patient because of her (non-obsessional) concerns about teratogenic effects of drugs. Behavioural therapy in the form of thought stopping was begun. The patient reported exacerbation of symptoms at the next consultation and subsequently disclosed that her obsessional thoughts also concerned the fear of contaminating her unborn baby. She repeatedly washed to avoid damage to her foetus. One month after her initial presentation, fluoxetine was started, at an initial dose of 20 mg/day gradually and gradually increased to 60 mg/day over the next four weeks. The patient reported reduction of obsessional and compulsive symptoms, and was able to resume her work. She remained on fluoxetine until the eighth month of pregnancy with no reports of exacerbation. However, the patient stopped the medication during the last month of gestation on the alleged advice of family members. The patient again experienced the relapse of intrusive obsessional thoughts followed by compulsions, but refused to resume pharmacotherapy until delivery. The patient returned to the outpatient clinic fifteen days postpartum. The patient described no obsessive thoughts or washing compulsions for the preceding one week. She was followed up for five visits in the next one year without any reports of obsessions or compulsions.
Discussion
There is evidence supporting the role of major life events including pregnancy and delivery in precipitating OCD. [4] However, to the best of our knowledge, there are no specific reports showing complete resolution of OCD after delivery in cases having onset during pregnancy. In two studies addressing the role of pregnancy in OCD, Neziroglu et al [5] and Williams et al [6] found pregnancy to be associated with onset of OCD in 39% and 13% patients, respectively. It occurred in primigravida in 52% of the patients. [5] Our case too had its onset in her first pregnancy at the fourth month of gestation. Our patient had major symptoms in the form of obsessions of contamination and compulsions with the underlying fear of contaminating her foetus. This phenomenology is in consonance with the literature. [4,11] Purely intrusive obsessional thoughts with the same underlying theme have also been described in a case series of postpartum OCD. [8]
The underlying mechanism can only be speculative at this stage. As full recovery was seen after delivery, our case report negates the proposed mechanism in postpartum OCD of adverse impact on serotonergic functions by rapid withdrawal of oestrogen and progesterone in postpartum period [9]. We propose it to be considered as equivalent to chorea gravidarum, which is also characterised by onset of involuntary movements during pregnancy with complete resolution after delivery. [12] Basal ganglia abnormalities are known to occur in pregnancy as in chorea gravidarum. Similarly, basal ganglia pathology, especially involving the caudate nucleus, has been implicated in OCD [13-15]. We hypothesize that similar mechanisms may underlie both chorea gravidarum and this case of pregnancy-induced OCD.
An underlying mechanism proposed for chorea gravidarum of enhanced dopaminergicsensitivity under the effect of elevated levels of female sex hormones due to pregnancy [16] could also be presumed as operating in this case but involving serotonin instead of dopamine. Our patient showed significant improvement with fluoxetine before being ceased by the patient, thus indirectly supporting serotonin dysfunction. Previous reports of post-partum OCD have also shown good response to fluoxetine. [9]
Careful prospective studies of pregnancy-associated OCD will help in understanding predisposing and aetiological factors involved in such cases. Comparison of chorea gravidarum and OCD in pregnancy by functional imaging techniques like PET/SPECT/fMRI might prove useful in understanding pathophysiological processes responsible for these disorders.
Competing interests
The author(s) declare that they have no competing interests.
Acknowledgements
The authors acknowledge the valuable comments made on earlier drafts by Dr Lindsay Allet.
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Kendall RE Mcguire RJ Connor Y Mood changes in the first three weeks of after childbirth J Affect Disord 1981 3 317 326 6459348 10.1016/0165-0327(81)90001-X
O'Hara MW Postpartum blues, depression, and psychosis: a review J Psychosom Obstet Gynecol 1987 7 205 227
Abramowitz JS Schwartz SA Moore KM Luenzmann KR Obsessive-compulsive symptoms in pregnancy and the puerperium: A review of the literature J Anxiety Disord 2003 17 461 478 12826092 10.1016/S0887-6185(02)00206-2
Neziroglu F Anemone R Yaryura-Tobias J Onset of obsessive-compulsive disorder in pregnancy Am J Psychiatry 1992 149 947 950 1609876
Williams K Koran L Obsessive-compulsive disorder in pregnancy, the puerperium, and the premenstrual J Clin Psychiatry 1997 58 330 334 9269260
Mania G Albert U Bogetto F Vaschetto P Ravizza L Recent life events and obsessive-compulsive disorder: the role of pregnancy /delivery Psychiatry Res 1999 89 49 58 10643877 10.1016/S0165-1781(99)00090-6
Sichel DA Cohen LS Dimmock JA Rosenbaum JF Postpartum Obsessive-compulsive disorder: a case series J Clin Psychiatry 1993 54 156 159 8486594
Sichel DA Cohen LS Rosenbaum JF Driscoll JD Postpartum onset of obsessive-compulsive disorder Psychosomatics 1993 34 277 279 8493313
World Health Organisation The ICD-10 classification of mental and behavioural disorders: clinical descriptions and diagnostic guidelines Geneva: WHO 1992
Buttolph ML Holland AD Jenike M, Baer L, Minichello WE Obsessive-compulsive disorder in pregnancy and childbirth Obsessive- compulsive disorder: Theory and Management 1990 Chicago: Yearbook Medical 89 97
Cordoso F Chorea gravidarum Arch Neurol 2002 59 868 870 12020275 10.1001/archneur.59.5.868
Baxter LR Phelps ME Mazziota JC Local cerebral metabolic rates in Obsessive-compulsive disorder Arch Gen Psychiatry 1987 44 211 218 3493749
Luxenberg JS Swedo S Flament M Neuroanatomic abnormalities in Obsessive-compulsive disorder detected with quantitative X-ray computed tomography Am J Psychiatry 1988 145 1089 1093 3414851
Swedo SE Shapiro MB Grady CL Cerebral glucose metabolism in childhood onset obsessive-compulsive disorder Arch Gen Psychiatry 1989 46 518 523 2786402
Unno S Iijima M Osawa M Uchiyama S Iwata M A case of chorea gravidarum with moyamoya disease Rinsho Shinkeigaku 2000 40 78 82
| 15955256 | PMC1164400 | CC BY | 2021-01-04 16:39:07 | no | Ann Gen Psychiatry. 2005 Jun 15; 4:12 | utf-8 | Ann Gen Psychiatry | 2,005 | 10.1186/1744-859X-4-12 | oa_comm |
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BMC AnesthesiolBMC Anesthesiology1471-2253BioMed Central London 1471-2253-5-61593263410.1186/1471-2253-5-6Research ArticleCarbon monoxide production from five volatile anesthetics in dry sodalime in a patient model: halothane and sevoflurane do produce carbon monoxide; temperature is a poor predictor of carbon monoxide production Keijzer Christiaan [email protected] Roberto SGM [email protected] Lange Jaap J [email protected] Department of anesthesiology, VU University medical center, Amsterdam, The Netherlands2005 2 6 2005 5 6 6 14 11 2004 2 6 2005 Copyright © 2005 Keijzer et al; licensee BioMed Central Ltd.2005Keijzer et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Desflurane and enflurane have been reported to produce substantial amounts of carbon monoxide (CO) in desiccated sodalime. Isoflurane is said to produce less CO and sevoflurane and halothane should produce no CO at all.
The purpose of this study is to measure the maximum amounts of CO production for all modern volatile anesthetics, with completely dry sodalime. We also tried to establish a relationship between CO production and temperature increase inside the sodalime.
Methods
A patient model was simulated using a circle anesthesia system connected to an artificial lung. Completely desiccated sodalime (950 grams) was used in this system. A low flow anesthesia (500 ml/min) was maintained using nitrous oxide with desflurane, enflurane, isoflurane, halothane or sevoflurane. For immediate quantification of CO production a portable gas chromatograph was used. Temperature was measured within the sodalime container.
Results
Peak concentrations of CO were very high with desflurane and enflurane (14262 and 10654 ppm respectively). It was lower with isoflurane (2512 ppm). We also measured small concentrations of CO for sevoflurane and halothane. No significant temperature increases were detected with high CO productions.
Conclusion
All modern volatile anesthetics produce CO in desiccated sodalime. Sodalime temperature increase is a poor predictor of CO production.
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Background
In 1990 first reports were published about carbon monoxide (CO) production in anesthetic circuits [1-3] followed by a few studies that concluded that there was no risk of CO intoxication in common anesthetic practice [4-6]. The potential risk of CO production, however, was clearly established in a laboratory study by Fang et al. [7]. This study was the first to prove that desflurane produced higher amounts of CO compared to enflurane and isoflurane respectively, when in contact with dry sodalime and Baralyme®. Furthermore, they found that Baralyme® produced higher amounts of CO compared to sodalime with all three volatile anesthetics. Frink et al. [8] and Bonome et al. [9] demonstrated in animal studies that desflurane produced high amounts of CO in dry carbon dioxide absorbents, with higher amounts in Baralyme® than sodalime.
In an in vitro study, Wissing et al[10] found high concentrations of CO production for enflurane and isoflurane as well, and to a lesser extent for sevoflurane and halothane. Wissing et al. further found temperature increase in all analyzed volatile anesthetics, which has been linked to higher production of CO [7]. However, as this study was performed using only a gas flow over a carbon dioxide absorber canister, these results cannot easily be extrapolated to a clinical situation. Furthermore CO measurements were performed with infrared absorption and electrochemical detection which are not as accurate as gas chromatography [11].
Therefore, the purpose of this study is to measure in a simulated patient model, the maximum amounts of CO production for all modern volatile anesthetics, with completely dry sodalime using a gas chromatograph. Also, temperature of the system was measured to establish the relationship between CO production and temperature increase.
Methods
Patient model
Two sample lines were connected to the Y-piece of the circle system: one to a small lumen gas chromatography sample line connected to the gas chromatograph, and one connected to the infrared anesthetic vapor analyzer (SAM, Marquette) sampling at 200 ml/min.
The volatile anesthetics and the sodalime (Drägersorb® 800 plus, composition: 0.003% KOH, 2% NaOH, 82% Ca(OH)2 and 16% H2O) were obtained from our own stock. The sodalime was dried completely by using an oxygen flow of 15 l/min in sealed glass containers until no more weight reduction could be measured. A 16% weight reduction was established confirming the producer's specifications.
Experiments
For each anesthetic vapor, an experiment was performed in which 950 grams of dry sodalime was used. The ventilator was set in IPPV mode with a tidal volume of 600 ml, a frequency of 14/min and 5 cm H2O PEEP. After an equilibration with 40 % oxygen and 60% nitrous oxide was established at a fresh gas flow (FGF) of 5 l/min, anesthetic vapor was introduced by a standard vaporizer. The dial was set until the vapor analyzer showed the target concentration of anesthetic vapor, after which the FGF was reduced to 500 ml/min. For the different anesthetic vapors equilibrium was maintained of 0.45 vol% halothane, 0.6% enflurane, 0.6% isoflurane, 0.8% sevoflurane and 3.0% of desflurane during an experiment.
Carbon monoxide measurements
A portable gas chromatograph (Varian Chrompack CP 2003P) with a TCD detector and a Mollsieve 5A column was used for CO quantification with a lower limit of 1 ppm. This gas chromatograph (GC) is capable of automatic sampling and was programmed to sample approximately every five minutes during an experiment (a total of 36 samples). The GC was calibrated with two calibration mixtures of 210 and 981 parts per million (ppm) CO in nitrogen (Hoekloos specialty gasses, Dieren). The GC was connected to a desktop PC for control of the GC and data recording, analysis and storage.
Temperature measurements
The sodalime container of the circle system was equipped with temperature probes in the upper and lower layer of the container, temperature data were continuously recorded (samplefrequency 30 Hz) during each experiment.
Analysis of data
The total amount of CO production and absorbent temperature were measured for each of the five volatile anesthetics. All experiments were performed in duplicate (ten experiments in total) in order to verify the reproducibility of the CO measurements. To verify that no CO was produced in normal circumstances, i.e. with fresh sodalime, these measurements were repeated with fresh sodalime.
Analyses were performed with SPSS 11.0. The Mann-Whitney-U was used to assess the reproducibility CO measurements, expressed as lack of significant differences between consecutive measurements, the Kruskal-Wallis and Mann-Whitney-U tests for comparison of CO productions between volatile anesthetics, and temperature change. Data were presented as peak, median and IQR (interquartile range) CO concentrations. For all analyses the significance level was set at 5%.
Results
Carbon monoxide measurements
When the first measurements started different amounts of CO were measured when the sodalime was not sufficiently dried. Therefore each experiment was performed twice and no significant difference was found between consecutive measurements. P-values were for desflurane, enflurane, isoflurane, halothane and sevoflurane respectively: 0.906, 0.481 . 1.00, 0.839, 0.725.
The control experiments with fresh sodalime showed no CO production.
Mean CO concentrations measured by the GC were calculated for each anesthetic vapor. Figure 1 shows the CO concentration for desflurane, isoflurane and enflurane. Because of distinctly lower CO productions for halothane and sevoflurane, both measurements were depicted in a separate figure (figure 2). A fast increase of CO concentration is seen with shortly after a slow exponential like decrease in concentration.
Figure 1 Carbon monoxide production of desflurane, enflurane and isoflurane in desiccated sodalime. Legend: Carbon monoxide was measured in parts per million (ppm).
Figure 2 Carbon monoxide production of halothane and sevoflurane in desiccated sodalime. Legend : Carbon monoxide was measured in parts per million (ppm),
Complete peak, median and interquartile range (IQR) CO concentrations of all experiments are shown in table 1. Highest CO concentrations in parts per million (ppm) were measured with peak concentrations of 14262 ± 694 for desflurane, followed by 10654 ± 510 for enflurane, 2512 ± 126 for isoflurane and 210 ± 11 for halothane and 121 ± 7 for sevoflurane. Significant differences were found between CO production of the five volatile anesthetics (Kruskall Wallis: p < 0.001). Except for the comparison between desflurane – enflurane (Mann-Whitney-U: p = 0.303) and halothane – sevoflurane (Mann-Whitney-U: p = 0.079) all paired comparisons were significantly different (Mann-Whitney-U: all p < 0.001)
Table 1 Carbon monoxide concentrations. Legend: Peak and median interquartile range carbon monoxide concentration [CO] in parts per million for each experiment: ex.1 = experiment 1, ex.2= experiment 2, both with desflurane 3.0 vol%, enflurane 0.6 vol%, isoflurane 0.6 vol%, halothane 0.45 vol% and sevoflurane 0.8 vol% in completely dry sodalime.
Anesthetic vapor Peak [CO] ex.1 Peak [CO] ex.2 Median [CO] ex.1 Median [CO] ex.2
Desflurane 13889 14262 1809 (1092–5947) 1816 (1050–6378)
Enflurane 10187 10654 1485 (793–4490) 2044 (892–4394)
Isoflurane 2512 2382 588 (329–1430) 664 (329–1311)
Halothane 185 210 28 (0–92) 31 (0–94)
Sevoflurane 113 121 0 (0–36) 5 (0–43)
Temperature measurements
The temperature measurements at the bottom of the sodalime container showed a mean temperature rise from 23.5 to 28.3°C in the experiments with fresh sodalime. In the experiments with dry sodalime (except for sevoflurane) a rise in mean temperature from 24.0 to 32.9°C was measured.
In the experiments with dry sodalime and sevoflurane a high increase in temperature from 26.0 to 67.7°C was measured during the first twenty minutes. In those twenty minutes the sevoflurane dial had to be set at maximum because otherwise 0.85 vol% sevoflurane could not be maintained in the circle system.
In all experiments a small difference in temperature was seen between the upper and lower layer of the sodalime container with a slightly higher temperature of 0.8 – 1.0°C at the bottom of the container.
Discussion
Carbon monoxide production
For this study we developed a method in which a gas chromatograph sampled automatically every five minutes during each experiment, therefore providing the most accurate and reliable CO measurement. To our knowledge this is the first time this kind of setup was used.
In this study the findings of Fang et al[7] concerning the fact that desflurane produces more CO than enflurane and isoflurane respectively, were confirmed. However, instead of using small vials of 30 ml, we used a patient model, therefore measuring the maximum amounts of CO in completely dry sodalime at a concentration equivalent of approximately 1 MAC of volatile anesthetic using a oxygen/nitrous oxide mixture. Regarding the toxicity of CO, the Henderson and Haggard's Index of Toxic effect[12] indicates that one hour of exposure of more than 1500 ppm of CO is dangerous to life. However one should also take into consideration that the CO is not continuously produced in this model in contrast with this index and that CO absorption by a patient is not included in this model. Therefore we can only conclude from our findings that in these extreme conditions very high CO concentrations can be reached for desflurane and enflurane and that isoflurane can produce significant concentrations of CO as well. One should take into consideration that the use of Baralyme® will produce higher levels of CO[7,13], and that carbon dioxide absorption, fresh gas flow and minute volume have small effects on CO production as shown by Woehlck et al. [13]. Because of the relative small effect of carbon dioxide absorption on CO production we didn't add carbon dioxide to our model.
As for the clinical relevancy, one could say that this model uses completely dry sodalime which is not seen very frequently in common anesthetic practice. However, there are reports of severe CO intoxications [2,3] recently published by Berry et al. [14] with desflurane as anesthetic agent. The highest risk develops when fresh gas flow is maintained in a anesthesia system for a few days. After 41 hours with a 7 l/min fresh gas flow, the soda lime will become critically dry as published by Soro et al. [15]. As there is always a potential risk, one should consider a safety protocol to maintain a proper humidity level inside the carbon dioxide absorbent as proposed by Woehlck et.al [16], especially when using anesthetic agents like desflurane and enflurane. One could also consider the use of more accurate electrochemical CO monitors [17,18] which can detect CO by continuous measuring in the anesthetic circuit. Another possibility is the use of different carbon dioxide absorbents, particularly absorbents with less Ba(OH)2, KOH and NaOH [19,20] that produce relatively safe amounts of CO or have no CO production at all[21,22].
During the desflurane experiments the infrared anesthetic vapor analyzer reported a concentration of enflurane up to 1.0 vol%, which correlated significantly with the measured CO concentration (Spearman's r: 0.805; p < 0.001). This reported enflurane concentration is probably attributable to the production of trifluoromethane that is simultaneously produced with CO[23] and is known to be detected as enflurane by this vapor analyzer[24]. The enflurane detection disappears below a CO concentration of 3400 ppm, which explains why in the isoflurane experiments no 'enflurane' was detected. In case of a 'mixed gas' warning or a unexpected 'enflurane' detection during anesthesia using desflurane, one should consider the possibility of a (high) CO production.
Contrary to reports in literature[7], we found significant amounts of CO with halothane and sevoflurane. Also CO production by both substances is not explained by the mechanism postulated by Baxter et al[23]. Previously, CO production was reported by Strauss et al. [25] for halothane and Wissing et al[10] for both sevoflurane and halothane. They reported higher concentrations of CO than found in our study, but at higher concentrations of these two volatile anesthetics and with use of a KOH containing absorber. Our reported amounts of CO are not dangerous for several hours in healthy individuals, but could be clinically relevant for anemic patients or small children[26,27].
Temperature measurements
No clinically relevant temperature increase was measured during the experiments with dry sodalime and desflurane, enflurane, isoflurane and halothane. This is not concurrent with findings of other authors [10,28]. Our explanation for these differences is the use of higher concentrations of vapor and a higher fresh gas flow used in the experiments of these studies which would give a more exothermic reaction than in our study. We did however measure a forty degrees Celsius temperature increase in the experiments with sevoflurane and dry sodalime. Simultaneously, we noticed a high degree of sevoflurane degradation because of the discrepancy between dial setting of the vaporizer and the measured sevoflurane concentrations in the circle system. This confirms the report of instability of sevoflurane in desiccated sodalime by Funk et.al[29]. We concluded that temperature measurement in the sodalime container is a very poor predictor of CO production because of the high CO production with desflurane with a small increase of temperature and the other way round for sevoflurane. However a study from Holak et.al. [27] demonstrated that clinically relevant CO concentrations with the use of Baralyme® do not occur until the absorbent temperature exceeds 80°C. Because of the use of a combination of sevoflurane and nitrous oxide in this study we cannot rule out that higher concentrations of sevoflurane without nitrous oxide would increase the absorbent temperature above a certain threshold where sodalime could also be capable of production of high concentrations of CO or even result in fire or explosions as recently reported with the use of dessicated Baralyme® and sevoflurane [30-32]. Further studies using sevoflurane and other absorbents with temperature measurement inside the absorbents [33] should be performed to determine if these reactions can also occur with other absorbents than Baralyme®.
Conclusion
In this patient model we demonstrated the possible production of very high amounts of CO in dry sodalime with desflurane and enflurane. CO production from isoflurane is less but still significant. Also sevoflurane and halothane can produce small amounts of CO. A report from the vapor analyzer that a mixed gas or a certain amount of enflurane is present when using desflurane suggests that more then 3400 ppm CO is already present in the anesthesia circle system.
When using desflurane one should consider implementing a safety protocol to prevent the sodalime from completely drying out. Another option is the choice for a 'safer' carbon dioxide absorber. Measurement of the soda lime temperature is a poor predictor for CO production in sodalime when using anesthetic vapor in combination with nitrous oxide.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
CK participated in the design of the study, performed all experiments, participated in the statistical analysis and drafted the manuscript. RP participated in the statistical analysis and helped to draft the manuscript. JL participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
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| 15932634 | PMC1164401 | CC BY | 2021-01-04 16:28:05 | no | BMC Anesthesiol. 2005 Jun 2; 5:6 | utf-8 | BMC Anesthesiol | 2,005 | 10.1186/1471-2253-5-6 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-1431594147310.1186/1471-2105-6-143Methodology ArticleDiscovery of protein-protein interactions using a combination of linguistic, statistical and graphical information Cooper James W [email protected] Aaron [email protected] Text Analytics, IBM Thomas J Watson Research Center, PO Box 704, Yorktown Heights, NY 10598, USA2 Bioinformatics, IBM Thomas J Watson Research Center, PO Box 218, YorktownHeights, NY 10598, USA2005 7 6 2005 6 143 143 3 11 2004 7 6 2005 Copyright © 2005 Cooper and Kershenbaum; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The rapid publication of important research in the biomedical literature makes it increasingly difficult for researchers to keep current with significant work in their area of interest.
Results
This paper reports a scalable method for the discovery of protein-protein interactions in Medline abstracts, using a combination of text analytics, statistical and graphical analysis, and a set of easily implemented rules. Applying these techniques to 12,300 abstracts, a precision of 0.61 and a recall of 0.97 were obtained, (f = 0.74) and when allowing for two-hop and three-hop relations discovered by graphical analysis, the precision was 0.74 (f = 0.83).
Conclusion
This combination of linguistic and statistical approaches appears to provide the highest precision and recall thus far reported in detecting protein-protein relations using text analytic approaches.
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Background
Scientists in molecular biology find that a significant technique for studying protein function is through the study of protein-protein interactions. While the actual experimental study of such interactions remains the most important manner of obtaining these data, the number of protein-protein interactions reported in the literature is substantial and growing rapidly. There are a number of tabulations of these interactions, such as that provided by the Munich Institute for Protein Sequence (MIPS); these tabulations are of necessity incomplete.
To address this problem, we have been developing a group of biology-specific computational annotators that work in conjunction with our group's text analytic software, for the discovery of protein-protein relations in text.
In this paper, we undertook a study that utilizes a combination of computational linguistics, statistics and domain-specific rules to detect protein-protein interactions in a set of Medline abstracts.
The system we describe here is particularly appealing because it can be used both to find known interactions and to find interactions not yet tabulated. According to the National Library of Medicine, Medline contains over 11 million abstracts, with about 40,000 being added each month. Thus, having a scalable, robust system for protein interaction discovery provides a major information tool for molecular biologists.
A number of workers have tackled portions of this problem previously with some partial success. The SUISEKI system [1] recognizes various grammatical frames which may describe protein interactions. They reported high precision (68%) for the shorter patterns and lower precision (21%) for the longer ones.
In a more narrowly focused experiment, Pustejovsky et. al [2] described a computational linguistic system for detecting inhibit relations, with 90% precision and recall of 57%.
Recently Leroy [3] described Genescene, a software package for detecting relations between genes. They used both rule-based detection and co-occurrence based methods, finding that rule-based relations were 95% correct and co-occurrence based relations 60% correct. Researchers at Ariadne Genomics [4] have quite recently described a system called MedScan, which they report as having 91% precision and 21% recall on human protein-protein interactions.
We [5] have previously described methods for detecting relations between noun phrases and methods for displaying them [6]. In this paper we propose using these techniques along with a combination of statistical and rule-based approaches to identify protein interactions in Medline abstract text. Ideally one would imagine constructing a protein interaction network much like the network that allowed Swanson to discover the relationship between "fish oil" and "Reynaud's disease" [7]. Swanson tabulated terms (chemicals, diseases) that occurred near the initial target name (Reynaud's disease) and formed a network through several papers that eventually led to the conclusion that fish oil was related to the reduction in the symptoms of that disease. The relations extracted in this paper can be used to form just such a network.
This paper discusses the text analytic tools used, and then describes our experiments against a gold standard of protein relations. Finally the results of mining relations across a large set of Medline abstracts are described.
Text analytic tools
The system used in these experiments is constructed using the TALENT (Text Analysis and Language Engineering Tools) text mining system [8]. The current version of this system is called TafTalent and operates in the Unstructured Information Management (UIMA) environment [9]. It consists of a series of document-level annotators that perform preliminary part-of-speech lookup, tag each word for part of speech, perform a shallow parse of each sentence, and annotate yeast proteins in a manner described below
In this work, the approach is to use tagger and shallow parser annotators primarily for sentence boundary recognition, and use a dictionary annotator derived from public sources to recognize the protein names. The goal of this approach is to be fast and scalable as well as to improve precision and recall over other methods.
After each Medline abstract is processed by the annotators, an annotation consumer program converts these annotations into entries in a database load file. This file contains all of the salient terms per document, their part of speech and their relative token positions in the document. An additional database load file contains the Medline document metadata: dates, titles, authors and ID numbers.
The flow of this process is show in Figure 1. The Medline documents are read one at a time by a collection reader and placed in the Common Annotation Structure provided by the UIMA system. Then a series of sequential annotators tokenize the text, perform part of speech tagging and shallow parsing, and identify the protein names using the dictionary described below. The results are written into a two database tables for further analysis.
Then it is possible to use a few simple database queries to construct a database table of all the unique terms in the document collection, and compute their frequencies, and the number of documents in which they appear once and more than once. Using these data the salience or IQ [10] of each term can be computed.
Results and discussion
We describe two experiments; one using a small amount of data derived from the MIPS tables on the web, and a larger experiment utilizing 125,000 Medline abstracts.
Preliminary experiments using MIPS data
The Munich Institute for Protein Sequences (MIPS) maintains a database of published yeast (saccharomyces cerevisiae) protein interactions along with a reference to the Medline abstract of the paper in which the interaction is reported. This table contains 2050 protein names and 2604 pairs of protein interactions and provides links to additional information on each protein. The interaction table was parsed and reduced to 959 unique relations, and the protein names and the 564 Medline abstracts downloaded.
An annotator was then developed that compared each lexical token found against the list of proteins and marked those that matched. Then, a simple program was designed to report the location of these proteins within each sentence in each document.
Initially, this was not particularly successful because each protein has a number of possible representations that needed to be matched to a common canonical form. For example, the protein SRV2 can also be represented as Srv2p, SRV2p, CAP and (CAP). Synonyms for most of these proteins are available on pages linked from the original page on the MIPS web site. The dictionary was expanded using these synonyms and the various allowed capitalizations and the analysis rerun, storing all terms and their document positions in a database table.
Even with the expanded protein synonym table, only 388 protein interactions were detected within single sentences that matched those in the MIPS interaction table, and 432 other interactions were detected which did not match those in the MIPS table. This amounted to a precision of 0.47 and a recall of 0.68. Further, there was no particular correlation between the computed strength (mutual information value) of the relation [12] and the likelihood that it agreed with those in the MIPS table.
Detecting relations in individual documents
In an effort to improve the accuracy of protein-protein interaction detection, a detailed study of 65 of the abstracts was undertaken to determine what algorithms and approaches would be most effective. In this study, each abstract was examined along with a list of the interactions reported by the MIPS table, including all of the synonyms for each protein. This process led to the following conclusions:
1. Some interactions were not reported in the abstracts, but only in the full papers. In fact some review articles contained no protein names at all in the abstracts. This finding is similar to that previously described [1].
2. Some interactions were described that were not tabulated by MIPS. For example, the abstract might mention prior work.
3. Protein complexes were frequently mentioned. For example references are made to dimers such as "Ddc2-Mec1" and trimers such as "Hap2p-Hap3p-Hap5p." Such complexes do, in fact, represent protein interactions and should also be detected and reported.
4. Proteins were frequently referred to by two synonyms separated by a slash, such as "GIM1/YKE2."
5. In all but one case, the interactions were described in the same sentence, and thus resolving co-reference issues would add only marginally to the quality of the interaction detection. Thus, the fact that two proteins occurred in the same abstract, but not in the same sentence was not a good metric for the number of relations we should be able to find.
6. No instances of negation were found.
7. A database query of verbs that lay between two proteins led to the small list shown in Table 1. We note that this list is virtually identical to that determined empirically by previous workers [11].
Accordingly, two additional annotators were written. One annotator recognized protein complexes: dimers and trimers, and the other recognized protein synonyms in the "slash notation" we illustrated in point 4 above. When the annotator found these synonyms, it only annotated one of the two mentions, to avoid skewing the mention statistics. All protein complexes were treated as reports of interactions and annotated as such.
We also annotated the verbs or their noun-equivalents in each sentence, that contained two or more different proteins.
Evaluation of revised annotations
Examination of protein interactions detected in a few documents showed that nearly all of the relations detected by our proximity algorithm actually existed in the document, whether tabulated by MIPS or not, and that of those our algorithm missed, nearly all were not discussed in the abstract at all.
Study of a larger set of Medline documents
With these encouraging preliminary results in hand, a study of a larger dataset was undertaken. It is recognized that the initial experiments were on the same data as the rules were developed on, and this second larger set is used to provide a more independent measurement of the accuracy of this approach. In the following experiments, no new protein interaction algorithms were developed, although we did introduce some further filtering to reduce spurious results. The dictionary of protein names and synonyms was the same one derived from MIPS tables. It is our assumption that this is a complete list, though if it is not, the precision and recall numbers would be slightly different.
The query "yeast protein" was submitted against our local indexing of Medline documents through 2002 and a list of the top 12,300 documents was obtained. The MIPS protein interaction table was enhanced by one from Stanley Fields [13]. These documents were annotated as above using the same series of annotators. The same database tables were created from the document ids, terms, and the proteins found in each of them.
The initial results of this experiment returned 912 relations, but only 133 (14%) agreed with the combined gold standard MIPS-Fields table. These and the following results in this section are summarized in Table 2. Considering the large number of abstracts examined, this small number of interactions indicates that the original data referred to by the MIPS table were a serendipitous set which referred specifically to protein-protein interactions. This larger dataset included a number of papers referring to genes which needed to be eliminated from consideration. Modifying the annotator to exclude sentences containing the words "gene," "express," and "encode," improved the accuracy to 17%.
In this larger set of data, protein names may co-occur in more ways that our initial approach allowed for. To reduce the error rate in these experiments, the annotator was further modified to exclude sentences which did not contain one of the verbs in Table 1, or their nominalizations. This resulted in improving the accuracy to 21%.
To further explicate the reasons for the remaining 75% apparent false positives, each relation reported was studied in each abstract where it was detected and conservatively rated either true or false. Of the 343 unmatched relations, 140 additional relations were discovered. While these relations were not in the combined MIPS-Fields table, they were definitely reported in the abstracts. In all questionable cases, the opinion of a biologist was obtained. This leads to 234 out of 437 (53%) relations being discovered correctly.
To further reduce the false positives, sentences containing any negation word (see Table 3) were also excluded from consideration, as were sentences containing the word "allele." It is possible that exclusion of sentences with "not" and the like will also exclude double negatives, but we found only one such case in the entire set of candidate abstracts. This improved the accuracy to 239 out of 381, or 62%.
Graphical study of secondary relations in the large dataset
We then undertook a study of the graphical relations between proteins, in a similar fashion to that described by Jeong [14]. In this study, we looked at two networks, one of the "true" relations described by the MIPS-Fields table and one described by the network of relations we discovered by text analytic methods. graph to contain only the nodes found in the experimental data.
In our experimental data, we found 385 interactions of which 239 were confirmed by the combined true relations table, while 146 were not, for a precision of 62%. These 385 interactions were among 266 proteins. However, our true relations table contained only 246 of these proteins. Of the 385 interactions found by our approach, 42 involved one of the 20 proteins not part of the true relations table. If we consider only interactions over the 246 proteins common to both tables, we find that 239 of 343 match and 104 do not, for a precision of 70%.
Results of the graphical study
In examining the experimental and true relations networks, we built a graph corresponding to each interaction found by our approach but not present in MIPS. We then compared the data to find out if relations which were not directly tabulated in the true relations graph but were found in the experimental data could be explained by indirect relations. For example, in Figure 2, there is no direct relationship between Ypt1 and Bet2 in the true relations network. However, our graphical study discovered such a relationship, and from examination of Figure 2, it is apparent that there is strong support for this relation. There are relations between Ypt1 and Sec4, Bet2 and Sec4, Bet2 and Mad2 and Mad2 and Sec4. Thus, there is a path of length 2 (Bet2-Sec4-Ypt1) and a path of length 3 (Bet2-Mad2-Sec4-Ypt1) between Ypt1 and Bet2. This lends considerable support for the relationship between Ypt1 and Bet2. These 2-hop and 3-hop paths are illustrated in Figure 2 using dotted and dashed lines.
If we then return to our database of documents and mined protein names, the document containing this relation is abstract 1903184, and the supporting text for this relation is:
"We propose that Bet2 modifies Ypt1 and Sec4 in an analogous manner."
Thus, our graphical analysis method discovered an actual relation missed by our text mining system. In this case, it was missed because the verb "modifies" was not one of those we tabulated in Table 1.
Formal description of algorithm
More formally, given a interaction between two proteins, P and Q, we define a neighborhood graph, GN(P,Q). We then analyze the cohesion of GN(P,Q) for each P and Q and collect statistics on the cohesion, as described in [15] .
The cohesion of a graph or subgraph is defined as the ratio of the number of edges present to the possible number of edges. In the case of a single node, n, in an undirected graph, if the degree (number of incident edges) of n is d we define the neighborhood of n as the set of nodes including the endpoints of these d edges, and all edges whose endpoints are in this node set. Say there are e such edges. The cohesion, C(n) is then defined in Equation (2).
In this paper, we are analyzing the cohesion of a subgraph defined over the union of the neighborhoods of two nodes, specifically P and Q above. There are also three types of edges in this graph. There are thus many possible definitions of cohesion. For simplicity, we take the conservative approach of only considering 2- and 3-hop paths (i.e., paths between P and Q which contain 2 or 3 edges).
In the 104 protein interactions found by our method, but not in the combined true relations table, 30 are related by at least one 2-hop path, 34 are related by at least one 3-hop path and 39 are related by at least one 2-hop or 3-hop path.
In 18 of the 30 cases where 2 hop paths were present, more than one path was present, with the average being 2.3, and similarly, 25 of the 34 occurrences of 3-hop paths were multiple occurrences, with the average being 4.1. Since the network contains 30,135 possible pairs, the assumption that these paths support new interactions found by our method seems statistically persuasive. These results are summarized in Table 2.
Validation of graphical computations
Computation of all relations having 2-hop and 3-hop paths which do not have direct reported interactions gave 30 relations deduced from a combination of 2-hop and 3-hop paths and 9 relations deduced only from 3-hop paths. Of the 2-hop path relations, 15 (50%) of them were found to be true by careful re-examination of the text of the abstracts. This gives us a precision of 74%.
We were unable to discover any abstracts supporting the interactions predicted by the 3-hop relations. However, of the 39 predicted interactions, 20 had 4 or more total 2-hop or 3-hop bridges, and of these 8 were validated by consulting our abstract collection. We suggest that the remaining 12 merit further investigation.
While protein interactions may not be the only reason for this close co-occurrence, other reasons for these graphical relations may be of interest. For example, FAR1 and STE5 are related by 1 2-hop bridge and 6 3-hop bridges. These proteins are reported to be homologous [16], that is having structural and functional similarity, and this may also be of significant interest.
Estimation of recall
Recall, of course, can only be approximated in such a large collection. In the 12,300 document collection, 451 documents were returned as containing one or more of the computed interactions. In reading these documents to validate these interactions, we found only one interaction which was missed by the algorithm because it was referred to across 2 sentences and the co-reference was not resolved by this system.
It is difficult to devise a method for measuring recall when 12,300 documents constitute the sample. We examined 100 randomly chose abstracts from this collection for description of any protein-protein interactions. Since none of the 100 we selected happened to be members of the set of 451 described above, we were looking for additional un-found interactions. None were discovered. On this basis, the recall was effectively 100%.
Since this seemed to be exceedingly optimistic, an experiment was devised which would return the most likely candidate documents where protein relations might have been missed. If we found many missed relations, the recall is reduced.
The method for finding protein interactions we have described above amounts to using a dictionary of yeast proteins names and their synonyms to find co-occurrences of two (or more) such names within a single sentence. Since this amounts to sentence parsing using a well-described tokenizer and parser, followed by simple string matching, and since by direct examination we found almost no cases where the statement of interactions spanned more than one sentence, it is extremely likely that we have found all such cases that exist within the entire 12,300 document collection, and the documents that are returned represent all that actually exist. The recall measurement thus amounts to examining the documents after suitable filtering to find how many actually describe (or do not describe) protein interactions.
Accordingly, we reduced the stringency of our filtering of sentences so that more candidate relations might be discovered. In this experiment, the verb filters (Table 1) were excluded. This approach will return documents containing at least one sentence with two proteins which does not include the word "gene." The other exclusion terms in Table 3 were not used. This resulted in 581 documents, of which 130 were additional to the original set of 451.
These additional 130 abstracts were examined in detail for the description of any protein interactions anywhere in the abstract, and 12 such interactions were found. Of these, 2 were discovered across sentence boundaries, requiring anaphora resolution and 2 more occurred in sentences containing the word "gene." This means that 118/130 documents were correctly identified as having no relations, or only 12/130 contained relations, resulting in a recall of at least 90.1%. If we use all 581 abstracts, the recall is much higher (97%). This gives us an F-measure of 0.83.
Rules used in finding protein interactions
This section summarizes the rules and techniques used in finding the protein interactions.
1. Exclude any sentence containing the terms in Table 3.
2. Recognize proteins from a dictionary of proteins and their synonyms and variant spellings. Exclude all lowercase spellings, which usually represent mutations.
3. Recognize protein complexes by hyphenation.
4. Recognize protein synonyms when separated by a slash.
5. Require any sentence with two or more proteins to contain one of the verbs in Table 1.
6. Allow any sentence containing "form" and "complex" along with two or more proteins.
7. Recognize secondary interactions based on those found by 2-hop and 3-hop connections in the primary table of correct interactions.
Conclusion
In a large set of 12,300 abstracts, the primary task is filtering out sentences in documents which describe genes and other non-protein interactions. Once this is done, 61% precision is possible, and if the graphical predictions of secondary interactions hold true, the precision is at least 0.74. Based on reading of the abstracts the recall is estimated to be 97%. The f measure is 0.74, based on a precision of 0.61, and is 0.83 based on the precision of 0.74.
These experiments result in respectable precision and considerably higher recall than previously reported methods and tend to indicate that a combination of statistical and linguistic methods [1-4] can give better results than linguistic (frame based) methods alone.
Finally, we note that there is apparently no "silver bullet" to improve detection of protein-protein relations. Instead, the process is one of incremental improvement based on rules and filters of data. However, the set of rules we report here appear to have the highest F-measure yet reported.
Methods
The MIPS data were downloaded and mined using custom Java programs. Sentence boundaries and tokenization was accomplished using JTalent, a Java wrapper for the TALENT system described above. The noun phrase and verb data were stored in a DB2 database, along with the document number, sentence number and offset, so that the verb co-occurrences listed in Table 1 could be deduced. All of the protein annotators were also written entirely in Java. The graphical network software was also written in Java by AK.
Authors' contributions
JC carried out the annotator design, text mining and database design. AK carried out the graphical analysis.
Acknowledgements
We thank Bhavani Iyer for writing the XML extractor from our database representation, Eric Brown for the use of his DictMatcher code for detecting dictionary terms, and Bob Mack for numerous helpful discussions. The graphical layout system in Figure 1 was developed by Daniel Tunkelang [17]. The UIMA annotation system is available at .
Figures and Tables
Figure 1 Logical flow of Medline document analysis.
Figure 2 The true relations network around the discovered YPT1-BET2 relation.
Table 1 Verbs Used to Describe Protein Interactions
act
activate
associate
bind
complex
co-precipitate
depend
inhibit
interact
mediate
phosphorylate
stabilize
Table 2 Summary of precision and recall in recognizing protein interactions under various conditions.
Matched relations All relations Precision Recall F measure Sample size
All sentences 133 912 0.14 1.00 * 0.24 451
Exclude genes 110 660 0.17 1.00 * 0.25 451
Require verbs 94 437 0.21 1.00 * 0.34 451
Discovering relations not in MIPS table 234 437 0.53 1.00 * 0.69 451
Exclude negatives, alleles 239 381 0.61 1.00 * 0.75 451
Including network analysis data
Exclude proteins not part of true relations table. 239 343 0.70 0.97 * 0.81 581
Include only validated 2-hop relations 254 343 0.74 0.97 * 0.83 581
* Assumes that the MIPS table of proteins and synonyms is complete
Table 3 Terms that cause a sentence to be excluded from protein interaction discovery
gene
express
encode
no
not
fail
mRNA
transcription
allele
* Assumes that the MIPS table of proteins and synonyms is complete
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| 15941473 | PMC1164402 | CC BY | 2021-01-04 16:02:51 | no | BMC Bioinformatics. 2005 Jun 7; 6:143 | utf-8 | BMC Bioinformatics | 2,005 | 10.1186/1471-2105-6-143 | oa_comm |
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-471590450710.1186/1471-2407-5-47Research ArticleReevaluation of the 22-1-1 antibody and its putative antigen, EBAG9/RCAS1, as a tumor marker Reimer Tatiana A [email protected] Ioannis [email protected] Bettina [email protected] Insa [email protected] Harald [email protected] Peter [email protected]örken Bernd [email protected] Armin [email protected] Max-Delbrück-Center for Molecular Medicine, Department of Hematology, Oncology and Tumorimmunology, Berlin, Germany2 Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Department of Pathology, Germany3 Max-Delbrück-Center for Molecular Medicine, Department of Electronmicroscopy, Germany4 Charité, Universitätsmedizin Berlin, Robert-Rössle-Klinik, Department of Hematology, Oncology and Tumorimmunology, Germany2005 17 5 2005 5 47 47 18 11 2004 17 5 2005 Copyright © 2005 Reimer et al; licensee BioMed Central Ltd.2005Reimer et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Tumor-associated antigens are appreciated as diagnostic markers, but they have also prompted tremendous efforts to develop tumor-specific immunotherapy. A previously cloned tumor-associated antigen, EBAG9, was initially defined by reactivity with the monoclonal antibody 22-1-1. Functionally, the EBAG9-encoded gene-product was believed to induce apoptosis in activated immune cells. However, using a cell-biological approach we identified EBAG9 as a Golgi-resident modulator of O-linked glycan expression, the latter product was then recognized by the 22-1-1 antibody. Secondly, EBAG9 expression was found physiologically in all murine tissues examined. This raised the question if EBAG9 is tumor-specific and mediates apoptosis itself or through O-linked glycans generated, among them the cognate 22-1-1 antigen Tn.
Methods
We have used immunohistochemistry to detect the expression of 22-1-1 and EBAG9 in various tissues. Correlation between expression of both antigens in cell lines was analysed by immunoblot and flow cytometry. Apoptosis was studied by using flow cytometry and Caspase-Glo™ 3/7 assay kit. Cellular distribution of EBAG9 was analysed by electron and confocal microscopy.
Results
Here, we compared expression of the 22-1-1 and EBAG9-defined antigens in normal and neoplastic tissues in situ. In contrast to 22-1-1 staining, EBAG9 is a ubiquitously expressed antigen in all normal and cancerous tissues. Functional studies on the role of 22-1-1 reactive material did not support any evidence for apoptosis induction. Employing electron and confocal microscopy, a refined subcellular localization of EBAG9 at the Golgi was obtained.
Conclusion
We suggest that the estrogen-inducible EBAG9 gene-product and the 22-1-1 defined antigen are structurally and functionally separate antigens.
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Background
In tumor immunology considerable effort has been made to discover tumor specific antigens. Numerous antigens were introduced and cancer vaccines based on these antigens have been shown in pre-clinical studies to elicit tumor-specific immunity and establish long-term memory without inducing an autoimmune response [1]. Other important clinical applications of tumor-associated antigens include a role as markers for diagnosis of onset and relapse of cancer.
Recently, the tumor-associated antigen RCAS1 has received considerable attention. Initially, RCAS1 was defined by the 22-1-1 monoclonal antibody (mAb), which was raised by immunization of mice with the human uterine cervical adenocarcinoma cell line SiSo [2]. Expression cloning led to the identification of a cDNA apparently encoding the 22-1-1 antigen. The gene product was termed " receptor binding cancer antigen expressed on SiSo cells" (RCAS1) and is identical with the estrogen-responsive protein EBAG9 (estrogen receptor-binding fragment-associated gene 9) [3,4]. In this report, we refer to the term EBAG9. Cell surface staining with 22-1-1 mAb was shown immunohistochemically in a large number of different tumor tissues [5-7]. Protein expression of EBAG9, as detected by immunoblotting with a polyclonal anti EBAG9 serum, was reported in ovarian cancer cell lines [8]. Functionally, cell culture supernatant from SiSo cells was proposed to inhibit proliferation of activated T lymphocytes and K562 cells and to induce apoptotic cell death in receptor bearing cells [3]. Therefore, EBAG9 was introduced as a new death receptor ligand involved in tumor immune escape, reminiscent of the Fas/Fas ligand system [9].
Since EBAG9 and 22-1-1 are broadly used as synonymous functional terms, a misleading picture emerged. We have recently reported that the EBAG9 encoded antigen is a predominantly Golgi-localized protein with a short transmembrane N-terminus and a large cytoplasmic C-terminus [10]. Upon reexamination, we found out that EBAG9 has a palmitoylation anchor, responsible for membrane attachment and functional protein-protein interactions [11]. EBAG9 is not recognized by the 22-1-1 mAb itself, instead we were able to show that EBAG9 overexpression leads to the generation of the normally cryptic O-linked glycan Tn, which is then recognized by the 22-1-1 antibody [10]. Of note, aberrant glycosylation of glycoproteins or glycolipids is often associated with neoplastic transformation [12,13].
Since many attempts are made to correlate mAb 22-1-1 reactivity and EBAG9 expression with clinical prognosis or even pathogenesis of tumors, these reports prompted us to revisit tumor-specificity of both antigens and their suggested role in induction of apoptosis.
Methods
Immunohistochemistry
The specimens analysed included 10 cases of each of squamous cell carcinoma from the oral cavity, adenocarcinoma of the lung, gastric, colorectal and prostate carcinomas. In addition to the invasive prostatic carcinoma 2 cases showed areas of high-grade prostatic intraepithelial neoplasia (PIN III). The carcinomas selected for investigation showed various degrees of differentiation and were always surrounded by various non-neoplastic tissues. All cases were retrieved from the files of the Institute of Pathology, Charité, Campus Benjamin Franklin, Medical University Berlin, Germany.
Four micrometer thick sections from paraffin-embedded tissue specimens were cut, dewaxed and subjected to antigen retrieval before incubation with primary antibodies. This consisted of a brief, high-temperature heating of the sections immersed in various solutions in a high-pressure cooker [14]. In the case of 22-1-1 (IgM; MBL, Göttingen, Germany, 1:200), 1 mM EDTA-NaOH at pH 8.0 and a heating time of 1 min was employed, while the conditions for Ab-1/clone 5E4 (IgG1; Oncogene, San Diego, CA, USA, 1:100) were a citrate buffer (10 mM, pH 6.0) and a heating time of 2 min. Appropriate control experiments involving isotype controls mouse IgG1 and IgM (DakoCytomation, Glostrup, Denmark) were performed. Bound antibodies were detected by using the streptavidin-biotin-alkaline phosphatase method and New-Fuchsin as chromogen. All reagents were purchased from DakoCytomation.
Cell lines
All cell lines employed and their tissue origin are listed in Table 1. Cell lines HBL-100, Colo-205, ZR-75-1, H-184 A1, T-47D, CAL-51 and MDA-MB-468 were kindly provided by Drs. U. Karsten and U. Jandrig (MDC, Berlin). The other cell lines were obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).
Table 1 Expression of EBAG9 and 22-1-1 on cancer cell lines.
Cell line Origin 22-1-1 expression Flow cytometry EBAG9 expression Immunoblot
MCF-7 breast carcinoma ++ +++
K562 chronic myelogenous leukemia - +++
Jurkat T cell leukemia +++ +++
Colo-205 colorectal carcinoma - +++
SiSo cervix carcinoma + +++
CAL-51 breast carcinoma - +++
T-47D breast carcinoma - +++
ZR-75-1 breast carcinoma - +++
HBL-100 breast carcinoma - +++
U-266 multiple myeloma - ++
CaCo-2 colon carcinoma - ++
MDA-MB-461 breast carcinoma - +
HEK293 A embryonal kidney - +
U-373 glioblastoma-astrocytoma - +
L-1236 Hodgkin's lymphoma - +
H-184 A1 normal breast - +
SW-480 colon carcinoma - +
MDA-MB-468 breast carcinoma - +
Expression of antigen on the cell surface was determined by incubation with 22-1-1 antibody and analysed by flow cytometry. Expression of EBAG9 was determined by immunoblot using the anti-EBAG9 serum. (+++, very high expression, ++, high expression, +, intermediate expression, -, no expression).
Gel electrophoresis and immunoblotting
Cell lysate preparation and electrophoresis were performed according to Engelsberg et al. [10]. Proteins transferred to nitrocellulose membranes were incubated with anti-EBAG9 serum (1:1000) and α-actin antibody (Sigma, Taufkirchen, Germany) overnight at 4°C. Following incubation with horseradish peroxidase-conjugated secondary antibody (Southern Biotechnology, Birmingham, AL, USA), bound proteins were visualized by chemiluminescence ECL (Amersham Biosciences, Freiburg, Germany).
Flow cytometric analysis
Flow cytometric analysis was performed according to Engelsberg et al. [10]. Briefly, cells were incubated with 22-1-1 or an isotype control IgM (1:100) for 1 h on ice and next with a biotinylated rat anti-mouse IgM secondary antibody (Dianova, Hamburg, Germany). Bound antibodies were detected with PE-conjugated streptavidin (Southern Biotechnology), and flow cytometric analysis was carried out on a FACSCalibur™ cytometer (BD Bioscience, Heidelberg, Germany).
Electron microscopy
HEK293 A cells were stably transfected with a GFP-tagged EBAG9 construct and selected with G418 (400 μg/ml). Cells were fixed with 4 % formaldehyde/0.5 % glutaraldehyde in 0.1 M phosphate buffer, 0.18 M sucrose for 1 h at room temperature. Following harvesting and washing with 0.1 M phosphate buffer/0.18 M sucrose cells were infiltrated with 1.8 M sucrose/20 % polyvinylpyrrolidone (K15, Fluka, Buchs, Switzerland) overnight. Ultrathin cryosections (70 nm) were obtained according to Tokuyasu [15] using an ultramicrotome (Reichert-Jung Ultracut S) attached to a cryosystem FC4S. GFP was detected with a polyclonal anti-GFP antibody (Abcam, Cambridge, UK, 1:400) diluted in a washing buffer containing 1 % BSA (fraction V; Serva, Heidelberg, Germany) and 0.12% glycine in phosphate buffered saline. For signal detection, 12 nm colloidal gold-AffiniPure goat anti-rabbit IgG, EM grade (Jackson Immuno Research Lab., Inc., West Grove, PA, USA) was used. Cryosections were contrasted and stabilized with a mixture of 3 % tungstosilicic acid hydrate (Fluka) and 2.5 % polyvinyl alcohol (Mr 10000, Sigma) according to Kärgel et al. [16]. Electron micrographs were taken with a Philips EM 400T at an acceleration voltage of 80 kV. As a control, non-transfected HEK293 A cells were used.
Apoptosiss assays
For the assessment of apoptosis, we used an annexin V-FITC apoptosis detection kit (Dako) according to the manufacturer's instruction. For coculture experiments, supernatant (RPMI 1640/10 % FCS) from subconfluent MCF-7 cells was obtained after 3–4 days. It was precleared from particulate matter by centrifugation and further concentrated by pressure filtration in an Amicon chamber (cut-off 15 kD). K562 cells were incubated with 10 × concentrated supernatant medium from MCF-7 cells diluted at a ratio of 1:4, normal or 10 × concentrated RPMI medium, diluted as described above. Cells were incubated for 48 h and apoptosis was assessed by annexin V-FITC/propidium iodide staining. For treatment with glycans, cells (1 × 105) were grown in a 24-well plate and incubated with β-GalNAc-PAA-biotin, β-GlcNAc-PAA, α-GalNAc-PAA (Tn) and Galβ1-2Galβ (TF) (Syntesome, Munich, Germany) in a concentration of 50 or 100 μg/ml for 48 h. Apoptosis was assessed by analysis of activation of caspase -3 and -7 using the substrate DEVD-aminoluciferin from Caspase-Glo™ 3/7 assay kit (Promega, Mannheim, Germany), according to the manufacturer's instruction. As a positive control, UV irradiated K562 cells were used. Data were analysed using Excel software. Student's t test was used to determine significance. Results are expressed as the mean ± SD.
Immunostaining and confocal microscopy
HEK293 A cells expressing EBAG9-GFP were grown on coverslips for 48 h and incubated with brefeldin A (BFA, 5 μg/ml) for 15, 30 and 60 minutes, or with nocodazole (Noc, 10 μg/ml) for 2 h. Next, cells were fixed with 3 % paraformaldehyde for 15 min, followed by permeabilization with PBS containing 0.25 % Triton X-100 for 5 min. Cells were stained with anti-mannosidase II (Chemicon Int., Temecula, CA, USA, 1:100) and anti-TGN38 (BD Transduction Lab., Heidelberg, Germany, 1:100) antibodies overnight at 4°C. Slides were washed, and bound antibodies were detected with biotinylated goat anti-rabbit or goat anti-mouse antibodies and streptavidin-conjugated Alexa Fluor™ 568 (Molecular Probes, Leiden, The Netherlands). Signals were visualized on a Zeiss LSM 510 inverted laser scanning microscope and processed in LSM 5 image browser.
Results
Immunohistochemical comparison of 22-1-1 and Ab-1 staining in non-neoplastic normal tissue
The clinical significance of EBAG9 expression has been assessed using the 22-1-1 antibody in immunohistochemistry. To our knowledge, only three reports make use of a polyclonal antibody generated against recombinant EBAG9 [8,17,18]. However, all conclusions drawn from these studies were based on the assumption that the 22-1-1 antigen was identical to the EBAG9 encoded antigen.
Since we recently identified the O-linked glycan Tn as the antigen recognized by mAb 22-1-1, we asked whether the 22-1-1 defined antigen was tumor-specific, and whether this mAb recognizes the same antigenic structure as a monoclonal anti-EBAG9 antibody, Ab-1. Our previous observations were made in cell lines, therfore we now applied both antibodies in a side-by-side immunohistochemical analysis to a variety of tumor entities and regular tissues.
In normal gastric epithelia 22-1-1 stained mucous epithelia of the surface and neck of gastric foveolae, displaying an intense cytoplasmic labeling (Figure 1 a, c). The antral glands and the chief and parietal cells of the body of stomach were negative. There was always a positive labeling of complete intestinal metaplasia areas in a pattern similar to that observed in enterocytes and goblet cells (see below). The neuroendocrine cells located in the basal parts of foveoles showed also a weak cytoplasmic labeling. In comparison, the Ab-1 mAb showed an intense cytoplasmic labeling of parietal and chief cells while all other cells displayed a weak cytoplasmic labeling. Also the antrum type glands showed a cytoplasmic positivity (Figure 1 b, d).
Figure 1 Examples of the different immunostaining patterns obtained using the antibody clones 22-1-1 and Ab-1 in normal glandular tissues. Mucosa of the corpus of stomach (a, b; magnification × 10) and (c, d; magnification × 45). Colonic mucosa (e, f; magnification × 25), (g; magnification × 60) and (h; magnification × 50). Prostatic glands (i; magnification × 60) and (j; magnification × 50).
In glandular epithelia, among them colonic enterocytes, mAb 22-1-1 exhibited granular labeling of the apical portion of cells (Figure 1 e, g). Only occasionally goblet cells showed an intense cytoplasmic labeling. In contrast, Ab-1 labeling was obtained in basal and apical portions of all cells except goblet cells which were constantly negative (Figure 1 f, h).
A third example with striking differences of staining patterns between both antibodies includes normal prostate tissue where staining with 22-1-1 was seen in glandular epithelia with a prominent labeling of the apical cytoplasmic portions (Figure 1i). Of note, also the secretions were intensely positive. Ab-1 stained prostate epithelia in a dot-like perinuclear pattern whereas secretions were essentially negative (Figure 1j). In summary, in healthy tissue Ab-1 invariably stains all cell types and is consistently negative for secreted matter from glandular tissues. In contrast, 22-1-1 reactivity is restricted to a characteristic apical cytoplasmic or plasma membrane pattern in glandular tissues being always positive in mucous secretions. Other normal tissues with striking differences between both antibodies included epidermis, salivary glands, pulmonary alveolar cells and hematolymphoid cells (data not shown).
Immunohistochemical comparison of 22-1-1 and Ab-1 staining in adenocarcinomas
Next, antibodies 22-1-1 and Ab-1 were compared for their reactivity towards adenocarcinomas derived from stomach, colon, prostate, and lung (Figure 2). With the 22-1-1 mAb, we observed that neoplastic cells usually exhibited strong cytoplasmic labeling (Figure 2 a, e, g, i). Of course, the labeling was not always homogeneous in the entire tumor specimen. In the case of more differentiated carcinomas the labeling was more intense in neoplastic cells adjacent to luminal structures (Figure 2c). Mucous secretions as well as intracellular mucin were always intensely positive (this being most prominent in signet ring cell carcinomas of the stomach, Figure 2a). Frequently the neoplastic infiltrate became easily visible at low magnification due to its more intense staining compared to the surrounding normal tissues. In case of prostatic intraepithelial neoplasia (PIN Grade III- high grade PIN) the cells were most intensely labeled (Figure 2g). In contrast, Ab-1 usually exhibited a weak ubiquitous, frequently dot-like labeling of neoplastic cells, which sometimes was more intense than that of the surrounding normal tissues. Mucous secretions were always entirely negative (Figure 2 d, f, h, j).
Figure 2 Examples of the different immunostaining patterns obtained using the antibody clones 22-1-1 and Ab-1 in various adenocarcinomas. Signet ring cell gastric carcinoma (a, b; magnification × 80). Colorectal adenocarcinoma (c, d; magnification × 60). Lymph node metastasis (e, f; magnification × 50). Prostatic adenocarcinoma (g; magnification × 60) and (h; magnification × 50): Adenocarcinoma of the lung (i, j; magnification × 60).
EBAG9 is a cis/medial Golgi protein
It has been demonstrated that EBAG9 colocalizes with the Golgi marker protein 1,4 galactosyltransferase [10]. To refine the subcellular localization of EBAG9, we first utilized electron microscopy. Ultrathin cryosections of HEK293 A cells showed a highly specific labeling of GFP-tagged EBAG9 on the entire Golgi stack (Figure 3b), including small vesicles surrounding the Golgi apparatus (Figure 3a). Sections of non-transfected control cells were always free of label (Figure 3c).
Figure 3 Subcellular localization of EBAG9 analysed by electron microscopy. Immunogold labeling of HEK293 A cells. EBAG9-GFP is found in Golgi cisternae and small vesicles in stably transfected cells. a) small vesicles surrounding the Golgi apparatus, b) Golgi stacks, c) control-non transfected cells. Bar; 0.5 μm.
Secondly, immunofluorescence staining in combination with confocal microscopy revealed that EBAG9-GFP was primarily localized to the juxtanuclear region of stably transfected HEK293 A cells, and colocalized with the cis/medial Golgi marker protein mannosidase II (Figure 4 a, b, c). TGN38, a prototypical trans-Golgi protein revealed only partial overlap (Figure 4 j, k, l). To more rigorously assess the colocalization of EBAG9 and mannosidase II, brefeldin A (BFA) [19] treatment was performed. After a 15 min BFA treatment, the Golgi-resident protein mannosidase II was detected in a characteristic ER pattern (Figure 4e), and EBAG9-GFP (Figure 4 d, f) was displaced from the Golgi cisternae to the cytosol in characteristic punctate like structures scattered throughout the cells. Similar observation was done with 60 min of incubation (Figure 4 g, h, i). Of note, the endogenous EBAG9 protein behaved similarly (data not shown). Nocodazole [20] was applied to explore the co-localization of EBAG9 with TGN38. This treatment revealed that EBAG9-GFP was only partially localized on the same Golgi fragments as TGN38 (Figure 4 m, n, o). We conclude that EBAG9 is predominantly expressed at the cis/medial Golgi complex, but also on vesicles derived thereof.
Figure 4 EBAG9 is sensitive to treatment with BFA and nocodazole. Displacement of EBAG9 from Golgi structures upon treatment with BFA. Cells stably transfected with EBAG9-GFP were left untreated (w/o) (a, b, c), incubated for 15 min (d, e, f) and 60 min (g, h, i) with BFA (5 μg/ml) or with nocodazole -Noc (10 μg/ml) (m, n, o) for 2 h. Cells were fixed and stained with anti-mannosidase and anti-TGN38 antibodies (red). Merged image, yellow. Bar; 10 μm
High endogenous expression of EBAG9 only partially correlates with cell surface expression of the mAb 22-1-1 defined antigen
Cancer cells often exhibit normally cryptic, O-linked glycan structures that can elicit humoral or cellular immune responses. Transient overexpression of the EBAG9 cDNA in HEK293 A cells leads to the generation of the antigen recognized by the mAb 22-1-1, which is identical to the Tn glycan antigen [10]. To investigate if expression of endogenous EBAG9 correlates with the occurrence of the 22-1-1 antigen, we have chosen several tumor cell lines and examined their EBAG9 protein content by immunoblot, whereas 22-1-1 surface staining was assessed by flow cytometry. Unfortunately, the mAb 22-1-1 was not applicable to immunoblot analysis.
Almost all tumor cell lines expressed EBAG9 in varying amounts, indicated by a characteristic double band and stained with our polyclonal anti EBAG9 serum (Figure 5). Strongest expression was obtained for MCF-7, K562, SiSo, and Jurkat cells. To test whether different endogenous protein expression levels of EBAG9 lead to surface display of the 22-1-1 antigen, we stained all tumor cell lines with mAb 22-1-1 and subjected them to flow cytometric analysis (Table 1, see Additional file 1). Only two cell lines, MCF-7 and Jurkat with high levels of EBAG9 protein expression were also strongly positive for staining with the anti-Tn antibody, 22-1-1. Additionally, SiSo cells exhibited moderate 22-1-1 antigen expression. In conclusion, endogenous expression levels of EBAG9 and Tn antigen were only partially correlated.
Figure 5 Endogenous expression of EBAG9 in cancer cell lines. Cells were lysed in NP-40 buffer and 50 μg of total protein was separated by SDS-PAGE and analysed by immunoblot using our polyclonal anti-EBAG9 antibody. α-actin served as control indicating protein load.
The antigen recognized by the 22-1-1 mAb does not induce apoptosis
Programmed cell death, or apoptosis, can be induced by multiple cell signaling mechanisms [21]. Recombinant EBAG9 has been previously suggested to induce apoptosis in activated T cells or K562 cells that express an unidentified EBAG9 receptor [3]. We were unable to repeat this experiment, because EBAG9 is only soluble in the presence of detergents. However, a soluble form of the 22-1-1 antigen was detected in supernatants from SiSo and MCF-7 cells, and this form was also suggested to cause apoptosis. To revisit whether an antigen present in culture supernatant of 22-1-1 positive MCF-7 cells can induce apoptosis, we incubated K562 cells with a dilution of concentrated culture supernatant [5]. To detect apoptosis induction, K562 cells were stained with annexin V-FITC and propidium iodide after 48 h of culture. In K562 cells treated with concentrated supernatant from MCF-7 cells in a dilution of 1:4, we observed less then 6% of cells positive for annexin V-FITC, which was similar to control cells (Figure 6 a). Incubation with concentrated regular medium also failed to induce apoptosis in K562 cells (Figure 6 a). Since we have established that mAb 22-1-1 recognizes the Tn antigen, we asked whether chemically defined glycans were able to mediate apoptosis. K562 cells were incubated with polyacrylamide-conjugated (PAA) αGlcNAc, αGalNAc (Tn), TF and βGalNAc in two different concentrations (50 and 100 μg/ml) and subjected to caspase -3 and -7 activity assay. As shown in Figure 6 b, essentially no caspase activity was observed in K562 cells incubated with glycans compared to control cells irradiated with UV light (P). In conclusion, we found no evidence for apoptosis induction with the antigen recognized by the 22-1-1 mAb.
Figure 6 The 22-1-1 antigen does not induce apoptosis in K562 cells. a) Cells were irradiated with UV-light as positive control for apoptosis, or incubated with regular non concentrated medium, or 1:4 dilution of concentrated regular medium, or concentrated MCF-7 cell culture supernatant. Apoptosis was assessed by annexin V-FITC staining. b) K562 cells were incubated with 50 μg, or 100 μg of (1) TF; (2) α-GalNAc-PAA; (3) β-GlcNAc-PAA; (4) β-GalNAc-PAA-biotin and subjected to caspase -3 and -7 activity assay. P-cells irradiated with UV; N-non treated cells.
Discussion
EBAG9 was originally described as a novel tumor associated antigen with a functional role in tumor-immune interactions [3]. Cell culture supernatant from SiSo cells inhibited the proliferation of activated T cells and induced apoptotic cell death in receptor bearing cells. Furthermore, recombinantly expressed EBAG9 was suggested to bind to a yet unidentified receptor on activated immune cells and on K562 cells. From these observations and from the detection of apoptotic tumor-infiltrating lymphocytes surrounding 22-1-1 mAb stained tumor lesions [3,22] it was inferred that EBAG9 is a new death receptor ligand involved in tumor immune escape [23]. In the present study, we have extended our previous cell biological characterization of EBAG9, resulting in conclusions that are not consistent with the currently held view on the functional and clinical role of EBAG9.
We first addressed the question whether EBAG9 is a tumor-specific marker. Recently, we and others have pointed out that EBAG9 is highly conserved in phylogeny, and the gene-product was found to be expressed in all murine tissues examined [24]. Applying immunohistochemistry on a representative selection of tumor and normal human tissue specimen, reactivity with the EBAG9-specific monoclonal antibody Ab-1, generated against a recombinant EBAG9 fusion protein, was seen in essentially all tissues and cell types examined. Staining in benign or malignant cell types was confined to a cytoplasmic pattern. In tumor infiltrating plasma cells a Golgi-like distribution could be clearly seen. Secondly, referring to our previous report [10] we asked whether the cognate 22-1-1 antigen and the EBAG9 antigen are distinguishable in situ. In a side-by-side comparison of both antibodies in immunohistochemistry, we confirmed significant differences between 22-1-1 and Ab-1 antibody reactivities. Most striking examples for differences in immunostaining included normal glandular tissues, among them gastric epithelia, colon and normal prostate tissue. Of note, 22-1-1 constantly stained secreted matter from glandular tissues, whereas Ab-1 was always negative for mucus. In agreement with previous reports [5,23], 22-1-1 staining is not limited to malignant tissue, but is also seen in normal gastric, colon and prostate epithelia. In case of adenocarcinomas, strong staining for the 22-1-1 mAb was observed in signet ring cell carcinomas with an intense labeling of the intracellular mucin. In sharp contrast those neoplastic cells remained almost negative with Ab-1. In most cases, the expression of the 22-1-1 antigen was enhanced in adenocarcinomas as compared to their normal counterpart. This was not the case for the antigen recognized by Ab-1. Other marked differences were seen with mucous secretions and with the subcellular distribution of the epitope recognized by Ab-1. This conclusion is substantiated by only partial correlation between endogenous EBAG9 protein levels and the corresponding expression profiles for the 22-1-1 antigen in human tumor cell lines, as determined by immunoblotting or flow cytometry, respectively.
The staining pattern obtained for 22-1-1 was almost identical to that described for the tumor-associated O-linked glycan Tn [25,26], thus confirming our epitope identification [10]. In addition, Tn antigen is found as soluble antigen in serum of tumor patients, an observation that is also shared with the occurrence of 22-1-1 soluble antigen [4,27].
We also refined the subcellular localization of the EBAG9-encoded antigen. EBAG9-GFP localized predominantly to the Golgi cisternae and to small vesicles surrounding the Golgi apparatus, as evidenced by immuno-electronmicroscopy. A more functional analysis revealed sensitivity to BFA and a corresponding colocalization with the cis/medial Golgi marker, mannosidase II. These findings identify EBAG9 as a predominantly Golgi localized protein which is unlikely to be secreted. Our results shed doubt on the hypothesis that soluble 22-1-1 reactive material induces apoptotic cell death in activated immune cells or other receptor bearing cells. This earlier conclusion rests on two different experimental approaches, either the incubation of activated T cells or K562 cells with culture supernatant, as obtained from SiSo or MCF-7 cells, or the exposure to recombinantly expressed EBAG9 protein [3,23]. Functional readout for both systems was the detection of apoptotic cell death. However, none of the apoptotic pathways have been elucidated yet. We have previously pointed out that full-length recombinant EBAG-GST is not soluble in aqueous solutions and requires the presence of detergent. Therefore, it was reasonable to suggest that cell viability in assays using recombinant EBAG9 was most likely affected by residual detergent [10]. In our hands, cell culture supernatant from 22-1-1 positive MCF-7 cells failed to induce apoptosis in K562 cells. Likewise, the O-linked glycan recognized by 22-1-1, Tn (αGalNAc), did not induce apoptosis in K562 cells. Presently, we cannot reconcile our data with those published earlier.
In conclusion, our data strongly suggest that the antigens recognized by 22-1-1 and the EBAG9 antibody, Ab-1, are different. It follows that studies on the correlation of EBAG9 expression and clinical prognosis were correct as long as their screens were based on RT-PCR or immunoblotting with a polyclonal anti EBAG9 antibody [8]. In contrast, functional and clinical studies on the 22-1-1 defined antigen (Tn) need to be revisited, and should be compared to other studies obtained with anti-Tn antibodies. At present, a direct link between the occurrence of Tn and expression levels of EBAG9 is still elusive, since we observed a correlation in some cell lines, but not in others. Provided that the physiologically occurring molecule EBAG9 is indeed dysregulated or mutated in tumors, this should prompt further investigations on the role of EBAG9 in the modulation of O-linked glycan expression.
List of abbreviations
EBAG9 (estrogen receptor-binding fragment -associated gene 9), RCAS1 (receptor binding cancer antigen expressed on SiSo cells), BFA (Brefeldin A), Noc (nocodazole)
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
TAR and IL conceived the study, participate in its design and experiments, and draft a manuscript. IA, PD and BE performed immunohistochemical and electron microscopy analysis. HS and BD helped to draft the manuscript and participate in interpretation of the data. AR participated in the design of the study, coordination and analysis and interpretation of the data and helped to draft the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional File 1
Expression of the 22-1-1 antigen on cancer cell lines. Expression of antigens on the cell surface was determined by incubation with mAb 22-1-1 antibody and analysed by flow cytometry.
Click here for file
Acknowledgements
We are grateful to Uwe Karsten, Uta E. Höpken and Constantin Rüder for critical reading of the manuscript. We thank S. Heydrich, C. Cieluch and M. Vannauer for expert technical assistance. This work was supported by a grant from the Deutsche Krebshilfe to A.R. (grant 10-2054-Re I)
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| 15904507 | PMC1164403 | CC BY | 2021-01-04 16:39:12 | no | BMC Cancer. 2005 May 17; 5:47 | utf-8 | BMC Cancer | 2,005 | 10.1186/1471-2407-5-47 | oa_comm |
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-481590450810.1186/1471-2407-5-48Research ArticleMinimum follow-up time required for the estimation of statistical cure of cancer patients: verification using data from 42 cancer sites in the SEER database Tai Patricia [email protected] Edward [email protected] Gábor [email protected] Georges [email protected] Melanie [email protected] Ian [email protected] Vincent [email protected] University of Saskatchewan, Faculty of Medicine, Saskatoon; Department of Radiation Oncology, Regina, Canada2 Radiation Oncology Program, London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada3 Bács-Kiskun County Teaching Hospital, Surgical Pathology, Kecskemét, Hungary4 Geneva University Hospitals, Department of Gynecology and Obstetrics, Senology and gynecologic oncology unit, Geneva, Switzerland5 University of New Mexico, Cancer Research and Treatment Center, Albuquerque, NM, USA6 Department of Clinical Oncology, Western General Hospital, Edinburgh, Scotland, UK7 AZ-VUB, Oncologisch Centrum, Jette, Belgium2005 17 5 2005 5 48 48 18 10 2004 17 5 2005 Copyright © 2005 Tai et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The present commonly used five-year survival rates are not adequate to represent the statistical cure. In the present study, we established the minimum number of years required for follow-up to estimate statistical cure rate, by using a lognormal distribution of the survival time of those who died of their cancer. We introduced the term, threshold year, the follow-up time for patients dying from the specific cancer covers most of the survival data, leaving less than 2.25% uncovered. This is close enough to cure from that specific cancer.
Methods
Data from the Surveillance, Epidemiology and End Results (SEER) database were tested if the survival times of cancer patients who died of their disease followed the lognormal distribution using a minimum chi-square method. Patients diagnosed from 1973–1992 in the registries of Connecticut and Detroit were chosen so that a maximum of 27 years was allowed for follow-up to 1999. A total of 49 specific organ sites were tested. The parameters of those lognormal distributions were found for each cancer site. The cancer-specific survival rates at the threshold years were compared with the longest available Kaplan-Meier survival estimates.
Results
The characteristics of the cancer-specific survival times of cancer patients who died of their disease from 42 cancer sites out of 49 sites were verified to follow different lognormal distributions. The threshold years validated for statistical cure varied for different cancer sites, from 2.6 years for pancreas cancer to 25.2 years for cancer of salivary gland. At the threshold year, the statistical cure rates estimated for 40 cancer sites were found to match the actuarial long-term survival rates estimated by the Kaplan-Meier method within six percentage points. For two cancer sites: breast and thyroid, the threshold years were so long that the cancer-specific survival rates could yet not be obtained because the SEER data do not provide sufficiently long follow-up.
Conclusion
The present study suggests a certain threshold year is required to wait before the statistical cure rate can be estimated for each cancer site. For some cancers, such as breast and thyroid, the 5- or 10-year survival rates inadequately reflect statistical cure rates, and highlight the need for long-term follow-up of these patients.
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Background
The normal distribution is often used to describe the random variation of data in many scientific disciplines. However some distributions are skewed with low mean values and large variance. The distributions may be exclusively positive, such as the duration of survival of cancer patients with chronic leukemias [1], the incubation time of infectious diseases [2], and the abundance of biological species [3], etc. These skewed distributions often fit the lognormal distribution [4-7]. A lognormal distribution is one with random variables whose logarithms follow a normal distribution. The lognormal survival time of cancer patients, who died of their disease, has been tested and applied for various anatomic sites [8-21].
The term survival rate is used commonly, yet it is inaccurate. Five- and ten-year survival rates are commonly used in the literature. Kaplan-Meier or life-table (actuarial) methods estimate the proportion (or fraction) of survivors. In this study, the term "survival rate" means "survival fraction" expressed in percentage, and the term "cure rate" means "cure fraction" expressed in percentage, to be proper.
It is difficult to know the statistical cure rate, which is an estimation based on statistical models [11,15], especially for slowly proliferating tumors. Because of this limitation, oncologists usually discuss survival in terms of 5-year or 10-year survival rates. In certain rapidly growing tumors, the cancer-specific survival rate [22] reaches a plateau within 5–10 years and approaches the statistical cure rate, which is the survival rate observed when no more risk of death from the disease. In the Kaplan-Meier method [23], a person with residual cancer but who died of reasons other than the specific cancer is censored for cancer-specific survival. In the present study we applied the analysis on the cancer-specific survival rates. The statistical cure was reached at the plateau of the Kaplan-Meier plot of the cancer-specific survival rate. For some fast proliferating cancers, such as pancreatic and stomach cancers, the plateau appears within 10 years. However for some slow proliferating cancers, such as thyroid and early breast cancers, the plateau does not appear even after decades. Hence the present commonly used five-year survival rates are not adequate to represent the statistical cure rate.
In the present study, we established the minimum number of years required for follow-up in order to estimate the statistical cure rate, by using a lognormal distribution of the survival time of those who died of their cancer by applying the result derived by Limpert et al.[24] This minimum number of years required for follow-up was defined as the threshold year by μ* × (σ*)2 where μ* is the median and σ* is the multiplicative standard deviation of a lognormal distribution (see Additional file 1 : Appendix). The follow-up time for patients dying from the specific cancer covers most of the survival data, leaving less than 2.25% uncovered. This is close enough to cure from that specific cancer.
Methods
Data sources
We analysed the 1973–1999 Database of the Surveillance, Epidemiology, and End Results (SEER) Program [25] of the United States National Cancer Institute. Data from registries of Connecticut and Detroit were used in this study and results were compared with the SEER-9 registries. The cancer sites were chosen according to the SEER codes. Primary site, vital status, cause of death, and survival time information were used. Those patients with unknown or zero survival time and unknown cause of death were excluded.
To test for lognormality of survival time of cancer patients who died of cancer-specific disease, patients diagnosed from 1973–1992 in the registries of Connecticut and Detroit were chosen so that a maximum of 27 years was allowed for follow-up to 1999. A total of 49 specific cancer sites were tested. For prostate, salivary gland, breast, and thyroid cancer, patients diagnosed from 1973–1977 were chosen to allow a long enough time for follow-up to 1999. For cancer sites with high frequencies of older patients, such as lung and bronchus, colon, prostate, and breast, younger patient ages less than 60 were chosen, because elderly patients are more likely to die of intercurrent diseases and so affect the distribution of cancer-specific deaths versus other deaths.
As an illustration for special cohorts of interest, breast cancer was further analyzed based on accepted prognostic factors such as stage, histologic type and tumor grade, to show that the threshold years were different for different cohorts of interest.
Statistical analysis
The survival times of the cancer patients who died of their disease were tested for goodness of fit for lognormality using a minimum chi-square method. The class intervals were in the powers of 2 in months of the survival time, such as 0–2, >2–4, >4–8, >8–16, and so on. The values of M, mean of the log (survival time), and S, standard deviation of the log (survival time), were varied in the tests so that a minimum chi-square value was obtained. The null hypothesis states that there is no difference between the observed data distribution and the lognormal distribution. It is rejected if P < 0.05. The values of M and S were obtained when the chi-square value reached a minimum.
Let τ be the threshold year at which statistical cure rate can be estimated, then the cancer-specific survival rate can be obtained by Kaplan-Meier method with follow-up time equal to τ. This cancer-specific survival rate at time τ was an estimation of the statistical cure rate of the disease. It was compared with the long-term cancer-specific rate calculated using Kaplan-Meier method with the actual long-term data available up to 1999 (Figures 1 and 2).
Results
Lognormality
The present study verified that, for 42 specific organ sites out of 49 cancer sites in the SEER database, the survival time of cancer patients who died of their disease followed different lognormal distributions approximately. For the cancer sites with cancer-specific survival time following lognormal distribution, the number of patients and values of S, multiplicative standard deviation, M, median and P at minimum chi-square are listed in Table 1. All the P-values in Table 1 are above 0.10.
The seven specific organ sites failed in the test for lognormality were: lip, oropharynx, rectosigmoid junction, rectum, testis, urinary bladder, and kidney & renal pelvis.
Estimation of statistical cure rate
The threshold years validated for statistical cure were found to range from 2.6 years for pancreatic cancer to 25.2 years for cancer of salivary gland. For these 40 cancer sites with survival time followed a lognormal distribution, their cancer-specific survival rates were obtained by Kaplan-Meier method at the threshold year, and they were compared with their corresponding long-term survival rates with follow-up to 1999. Out of the 42 cancer sites with survival time followed a lognormal distribution approximately, the statistical cure rates for 40 cancer sites were found to match the actuarial long-term survival rates estimated by the Kaplan-Meier method within six percentage points, at threshold year. For the two remaining cancer sites: breast and thyroid, the values of their threshold years were so long that the cancer-specific survival rates could not be obtained because the SEER data up to 1999 do not provide sufficient long-term follow-up. Table 2 shows the comparison of the cancer-specific survival rates at τ year and at long-term follow-up.
Breast cancer was further analyzed according to accepted prognostic factors. The corresponding threshold years according to stages in the SEER classification: localized (age < 50), regional (age < 50) and distant were 30.8, 27.9, and 13.1 years respectively. For histologic types: medullary, ductal combined with adenocarcinoma not otherwise specified (NOS), and lobular, the threshold years were 17.0, 30.6, and 46.0 years respectively. The threshold years according to breast tumor grades were 39.8, 26.3, and 20.7 years for Grades I, II, and III+IV respectively. The breast cancer-specific survival rates at their threshold years were 9% for distant stage, 68% for medullary histology, and 40% for Grades III+IV breast cancer. These three survival rates were only one percentage point higher when compared with the available long-term actuarial survival rates. For regional stage and Grade II breast cancer, their threshold years were 27.9 and 26.3 respectively. It can be predicted that a few more years of follow-up are needed to see the plateau and their statistical cure rates are close to 39% and 47% respectively. The most recently available SEER database is now up to end of 2001. With 2 additional years of follow-up from the end of 1999, the cancer-specific survival rates were 39% and 44% respectively. For those with τ values longer than 27 years, the cancer-specific survival rates could not be obtained because the SEER data to date do not provide sufficiently long enough follow-up time.
Discussion
The cancer-specific survival has not included the deaths due to other causes. The cancer-specific rates also depend on the reliability of the assignment of the cause of death. Generally, cancer-specific death rates underestimate the mortality associated with a diagnosis of the specific cancer, because some patients died of other causes[26]. SEER is a set of geographically defined, population-based, central cancer registries in the United States, operated by local non-profit organizations under contract to the National Cancer Institute (NCI). Registry data are submitted electronically to the NCI on a biannual basis, and the NCI makes the data available for analysis. The SEER Program is considered the standard for quality among cancer registries around the world. Quality control has been an integral part of SEER since its inception. Every year, studies are conducted in SEER areas to evaluate the quality and completeness of the data being reported.
Gamel and Vogel [27] have compared the advantage of lognormal distribution over other distributions such as Weibull and log logit.
The 1973–1992 data were used so that the data were not out-dated. For a lognormal distribution of the survival time of those patients died of the specific cancer, there were only a very small proportion dying at the tail of follow-up (Figure 1), so it would not cause much change to the lognormal distribution, even with only 7 years of follow-up to 1999 at the tail. For those cancer sites with threshold years longer than 24 years, the 1973–1977 data were used so as to allow a long enough time for follow-up to 1999.
For 42 sites in the SEER database, the survival times of cancer patients who died of their disease followed different lognormal distributions. For 40 cancer sites, the ultimate cure rate can be roughly estimated from the cancer-specific survival rates at τ years. These are the required minimum number of follow-up years for the estimation of the cure rates. They are different for different cancer sites. For pancreatic cancer, with its typically short natural history, the cure rate can be estimated after only 2.6 years. For cancers with a longer natural history, longer follow-up periods are required; such as breast (36.2 years). These long periods are cancer-specific survival times and in reality patients may die from intercurrent non-cancer causes before τ years. For thyroid cancer, the estimated threshold year was 134.1 years. It seems that for some slow proliferating cancer types, the cure can never be estimated due to the limit of human lifetime.
We also find that the required minimum number of years of follow-up, τ, is independent of cure rate (correlation coefficient of determination, R2 = 0.10). Even for cancer sites where the cure rates were >50%, the required follow-up time τ could be less than 10 years. On the other hand, for other cancer sites, the cure rates could be < 50%, and the required follow-up time were >10 years. It shows that 5- or 10-year survival rates are inadequate to reflect the statistical cure rates.
If there are more patients dying due to other causes than dying due to the specific cancer, then the cause specific survival time distribution will not be lognormal. Hence it is not expected that all cancer-specific survival time distributions will follow lognormal distributions.
According to the bell-shaped property of a normal distribution, from 0 to τ year covers 97.75% of the lognormally distributed survival time of those cancer patients who died from their specific cancer. The cancer-specific survival rates estimated at τ years, generally, slightly overestimate the long-term cure rates compared to the Kaplan-Meier method, but the differences are reasonably small, by less than six percentage points as verified empirically. We still need to follow the patients to τ year to know the actual value of the estimated cure rates.
For both rapidly and slowly proliferating cancers, we have shown that the statistical cure rates can be estimated before a stable plateau is reached in the Kaplan-Meier survival curve. It may take decades to see a stable plateau, during this waiting time many patients might be lost to follow-up or die of intercurrent diseases.
Gamel and Vogel [28] used cause-specific survival and relative survival to determine actuarial survival in breast cancer patients from the SEER database. They found that there was only minimal deviation between the two survival methods.
Ries et al. [29] reported up to 20-year relative survival rates (RSR) from 9 registries of the SEER database. For cancers of pancreas, esophagus and stomach, the RSR were slightly decreased after five years since diagnosis. These are consistent with the threshold years of the present study varying from 2.6 years for pancreas, to 3.9 years for esophagus, and to 5.8 years for stomach. Dickman et al. [30] reported similar results with 10-year RSR for the Finnish Cancer Registry. Talback et al.[31] also showed the similar results on two RSR graphs up to 30 years for pancreas and stomach for the Swedish Cancer Registry.
The threshold year of statistical cure for ovarian cancer was 10.4 years. It was consistent with the results of Ries et al. and Dickman et al. up to 10 years. After 10 years, there was only a slight decrease in RSR as reported by Ries et al. Talback et al. showed the same results on a RSR graph for ovarian cancer. Their results were also consistent with the present study for the cancer sites of lung, colon and skin melanomas, which have threshold years of 9.0, 12.2 and 18.2 years respectively. These consistencies show that the results obtained from two SEER registries in the present study are similar to those from 9 registries and from the Finnish and Swedish Cancer Registries.
For prostate cancer, the threshold year for statistical cure was 24.6 years. The RSR graph started to level after 24 years since diagnosis in the article of Talback et al.
For breast cancer, the threshold year was 36.2 years, RSR leveling was not seen even 30 years after diagnosis in the article of Talback et al. Leveling of RSR in breast cancer was also not seen in two separate studies by Schairer et al. [32] and Brenner and Hakulinen [33]. Kerr et al. [34] reported that the ratio of observed to expected mortality remained significantly greater than unity for at least 25 years following diagnosis and treatment, indicating a failure to demonstrate cure of the disease in a statistical sense for a median of 32 years of follow-up.
Conclusion
The present study suggests a certain threshold year is required to wait before the statistical cure rate can be estimated for different cancer sites. Although the often used 5- or 10-year survival rates may adequately reflect statistical cure rates for cancers with short natural history, such as pancreatic cancer, this is not the case for many other cancers. This highlights the need for continued long-term cancer surveillance, especially for cancers with long natural histories such as thyroid cancer and early stage, well-differentiated breast cancer. This study is relevant for public health and cancer control. Whether knowledge of the threshold year will have any impact on decisions regarding therapy for cancers thought to have a good prognosis remains to be investigated.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
PT: Data analysis and writing of the manuscript.
EY, GCs, GV, MR, IK, VVH: Critical appraisal of the manuscript.
All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional File 1
Appendix
Click here for file
Acknowledgements
PT: Saskatchewan Cancer Agency Research Grant Award 2792.
GCs: János Bolyai Research Fellowship from the Hungarian Academy of Sciences.
Figures and Tables
Figure 1 The survival time for cancer patients who died of their disease. The single arrow shows the location of the threshold year (τ). This diagram uses stomach cancer data as an example.
Figure 2 Cancer-specific survival rate of cancer patients who died of their disease calculated by Kaplan-Meier method. Note the small difference between the cancer-specific survival rate at τ year as compared to the long-term survival rate. The single arrow shows the location of the threshold year (τ). The pair of opposing arrows at the long-term follow-up shows that the difference between the estimated and actual cancer-specific rate is small, less than six percentage points. The survival time for cancer patients who died of their disease for stomach cancer is shown as an example.
Table 1 List of cancer sites following lognormal distribution and their S (standard deviation of the log-survival), multiplicative standard deviation, M (mean of the log-survival), and median values at minimum chi-square with maximum P-value
Site S SD* M† Median‡ Pt. No. P-value
Pancreas 0.45 2.82 0.60 3.98 11185 0.57
Gallbladder 0.54 3.47 0.52 3.31 1196 0.60
Liver 0.62 4.17 0.41 2.57 1701 0.22
Esophagus 0.44 2.75 0.80 6.31 4915 0.35
Hypopharynx 0.41 2.57 0.96 9.12 298 0.57
Stomach 0.51 3.24 0.82 6.61 8608 0.13
Intrahepatic Bile Duct 0.61 4.07 0.63 4.27 105 0.50
Tonsil 0.46 2.88 1.02 10.47 137 0.70
Ureter 0.41 2.57 1.15 14.13 125 0.65
Tongue 0.47 2.95 1.05 11.22 684 0.44
Brain 0.64 4.37 0.74 5.50 2870 0.16
Lung & Bronchus (age<60) 0.63 4.27 0.78 6.03 18913 0.62
Floor of Mouth 0.42 2.63 1.20 15.85 126 0.95
Small Intestine 0.55 3.55 0.99 9.77 364 0.44
Ovary 0.48 3.02 1.13 13.49 4533 0.58
Vagina 0.49 3.09 1.13 13.49 108 0.30
Cecum 0.52 3.31 1.08 12.02 2702 0.16
Nasopharynx 0.46 2.88 1.19 15.49 151 0.90
Corpus Uteri 0.45 2.82 1.22 16.60 1446 0.73
Cervix Uteri 0.46 2.88 1.21 16.22 2256 0.72
Large Intestine, NOS 0.61 4.07 0.91 8.13 622 0.72
Splenic Flexure 0.48 3.02 1.19 15.49 506 0.43
Other Nervous System# 0.50 3.16 1.14 13.80 31 0.88
Colon (age<60) 0.45 2.82 1.26 18.20 2868 0.49
Hepatic Flexure 0.55 3.55 1.06 11.48 455 0.42
Penis 0.61 4.07 0.97 9.33 107 0.39
Uterus, NOS 0.62 4.17 0.96 9.12 81 0.40
Anus, Anal Canal & Anorectum 0.44 2.75 1.33 21.38 129 0.87
Nasal Cavity, Middle Ear & Accessory Sinuses 0.52 3.31 1.20 15.85 109 0.58
Soft Tissue (with heart) 0.50 3.16 1.25 17.78 445 0.52
Bones & Joints 0.54 3.47 1.21 16.22 208 0.78
Larynx 0.50 3.16 1.30 19.95 1752 0.43
Skin Melanomas 0.42 2.63 1.49 30.90 1744 0.24
Appendix 0.48 3.02 1.40 25.12 49 0.64
Vulva 0.62 4.17 1.15 14.13 235 0.11
Retroperitoneum 0.69 4.90 1.00 10.00 100 0.59
Pleura 0.75 5.62 0.90 7.94 47 0.47
Eye & Orbit 0.43 2.69 1.59 38.90 106 0.88
Prostate (age<60, 1973–77) 0.40 2.51 1.67 46.77 413 0.44
Salivary Gland (1973–77) 0.58 3.80 1.33 21.38 65 0.81
Breast (age<60, 1973–77) 0.47 2.95 1.71 51.29 4047 0.23
Thyroid (1973–77) 0.97 9.33 1.28 19.05 123 0.15
The order of organ sites follows Table 2 in ascending order of the threshold years;
SD* Mutiplicative standard deviation of the lognormal distribution;
† M values in log-months;
‡ Median in months;
Pt. No. denotes the number of patients died of the specific cancer;
# Nervous system other than brain.
Table 2 List of cancer sites following lognormal distribution in ascending order of τ (the threshold year for estimation of statistical cure rate)
Site τ (yr.) CSSR (τ) CSSR (LT) Diff. Pt. No.
Pancreas 2.6 7.1 3.3 3.8 14926
Gallbladder 3.3 24.6 20.1 4.5 2123
Liver 3.7 13.7 10.6 3.1 3244
Esophagus 3.9 14.5 9.9 4.6 7021
Hypopharynx 5.1 66.9 62.2 4.7 1869
Stomach 5.8 27.3 22.3 5.0 15310
Intrahepatic Bile Duct 5.9 23.7 20.3 3.4 327
Tonsil 7.4 82.5 79.8 2.7 1137
Ureter 7.8 86.2 85.3 0.9 1122
Tongue 8.0 61.0 56.1 4.9 2264
Brain 8.6 24.4 22.0 2.4 5019
Lung & Bronchus (age<60) 9.0 17.0 11.2 5.8 24408
Floor of Mouth 9.3 80.3 75.1 5.2 1430
Small Intestine 10.3 65.7 65.2 0.5 1633
Ovary 10.4 38.0 34.9 3.1 8400
Vagina 10.6 81.0 81.0 0 618
Cecum 10.8 53.4 48.5 4.9 6766
Nasopharynx 10.8 55.4 49.5 5.9 456
Corpus Uteri 11.1 99.0 98.3 0.7 14894
Cervix Uteri 11.2 69.7 68.2 1.6 8166
Large Intestine, NOS 11.2 41.5 37.8 3.7 1466
Splenic Flexure 11.6 49.5 47.2 2.3 1223
Other Nervous System# 11.7 85.8 83.6 2.1 293
Colon (age<60) 12.2 60.1 57.1 3.0 7556
Hepatic Flexure 12.3 52.0 49.2 2.8 1184
Penis 13.0 83.7 83.7 0 766
Uterus, NOS 13.4 70.5 70.5 0 423
Anus, Anal Canal & Anorectum 13.5 85.3 85.3 0 1254
Nasal Cavity, Middle Ear & Accessory Sinuses 14.6 65.2 65.2 0 449
Soft Tissue (with heart) 15.1 69.3 66.1 3.2 1938
Bones & Joints 16.2 63.8 63.3 0.5 728
Larynx 16.8 73.7 68.6 5.1 8899
Skin Melanomas 18.2 77.8 75.8 2.0 10905
Appendix 19.7 66.8 66.8 0 199
Vulva 20.2 84.5 83.6 0.7 1931
Retroperitoneum 20.2 74.3 74.3 0 681
Pleura 21.2 72.1 72.1 0 376
Eye & Orbit 23.8 84.4 82.7 1.7 641
Prostate (age<60, 1973–77) 24.6 36.7 33.9 2.8 809
Salivary Gland (1973–77) 25.2 73.5 73.5 0 309
Breast (age<60, 1973–77) 36.2 xxx 52.5 xxx 9549
Thyroid (1973–77) 134.1 xxx 88.7 xxx 1259
CSSR (τ) denotes cancer-specific survival rate at τ year in percent, calculated by Kaplan-Meier method;
CSSR (LT) denotes long-term cancer-specific survival rate in percent, calculated by Kaplan-Meier method up to 1999;
Diff. denotes difference between CSSR (τ) and LT CSSR in percentage point;
Pt. No. denotes number of all patients, alive or dead, with or without disease;
NOS denotes not otherwise specified;
# Nervous system other than brain;
xxx denotes data not available as at 1999.
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| 15904508 | PMC1164404 | CC BY | 2021-01-04 16:03:03 | no | BMC Cancer. 2005 May 17; 5:48 | utf-8 | BMC Cancer | 2,005 | 10.1186/1471-2407-5-48 | oa_comm |
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-531591345310.1186/1471-2407-5-53Research ArticleEfficacy of Wnt-1 monoclonal antibody in sarcoma cells Mikami Iwao [email protected] Liang [email protected] Biao [email protected] Zhidong [email protected] Sonny [email protected] Amie Y [email protected] Julien [email protected] Noemi [email protected] Kazutsugu [email protected] Kiyoshi [email protected] David M [email protected] Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California, San Francisco, CA 94115, USA2 Department of Surgery II, Nippon Medical School, Tokyo 113-8602, Japan2005 24 5 2005 5 53 53 16 8 2004 24 5 2005 Copyright © 2005 Mikami et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Sarcomas are one of the most refractory diseases among malignant tumors. More effective therapies based on an increased understanding of the molecular biology of sarcomas are needed as current forms of therapy remain inadequate. Recently, it has been reported that Wnt-1/β-catenin signaling inhibits apoptosis in several cancers. In this study, we investigated the efficacy of a monoclonal anti-Wnt-1 antibody in sarcoma cells.
Methods
We treated cell lines A-204, SJSA-1, and fresh primary cultures of lung metastasis of sarcoma with a monoclonal anti-Wnt-1 antibody. Wnt-1 siRNA treatment was carried out in A-204. We assessed cell death using Crystal Violet staining. Apoptosis induction was estimated by flow cytometry analysis (Annexin V and PI staining). Cell signaling changes were determined by western blotting analysis.
Results
We detected Wnt-1 expression in all tissue samples and cell lines. Significant apoptosis induction was found in monoclonal anti-Wnt-1 antibody treated cells compared to control monoclonal antibody treated cells (p < 0.02). Similarly, we observed increased apoptosis in Wnt-1 siRNA treated cells. Blockade of Wnt-1 signaling in both experiments was confirmed by analyzing intracellular levels of Dishevelled-3 and of cytosolic β-catenin. Furthermore, the monoclonal anti-Wnt-1 antibody also induced cell death in fresh primary cultures of metastatic sarcoma in which Wnt-1 signaling was active.
Conclusion
Our results indicate that Wnt-1 blockade by either monoclonal antibody or siRNA induces cell death in sarcoma cells. These data suggest that Wnt-1 may be a novel therapeutic target for the treatment of a subset of sarcoma cells in which Wnt-1/β-catenin signaling is active.
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Background
Sarcomas are highly malignant neoplasms that arise from mesenchymal tissues and the mechanism by which mesenchymal tissues undergo neoplastic transformation is largely unknown. Despite progress in the multidisciplinary treatment (surgery, chemotherapy, and radiation) of sarcomas, the results of these treatments in advanced disease remain unsatisfactory and the majority of these patients die from disseminated metastatic disease. Approximately 11,000 cases are diagnosed in the United States annually and 45 % of these patients will go on to die of their disease [1]. New therapies based on an improved molecular understanding of sarcomas are needed.
Recently, it has been reported that the Wnt pathway may be activated in several sarcomas [2-5]. Wnt signaling is essential for development and organogenesis [6,7]. It has been shown that the Wnt pathway is associated with tumor development and/or progression. We previously identified the overexpression of Dishevelled-3 (Dvl-3), a critical mediator of Wnt signaling, in non-small cell lung cancer and malignant pleural mesothelioma [8,9]. Wnt proteins, including Wnt-1, have been shown to be expressed in several cancers. We have developed a monoclonal anti-Wnt-1 antibody. In previous studies, we have demonstrated its efficacy in induction of apoptosis in human cancer cell lines and shown that the antibody lacks general toxicity in cells lacking the Wnt-1 protein [10-12]. This report suggests that the monoclonal anti-Wnt-1 antibody may also be efficacious in refractory sarcomas if Wnt-1/β-catenin signaling exists in these sarcomas. In the present study, we address this hypothesis and demonstrate a possible therapeutic role of this monoclonal anti-Wnt-1 antibody in the treatment of sarcoma cells.
Methods
Cell lines and tissue samples
Human sarcoma cell lines, A-204 (origin: muscle) and SJSA-1 (origin: bone) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). A-204 and SJSA-1 were cultured in McCoy's 5a medium and RPMI 1640 respectively, with 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 μg/ml). Both cells were cultured at 37°C in a humid incubator with 5% CO2. Fresh tissue samples of lung metastasis of sarcoma were obtained with consent from patients undergoing resection. They were cut into small pieces (1–2 mm in diameter), and digested with Collagenase A (Roche Applied Science, Indianapolis, Indiana) at room temperature for 2 hours according to manufacture's protocol. Single cells from the digestion were spun down and the cell pellets were washed twice using RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 IU/ml) and streptomycin (100 μg/ml). Then, the cells were resuspended in the same medium and cultured in 6-well plates at 37°C in a humid incubator with 5% CO2 until they were ready for treatments. Other fresh tissue samples of lung metastasis of sarcoma were immediately snap-frozen in liquid nitrogen. They were kept at -170°C in a liquid nitrogen freezer before use.
Western blotting
Whole cell lysates in tissue samples were obtained with T-Per Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Whole cell lysates in A-204 and SJSA-1 cell lines were obtained with M-Per Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Cytosolic proteins were prepared according to a previously described protocol [9]. The aliquots were separated on 4–15% gradient SDS-polyacrylamide gels and transferred to Immobilion-P (Millipore, Bedford, MA) membranes. Antigen-antibody complexes were detected by ECL blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ). The following primary antibodies were used: Wnt-1 (custom-made, Rockland Inc., Gilbertsville, PA); Dvl-1, Dvl-2, Dvl-3, Survivin (Santa Cruz Biotechnology, Santa Cruz, CA); β-catenin (Transduction Laboratories, Lexington, KY); and β-Actin (Sigma Chemical Co., St Louis, MO).
Antibody incubation with cells
The anti-Wnt-1 mouse monoclonal antibody (IgG1) was custom-made at Rockland Inc. (Gilbertsville, PA). The control monoclonal antibody (Sigma-Aldrich Co., St Louis, MO) used was the same subtype as the anti-Wnt-1 monoclonal antibody (IgG1). Cells were plated into six-well plates one day before experiments. Then normal medium was replaced by media containing antibodies at 10 μg/ml concentration and the cells were incubated at 37°C in a humid incubator with 5% CO2. Each experiment was performed at least 3 times.
Apoptosis analysis
Cells were harvested by trypsinization and stained using an Annexin V-FITC Apoptosis kit (BioSource, Camarillo, CA), according to the manufacturer's protocol. Stained cells were immediately analyzed by flow cytometry (FACScan; Becton Dickinson, Franklin Lake, NJ). Early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to Annexin V-FITC but excluded propidium iodide (PI). Cells in necrotic or late apoptotic stages were labeled with both Annexin V-FITV and PI. Experiments were performed in triplicate and a total of 20000 cells were analyzed in each individual experiment.
RNA interference
Cells were plated into a six-well plate with fresh media without antibiotics 24 hours before testing. The ion exchange high-performance liquid chromatography-purified siRNAs (Wnt-1 siRNA and nonsilencing siRNA control, >97% pure) were purchased from Qiagen-Xeragon (Germantown, MD). The siRNA-targeted human Wnt-1 is derived from a mRNA sequence (GGTTCCATCGAATCCTGCA) of human Wnt-1. The control (nonsilencing) siRNA does not target any known mammalian gene (the targeted sequence is AATTCTCCGAACGTGTCACGT). The lyophilized siRNA were dissolved in annealing buffer and reheated to 95°C for 1 minute followed by 1 hour at 37°C incubation. We followed the protocol described by Elbashir et al with some modifications [13]. After siRNA transfection, we incubated plates at 37°C before further analysis.
Statistical analysis
Data shown represent mean values (± S.D.). Student's t-test was used for comparing different treatments and cell line.
Results
Detection of Wnt-1/ β-catenin signaling in tissue samples of metastatic sarcoma and sarcoma cell lines
The expression of Wnt-1, Dvl-1, 2, 3 and cytosolic β-catenin was analyzed in 6 tissue samples of metastatic sarcoma, A-204 and SJSA-1 cell lines. The expression of Wnt-1, Dvl-3 and cytosolic β-catenin was found in all tissue samples and both cell lines (Figure 1). Dvl-1 and Dvl-2 were minimally expressed or not expressed in all tissue samples (data not shown). These results indicate that Wnt-1/β-catenin signaling may be activated in sarcoma cells.
The monoclonal anti-Wnt-1 antibody induces apoptosis in A-204 cell line
Next, the monoclonal anti-Wnt-1 antibody [10,11] was utilized for treating the sarcoma cell lines A-204 and SJSA-1. Cell death was found after 7 days of treatment with the monoclonal anti-Wnt-1 antibody (Figure 2A). By flow cytometry analysis, we found significant apoptosis induction in A-204 cell line (Figure 2B)(p < 0.02). In contrast, no noticeable effect was found after control monoclonal antibody treatment.
The monoclonal anti-Wnt-1 antibody inhibits Wnt-1/β-catenin signaling
The blockade of Wnt-1 signaling in A-204 and SJSA-1 cell lines was confirmed by analyzing the levels of Wnt-1 downstream mediators (Figure 2C). Dvl-3 and β-catenin protein levels decreased after the monoclonal anti Wnt-1 antibody treatment. Survivin, an inhibitor of apoptosis, was also downregulated.
RNA interference
Next, we investigated the effects of silencing Wnt-1 expression through RNA interference. Apoptosis induction by Wnt-1 siRNA was similar to that by anti-Wnt-1 monoclonal antibody (Figure 3A). Moreover, we confirmed the blockade of Wnt-1 signaling in A-204 cells after Wnt-1 siRNA treatment (nonsilencing siRNA as control) by western blot analysis (Figure 3B).
The monoclonal anti-Wnt-1 antibody induces cell death in fresh primary cultures of metastatic sarcoma
After confirmation of the efficacy of monoclonal anti-Wnt-1 antibody treatment in A-204 and SJSA-1 cells, we tested its ability to induce cell death in fresh primary cultures of metastatic osteosarcoma that has activated Wnt-1/β-catenin signaling (Cases 3 and 6). The cell death was found after 5 to 7 days of the antibody treatment, whereas no noticeable effect was found in these fresh samples after control monoclonal antibody treatment (Figure 4).
Discussion
Wnt signaling consists of an intracellular cascade that involves Frizzled, Dvl, glycogen synthase kinase-3β, β-catenin and T Cell Factor (TCF)/Lymphocyte Enhancer Binding Factor (LEF) [14,15]. It is thought that a component of cancer induction and progression results from elevated intracellular β-catenin levels [16]. It has been reported that Wnt signaling is associated with the progression of osteosarcoma [17]. Translocation of β-catenin to the cytoplasm and/or nucleus of osteosarcoma cells was detected in five resected cases of pulmonary metastasis, although seven primary osteosarcoma cells that did not metastasize for more than five years did not show β-catenin expression [18].
Wnt-1 was originally described as a proto-oncogene in mouse mammary tumor induced by mouse mammary tumor virus [19]. Chen et al. reported that Wnt-1 signaling inhibited apoptosis through β-catenin and that cells expressing Wnt-1 resisted apoptosis induction by chemotherapy [20]. Recently, it has been reported that a polyclonal anti-Wnt-1 antibody can downregulate β-catenin/TCF activity and induce apoptosis in head and neck squamous cell carcinoma [21]. In addition, we demonstrated that both a monoclonal anti-Wnt-1 antibody and Wnt-1 siRNA induced apoptosis in human cancer cells [10-12]. These reports strongly suggest that Wnt-1 signaling plays a critical role in the survival of malignant cells. We treated A-204, SJSA-1 cells and fresh primary cultures with the monoclonal anti-Wnt-1 antibody after confirmation of Wnt-1/β-catenin signaling in sarcoma cells. The antibody induced cell death in these sarcomas and decreased levels of Dvl-3 and β-catenin in A-204 and SJSA-1 cell lines. Furthermore, Wnt-1 siRNA induced similar effects. These results indicate that Wnt-1 signaling may play a critical role in the survival of a subset of sarcoma cells. However, much work remains in order to gain a clear understanding of the pathway.
Conclusion
Our results indicate that the monoclonal anti-Wnt-1 antibody induces cell death in sarcoma cells. These findings suggest that Wnt-1 inhibition may be of therapeutic interest in a subset of sarcomas in which Wnt-1/β-catenin signaling is active.
Competing interests
We have not received reimbursements, fees, funding, or salary for our work. We have licensed our Wnt antibody to a start-up company commercializing Wnt based therapeutics (David M. Jablons, Liang You, Zhidong Xu, and Biao He). Our monoclonal Wnt-1 antibody is pending patent.
Authors' contributions
IM carried out western blotting, cell staining, RNA interference and apoptosis analysis. LY, BH, ZX, KU, KK and DJ conceived of the study, and participated in its design and coordination. SB, AY, JM and NR drafted the manuscript.
All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was supported by the Larry Hall memorial trust and the Kazan, McClain, Edises, Abrams, Fernandez, and Lyons & Farrise Foundation.
Figures and Tables
Figure 1 Tissue samples of metastatic sarcoma and sarcoma cell lines express Wnt-1 signaling mediators. Cases 1–6: tissue samples (1,3 and 6;osteosarcoma, 2;synovical sarcoma, 4;liposarcoma, 5;leiomyosarcoma).
Figure 2 (A) 0.5% Crystal Violet staining of A-204 and SJSA-1 cell lines after the monoclonal anti-Wnt-1 antibody treatment (7 days after treatment). The concentration of both control and monoclonal anti-Wnt-1 antibodies used was 10 μg/ml. The monoclonal anti-Wnt-1 antibody induced cell death. (B) Apoptosis analysis using flow cytometry after the monoclonal anti-Wnt-1 antibody treatment (10 μg/ml for 6 days) in A-204 cell line. The results (percentage of apoptotic cells) are the mean ± SD (error bars). Significant apoptosis induction was observed in the monoclonal anti-Wnt-1 antibody treatment compared to the control monoclonal antibody treatment (p < 0.02). (C) Western analysis after the monoclonal anti-Wnt-1 antibody treatment (10 μg/ml for 6 days) in A-204 and SJSA-1 cell lines. β-Actin served as a loading control. Decreased levels of Dvl-3 and of cytosolic β-catenin confirmed the blockade of Wnt-1 signaling.
Figure 3 (A) Apoptosis and (B) expression of Wnt-1 signaling mediators analysis after Wnt-1 siRNA treatment (200 nM for 4 days) in A-204 cell line. The results were similar to that of the monoclonal anti-Wnt-1 antibody treatment.
Figure 4 0.5% Crystal Violet staining of fresh primary cultures of metastatic osteosarcoma after the monoclonal anti-Wnt-1 antibody treatment. The concentration of both control and monoclonal anti-Wnt-1 antibodies used was 10 μg/ml. The monoclonal anti-Wnt-1 antibody also induced cell death in these fresh samples.
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| 15913453 | PMC1164405 | CC BY | 2021-01-04 16:03:04 | no | BMC Cancer. 2005 May 24; 5:53 | utf-8 | BMC Cancer | 2,005 | 10.1186/1471-2407-5-53 | oa_comm |
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-581593510010.1186/1471-2407-5-58Research ArticlePreponderance of the oncogenic V599E and V599K mutations in B-raf kinase domain is enhanced in melanoma cutaneous/subcutaneous metastases Deichmann Martin [email protected] Marianne [email protected] Axel [email protected] Martin [email protected] Judith [email protected] Hjalmar [email protected] Department of Dermatology, Heidelberg University Clinics, Voßbstraße 2, 69115 Heidelberg, Germany2 Central Unit of Biostatistics, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany3 Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karls University of Heidelberg, Theodor-Kutzner-Ufer 1–3, 68135 Mannheim, Germany2005 3 6 2005 5 58 58 1 4 2004 3 6 2005 Copyright © 2005 Deichmann et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of mutations within the kinase domain including the activating V599E and V599K transitions.
Methods
We here investigated a representative series of 60 resection specimens of cutaneous and subcutaneous melanoma metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) gel electrophoresis.
Results
Sequencing of cloned PCR-SSCP amplicons resulted in 24 (40%) samples harbouring somatic mutations which is not exceeding the mutation frequency in recently investigated primary melanomas. The activating mutation T1796A was present in 24/60 (40%) resection specimens, followed in frequency by the oncogenic g1795A mutation in 8/60 (13%) cases. As to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 19/60 (32%) and 6/60 (10%) cases, resepectively, but were not associated with enhanced risk for subsequent metastasis in patients' follow up. In comparison to the primary melanomas that we recently investigated, the spectrum of predicted B-raf protein mutations narrowed significantly in the cutaneous/subcutaneous metastases. Unexpectedly, V599 and V599E mutations were absent in cutaneous/subcutaneous metastases derived from acrolentiginous melanomas as preceding primary tumours.
Conclusion
During transition from primary melanomas towards cutaneous/subcutaneous metastases, the spectrum of predicted B-raf mutations narrows significantly. Focusing on the V599E and V599K, these oncogenic mutations are likely to affect melanocyte-specific pathways controlling proliferation and differentiation.
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Background
Melanomas are one of the most aggressive of the skin cancers and have shown a dramatic increase in both incidence and mortality over the past decades [1]. Several genes implicated in the development of various malignancies, among them the tumour suppressor genes p53, CDKN2A and PTEN and the Ras oncogenes, have been extensively studied in melanoma and found to be rarely mutated in resection specimens [2]. Recently, the B-raf oncogene has been reported to be mutated, among other carcinomas, in a majority of melanoma cell lines [3,4]. Remarkably, all mutations were within the kinase domain, with a single amino acid substitution (V599E) accounting for 95% of B-raf mutations in melanoma cell lines [3,4] and leading to constitutive kinase activity [3]. In preliminary series of primary melanomas, 5/9 [3] and 4/5 [5] of the resection specimens harboured this V599E mutation, and only a single different mutation occurred [3]. Investigating a representative series of 50 resection specimens of primary cutaneous melanomas, we detected 12/50 cases (24%) to harbour this V599E transition [6].
As limited attention has been given to metastatic melanoma clinical specimens yet [7,8], we decided to analyse a representative number of resection specimens for the presence of mutations in the activation segment (exon 15) of the B-raf kinase domain in cutaneous/subcutaneous melanoma metastases. Applying polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) gel electrophoresis, followed by DNA cloning and sequencing, we here describe B-raf kinase domain mutations in a substantial subset of cutaneous/subcutaneous melanoma metastases, the oncogenic T1796A substitution being the most frequent followed by the neighboured g1795A transition.
Methods
Isolation of genomic DNA from paraffin embedded tissues
Total cellular DNA was extracted from paraffin embedded tissues of 60 melanoma cutaneous and subcutaneous metastases from 60 different patients from which melanoma primary tumours of the skin had been excised (previous primary tumours: 16 superficial spreading melanomas, 8 nodular melanomas, 8 acrolentiginous melanomas, 4 lentiginous melanoma, 18 melanomas not further classified, 4 no specification, 2 no primary melanoma). Using archival tissues for this study from previous patients' therapeutic surgery, there were no objections raised by the local ethics committee. DNA was isolated from both tumour and surrounding skin tissues by microdissection. Slides with 15 μm tissue sections were incubated in xylene for 30 min and in a series of 100%, 80%, 60%, 40% ethanol and in aqua, 10 s each, at room temperature. Tissue was scratched from the slides under microscope, put in tubes, and isolation of DNA was performed using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The quality of the isolated DNA was assessed by agarose gel electrophoresis.
Polymerase chain reaction (PCR)
Prior to PCR-SSCP analysis of B-raf exon 15 in melanomas, a 184 base pair (bp) fragment of the human A-myb gene was amplified by primers A-Myb-1 and A-Myb-2 to ensure the DNA integrity of the genomic DNA samples as well as the absence of major Taq polymerase inhibitors. PCR was performed in a final volume of 25 μl containing 0.6 units proofreading FailSafe Taq DNA polymerase and 12.5 μl FailSafe PCR 2X PreMix F (Biozym, Oldendorf, Germany), containing 100 mM Tris-HCl pH 8.3, 100 mM KCl, 400 μM of each dNTP, supplemented by 0.25 μl of each primer (100 pmol/μl). PCR conditions were: 95°C 2 min, 46 cycles of 95°C 30 sec, 58°C 30 sec, 72°C 30 sec, followed by 72°C 7 min. All PCRs were performed on a GeneAmp 2400 thermal cycler (Perkin Elmer, Norwalk, CT, USA). PCR products were analysed by 1.5% agarose gel electrophoresis.
To screen for mutations in the activation segment (exon 15) of the B-raf kinase domain, 100 ng of each melanoma DNA sample were PCR amplified by primers B-raf-6998-for and B-raf-70221-rev in 25 μl PCRs as described above. 5 μl aliquots of each reaction were electrophoresed on 1.5% agarose/ethidiumbromide gels using highly resolving NuSieve 3:1 agarose (Biozym). Positive controls were generated by a primer-mediated PCR mutagenesis-based protocol as described (Ruiz et al, 1997). Here, we incorporated a third longer primer, B-raf-70009-A-for or B-raf-70006-T-for, into the PCR along with the two wild-type primers B-raf-6998-for and B-raf-70221-rev. Longer mismatched primers (LMP) share the sequence of the wild-type primers but also contain additional bases, which include a mismatched base. Primer sequences according to the database of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA, ) were: A-Myb-1, 5'-CAT ggA ATg CCA ATT TAA Cg-3'; A-Myb-2, 5'-CAT CCC TAA gTT CgC TgC C-3' (gene database accession number X66087); B-raf-6998-for, 5'-ggC CAA AAA TTT AAT Cag Tgg A-3' (identical with primer sequence exon-15-rev according to [3]); B-raf-70221-rev, 5'-TCA TAA TgC TTg CTC TgA TAg gA-3' (identical with primer sequence exon-15-for according to [3]; gene database accession number NM_004333); B-raf-70009-A-for, 5'-ggC CAA AAA TTA AAT Cag Tgg A-3'; B-raf-70006-T-for, 5'-ggC CAA AAT TTT AAT Cag Tgg A-3'.
SSCP gel electrophoresis
The amplified DNA was mixed with an equal volume of formamide loading dye (94% formamide, 0.05% xylene cyanol and 0.05% bromphenol blue), denatured at 95°C for 5 min, chilled on ice for 1 min and loaded onto Gene Excel 12.5/24 polyacrylamide gels (Pharmacia Biotech, Freiburg, Germany). Following non-denaturing electrophoresis at 600 Volt, 25 mA, 15 W, 6°C for 80 min, DNA fragments were stained by silver using the DNA silver staining kit according to the manufacturer's protocol (Pharmacia Biotech). DNA fragments that reproducibly showed mobility shifts according to independent two-repeated SSCP analyses were cut out from the acrylamide gel and subjected to semi-nested PCRs performed with primers B-raf-70023-for and B-raf-70221-rev using the conditions described above. Sequence of primer B-raf-70023-for was: 5'-ATA gCC TCA ATT CTT ACC ATC C-3'.
Cloning of PCR amplicons and sequencing of plasmid inserts
Nested PCR products were then purified using the QIAquick PCR purification kit (QIAGEN) and cloned into pCRR2.1-TOPO vector (Invitrogen, NV Leek, Netherlands) according to the manufacturer's instructions. Briefly, 10 ng of the secondary PCR product were ligated into 10 ng vector and the ligation mixture was introduced into competent TOP10 bacteria by heat shock. The library was plated onto LB plates containing 50 μg/ml ampicillin. Single bacterial transformants that appeared positive in blue/white screening were picked randomly and grown in 2 ml LB medium containing 50 μg/ml ampicillin at 37°C overnight. Following alkaline lysis of bacterial cultures neutralized lysates were loaded onto silica-gel membranes (QIAprep spin miniprep kit, Qiagen) and plasmid DNAs were eluted in low-salt buffer.
Plasmid inserts were sequenced at a concentration of 50 ng/μl with a GeneAmp PCR system 9600 using ABI Prism dGTP BigDye Terminator Ready Reaction Kits and the AmpliTaq DNA polymerase FS according to the manufacturer's protocol (Perkin Elmer; Seqlab, Göttingen, Germany). For sequencing B-raf exon 15 fragments from both sides, M13 forward and M13 reverse primers were used. PCRs consisted of 25 cycles including a denaturation step at 96°C for 10 sec, a primer annealing step at 50°C for 5 sec and a chain elongation step at 60°C for 60 sec. Cycle sequencing products were then ethanol precipitated, run on a 4% polyacrylamide 7 M urea gel and analysed with the ABI Prism 377 Genetic Analyser (Perkin-Elmer; Seqlab). Primer sequences were: M13 forward primer, 5'-CAA AAg ggT CAg TgC Tg-3'; M13 reverse primer, 5'-gTC CTT TgT CgA TAC Tg-3'. The resulting sequences were aligned to the known B-raf sequence in the NCBI database (accession number NM_004333). The numbering began with the start codon ATG, corresponding to nucleotide positions 1–3. B-raf protein sequences predicted by the cDNA sequences were compared with B-raf wild type protein sequence (NCBI accession number NP_004324) using the BLASTX software at the NCBI.
Statistical Analysis
Fisher's exact test was used to assess clinical data and histologic characteristics of the preceding primary melanomas as possible prognostic factors for the occurance of B-raf exon 15 mutations and to compare the results in cutaneous/subcutaneous melanoma metastases with data very recently reported from primary melanoma resection specimens [6]. The exact Wilcoxon rank sum test [10] was applied to test the hypothesis of B-raf exon 15 mutations being associated with tumour thickness according to Breslow in the preceding primary melanomas. Length of time between excision of primary melanomas and the occurance of cutaneous/subcutaneous metastases which we investigated was compared with length of time between excision of the cutaneous/subcutaneous metastases that we analysed and subsequent metastases by the exact Wilcoxon signed rank test. For all statistical analyses the statistical software package R, version 1.7 [11], was used. All statistical tests were 2-sided. An effect was considered statistically significant at P < 5%.
Results
Pattern of B-raf exon 15 mutations
Assessing the presence of amplifyable DNA by PCR amplification of a 184 bp human A-myb gene fragment, 60 resection specimens of melanoma cutaneous/subcutaneous metastases were positive for both cancerous and matched noncancerous tissues. Positive results in A-myb PCR were in accordance with agarose gel electrophoresis showing the integrity of the high molecular weight DNA. These melanoma resections specimens were then analysed by PCR and SSCP gel electrophoresis for mutations in exon 15 of the B-raf gene. 24/60 (40%) cases exhibited SSCP patterns distinctly different obtained from corresponding adjacent normal tissues (Figure 1). The total cellular DNA samples amplified with elongated primers that harboured mutations were identified as positive controls in all PCR-SSCP analyses (data not shown).
Sequencing of cloned PCR-SSCP amplicons resulted in 24 positive melanoma samples harbouring a total of 44 DNA point mutations of B-raf exon 15 (Table 1). The activating mutation T1796A [3] was present in 24 (40%) of the 60 investigated melanoma metastases (Figure 2), followed in frequency by the g1795A mutation in 8 cases (13%). Altogether, 16 different B-raf exon 15 mutations (T1749C, g1757A, C1758A, T1760C, T1779C, T1787A, A1794T, g1795A, T1796A, T1803g, A1810g, A1823g, g1824A, A1830C, T1837C, T1847C) were detected in the 60 investigated cutaneous melanoma resection specimens. In all melanomas, the mutations in neoplastic tissues were shown to be somatic by cloning and sequencing of SSCP gel bands from the corresponding normal tissues.
It is remarkable that CC to TT or C to T transitions, which occur in the p53 gene in non-melanoma skin cancers following exposure to ultraviolet light [12-15] were not detected in any of the investigated melanoma resection specimens.
Predicted changes of the B-raf protein sequence
Next, the B-raf protein sequences predicted by the nucleotide sequences that we found were compared with the known B-raf protein sequence (NCBI accession number NP_004324) using the BLASTX software. All melanomas harbouring B-raf point mutations were predicted to exhibit alterations of the protein sequence (Table 1). Within the kinase domain, the oncogenic V599E amino acid substitution occurred in 19 of the 60 tested melanomas (32%), followed in frequency by V599K in 6/60 (10%) cases, both protein mutations occurring in two separate DNA fragments from melanoma case 46 (Table 1). Valine at protein sequence position 599 was replaced in all (100%) of the melanoma resection specimens positive for B-raf exon 15 mutations. No nonsense mutation of B-raf exon 15 DNA, causing a predicted protein truncation, occurred in our analysis.
Altogether, 9 different B-raf exon 15 protein mutations (D586N, L587P, F594L, V599E, V599K, S604G, E610D, S613P, I616T) were detected in the 60 investigated cutaneous/subcutaneous melanoma resection specimens. In comparison to the 16 different protein mutations very recently described in 19/50 B-raf mutations bearing primary melanomas [6], a narrowing of the spectrum of predicted B-raf protein mutations is observed during transition from primary melanomas towards cutaneous/subcutaneous metastases (p = 0.04 upon Fisher's exact test; estimated odds ratio 2.64, 95% confidence interval 0.97–7.63).
Applying Fishers's exact test, Clark's level of the primary melanoma (p = 0.46 and p = 0.55, respectively), level of UV irradiation of the preceding primary tumours (localization of the primary melanomas with chronic, intermittent or none UV exposition, p = 0.72 and p = 0.54, respectively) and type of subsequent metastasis (none, cutaneous, lymph node, visceral; p = 0.52 and p = 0.75, respectively) were no statistically significant prognostic factors for the occurence of valine substitution at B-raf amino acid position 599 (V599) or for the detection of the oncogenic V599E mutation, respectively. The occurence of valine substitution at B-raf amino acid position 599 (V599) or the detection of the V599E mutation were not associated with a change in the distribution of tumour thickness according to Breslow of the prededing primary melanomas (exact Wilcoxon rank sum test: p = 0.40 and p = 0.46, respectively). Unexpectedly, the histologic diagnosis of acrolentiginous melanoma in the preceding primary tumour (Fishers's exact test: p = 0.02 and 0.04, respectively) was associated with the absence of V599 and V599E in the cutaneous/subcutaneous metastases.
B-raf mutations and clinical outcome
56 of the 60 patients were seen in follow up examinations. Following cutaneous/subcutaneous metastases, subsequent metastasis occurred in 55 patients including 11 cutaneous, 9 lymph node and 35 visceral decays (lung 15, bone 7, liver 6, brain 6, kidney 1). The median follow up period for all patients was 17 months (lower quartile 5.75 months; upper quartile 25 months).
Addressing the 11 patients with only subsequent cutaneous metastasis and the single patient which remained free of disease for 50 months (case 18), the follow up period of this subgroup was longer with a median of 22 months (lower quartile 17.25 months; upper quartile 25.25 months). The time between excision of primary melanomas and the occurance of cutaneous/subcutaneous metastases which we investigated (median 51 months; lower quartile 20 months; upper quartile 111.5 months) was longer in comparison to the length of time between excision of the cutaneous/subcutaneous metastases that we analysed and subsequent metastasis during follow up (exact Wilcoxon signed rank test: p < 0.001 ; estimated median difference 49 months with 95% confidence interval ranging from 29 months to 70.5 months). Following a long period free of disease after excision of the primary tumour, cutaneous/subcutaneous metastasis may therefore be regarded as a first indicator of melanoma progression, shortly followed by subsequent visceral metastasis in most patients.
Looking at the 24 cutaneous/subcutaneous melanoma metastases harbouring the oncogenic V599E or V599K amino acid substitutions, 21 patients were seen in follow up examinations and all of them developed subsequent metastasis. In comparison, 35/36 patients with B-raf exon 15 wild type metastases were monitored in follow up examinations from which 34 developed further metastasis. A single patient (melanoma metastasis case 18) remained free of tumour in a follow up interval of 50 months.
Comparing mutations of B-raf exon 15 in cutaneous/subcutaneous metastases with those in primary melanomas [6] by Fisher's exact test, no statistically significant differences in the proportion of mutations in general (p = 0.85; odds ratio = 0.92, 95% confidence interval 0.39–2.13), T1796A (p = 0.56; odds ratio = 0.77, 95% confidence interval 0.33–1.81), V599 (p = 0.56; odds ratio = 0.77, 95% confidence interval 0.33–1.81), V599E (p = 0.40; odds ratio = 0.68, 95% confidence interval 0.26–1.72) and V599K (p = 0.77; odds ratio = 1.22, 95% confidence interval 0.30–4.94) could be observed.
Discussion
Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. One important pathway mediating cellular responses to growth signals is the RAS-RAF-mitogen-activated protein (MAP)-kinase kinase (MEK)-extracellular signal-regulated kinase (ERK)-MAP kinase cascade whose activation can be achieved by mutation at various levels [16]. RAS is mutated to an oncogenic form in about 15% of human cancer. In a mouse model null for the tumour suppressor INK4a, melanoma initiation and maintanance were dependent upon expression of H-RasV12G [17]. Still, H-Ras as well as K-ras mutations rarely occur in human cutaneous melanomas [18,19] and are even less frequent in comparison to N-Ras which is mutated in less than 15% of uncultured melanoma tissue specimens [19-22]. Neither quantitative nor qualitative alterations of ras-p21 expression were found to correlate with tumour progression [20]. Downstream of Ras and upstream of MEK lies B-raf, which is highly present in neural cells and tissues [23-25]. This kinase belongs to the Raf family of serine/threonine kinases regulated by binding RAS which is composed of the ubiquitously expressed Raf-1 and by A-Raf and B-raf [16]. RAF proteins phosphorylate MEK1/2, which in turn phosphorylate ERK1/2. Very recently, B-raf somatic missense mutations have been reported in 59% of 34 melanoma cell lines [3]. All mutations were within the kinase domain, with a single substitution (V599E) accounting for 95% of all B-raf mutations in the melanoma cell lines.
In preliminary series of primary melanomas, 5/9 [3] and 4/5 [5] of the specimens harboured this V599E mutation, and only a single different mutation occurred in the B-raf oncogene outside of exon 15 [3,7]. Investigating a representative series of 50 resection specimens of primary cutaneous melanomas, we recently detected 12/50 cases (24%) to harbour the V599E transition independent from risk for further metastasis [6]. In contrast to cutaneous melanoma, V599E was detected in none of 46 familial melanomas [26], 21 multiple melanomas [26], 29 [27] and 48 [28] uveal melanomas. In 80 melanoma families, V599E was no germline mutation [29].
Addressing melanoma metastases, the V599E transition has recently been reported for 83% of 12 [5] and 50% of 28 [7] lymph node and in 64% of 12 [5] and 52% of 27 [7] visceral melanoma metastases. Looking at cutaneous/subcutaneous metastases, 63% of 29 cutaneous/subcutaenous cases were reported to harbour this oncogenic mutation [5]. These reports are in line with our data, the mutation rate in melanoma metastases similar to primary melanomas [6] suggesting V599E and V599K as early events in melanocyte transformation, possibly contributing to proliferation and survival, but not to metastatic spread. This postulate is supported by the finding of B-raf exon 15 mutations in a major subset of nevi [5,30].
As a novel finding, we here describe the spectrum of predicted B-raf protein mutations narrowing significantly during transition from primary melanomas towards cutaneous/subcutaneous metastases. Focusing on V599E and V599K in cutaneous/subcutaneous metastases, these oncogenic transitions are nonetheless not associated with enhanced risk for subsequent further metastasis. As the B-rafV599E mutant possesses tenfold greater basal kinase activity and induces focus formation in NIH3T3 cells 138 times more efficiently than does wild-type B-raf [3], relevance of this alteration in the development of melanomas is very likely. For the growth of cancer cell lines with the V599E mutation, RAS function was not required any more [3]. The V599E mutation is thought to mimic phosphorylation of threonine 598 and serine 601 within the activation loop of B-raf at the plasma membrane, resulting in a protein with high activity and leading to constitutive ERK activation [31].
Whereas the missense mutation T1796A alone predicts the activating amino acid substitution V599E within the B-raf kinase domain, T1796A together with the g1795A nucleotide transition predict another acidic amino acid substitution, namely V599K with transforming activity in NIH3T3 cells comparable to V599E transfectants [32]. As a novel finding, we here describe the V599K mutation in 10% (6/60) of the tested cutaneous/subcutaneous metastases which is identical to the recently reported frequency of 10% (5/50) in primary melanomas [6]. In contrast to our data, V599K has been detected by other investigators in noticeable lower frequencies of 1% (1/77; [7]) and 3% (2/60; [30]) of melanoma metastases which may be due to higher sensitivity of the PCR-SSCP approach that we applied when compared to direct sequencing of tumour DNA samples.
As we found mutations within the activation segment (exon 15) of the B-raf kinase domain in a total of 24 (40%) of all investigated metastatic resection specimens which unexceptionally affected valine at protein sequence position 599, this protein sequence position seems to be a hot spot for alterations. The high frequency of B-raf mutations in melanomas may be related to a principal melanocyte-specific signalling pathway controlling proliferation and differentiation: α-melanocyte stimulating-hormone (α-MSH) and proopiomelanocortin-derived peptides, secreted by keratinocytes, bind to the melanocortin receptor I on melanocytes, leading to increased proliferation and melanogenesis in response to UVB radiation [33]. This signalling cascade via stimulation through G-protein coupled receptors (GPCRs) and upregulation of cyclic AMP (cAMP) does not require RAS but also activates B-raf and subsequently ERK [34]. When activated, extracellular signal-regulated kinases (ERKs) translocate to the nucleus where they regulate gene expression, leading to cell proliferation. The activation of ERKs by cAMP has been reported in a limited number of cell systems, including B16 melanoma [35].
Endothelin-1 (Et-1), a strong melanocyte mitogen [36,37], is another candidate for signaling through B-raf. Et-1, produced by keratinocytes and accentuated by exposition of keratinocytes to UVB radiation, can activate the MAP kinase pathway, although the role of B-raf in transducing this signal has not been demonstrated in melanocytes [38]. Besides α-MSH and Et-1, the RAS-RAF-MEK-MAP kinase cascade is also activated by the growth factors basic fibroblast growth factor [39] and stem cell factor [40], leading to increased proliferation of cultured human melanocytes.
That principal melanocyte-specific signalling pathways controlling proliferation and differentiation operate through activation of B-raf possibly explains the high frequency of B-raf mutations in melanomas when compared to colon (18% of 40 cell lines and 12% of 33 tumours) or ovarian cancers (4% of 26 cell lines and 14% of 35 tumours, according to [3]), an exception being papillary thyroid carcinomas with 69% (24/35 cases positive, [41]) and 36% (28/78 cases positive, [42]) of the investigated cases, respectively. The attempt of inhibiting B-raf activity in melanoma has been inspired by the demonstration that the ERK1/2 inhibitor U0126 [43,44] decreases proliferation of melanoma cell lines bearing B-raf mutations [3].
Conclusion
Altogether, the serine/threonine kinase B-raf, involved in the Ras-Raf-MEK-ERK-MAP kinase pathway of signal transduction, harbours mutations of it's kinase domain in 40% of 60 melanoma metastatic resection specimens which is not exceeding the mutation frequency in primary melanomas [6] and which is not associated with enhanced risk for subsequent further metastasis. The oncogenic V599E and V599K transitions account for 79% and 25% of these positive metastases, respectively, one melanoma resection specimen (case 46) bearing both mutations. Acrolentiginous melanoma as preceding primary tumour was associated with the absence of V599 and V599E. During transition from primary melanomas towards cutaneous/subcutaneous metastases, the spectrum of predicted B-raf protein mutations narrows significantly. The B-raf mutations that we detect in uncultured primary melanoma samples are distinct from C to T or CC to TT mutations associated with pyrimidine dimer formation following UV radiation [12,13]. As B-raf alterations possibly affect melanocyte-specific pathways controlling proliferation and differentiation, inhibition of B-raf activity may be a future strategy in the treatment of melanoma as is currently addressed in phase I and II studies [45].
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Martin Deichmann designed the study and wrote the manuscript. Martin Deichmann and Marianne Thome carried out the PCR-SSCP analyses. Hjalmar Kurzen collected the melanomas and participated in DNA extraction. Axel Benner did the statistical analyses. Michael Kirschner and Judith Hassanzadeh participated in DNA cloning and DNA and protein sequence comparisons. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Figures and Tables
Figure 1 PCR-SSCP analysis of B-raf exon 15 in resection specimens from cutaneous/subcutaneous melanoma metastases. Cases 7, 23, and 54 exhibit different DNA fragment mobilities when compared to the respective normal tissues from patients. Suspecting these tumour specimens to harbour mutations, these samples were subjected to DNA sequencing.
Figure 2 Part of the DNA sequence of the activation segment of the B-raf kinase domain. Representative melanoma resection specimens are shown which harbour missense mutations. In comparison to wild type DNA sequence (upper electropherogram, forward sequence on the left, reverse sequence on the right), melanoma cases 7, 23 and 54 exhibit nucleotide transitions, among them the activating T1796A mutation.
Table 1 B-raf exon 15 mutations in resection specimens of melanoma cutaneous/subcutaneous metastases. Predicted protein sequence alterations are listed together with the clinical course of the disease in patients' follow up. Distinct mutations in two different abnormal bands in SSCP gels are specified by two lines for one melanoma.
Case Previous primary (months before) DNA mutations Predicted Protein changes Subsequent Metastasis (months later)
1 MM-2.7-IV (30 m) T1796A V599E LN (1 m)
g1757A D586N
7 NM-2.5-IV (11 m) T1796A V599E liver (simult)
A1810g S604G
12 SSM (18) T1796A V599E liver (74 m)
13 NM-2.1-V (50) T1796A V599E liver (2 m)
20 NM-4-IV (51) T1796A V599E none (74 m)
T1837C S613P
22 MM (19) T1796A V599E sc (7 m)
23 MM-1.9-III (37) g1795A, T1796A V599K lung, liver (simult)
24 MM-2.5-IV (84) T1796A V599E bone (simult)
28 no data g1795A, T1796A V599K no follow up
C1758A, T1760C, g1795A, T1796A V599K, L587P
29 NM-3.6-IV (13) g1795A, T1796A V599K liver (3 m)
32 no data g1795A, T1796A V599K no follow up
g1795A, T1796A, T1803G V599K
34 no data T1779C, T1796A F594L, V599E no follow up
37 SSM-1.8-IV (120) T1796A V599E sc (2 m)
T1796A, A1823g V599E
41 MM (144) T1796A V599E lung (simult)
43 SSM-1.8-IV (21) T1796A V599E lung (3 m)
44 MM (71) T1796A V599E lung (simult)
46 LMM-2.7-IV (53) T1796A V599E bone (20 m)
g1795A, T1796A V599K
47 MM-1.8-IV (126) T1796A V599E none (42 m)
T1787A, T1796A V599E
50 SSM-0.6-III (157) T1749C, T1796A V599E brain, bone (simult)
G1824A WT
53 no primary T1796A V599E bone (simult)
T1796A, T1847C V599E, I616T
54 LMM-III (290) T1796A V599E sc (8 m)
T1796A, A1794T V599E
55 MM-10-V (5) T1796A, A1830C V599E, E610D lung (22 m)
56 MM (1) T1796A V599E lung (1 m)
58 LMM-1.2-V (6) g1795A, T1796A V599K sc (12 m)
Abbreviations used in Table 1: A, adenine; C, cytosine; D, asparagin acid; E, glutamic acid; F, phenylalanine; G, glycine; g, guanine; I, isoleucine; K, lysine; L, leucine; LMM, lentiginous malignant melanoma; LN, lymph node; m, month(s); MM-2.7-IV, melanoma, not further classified, tumour thickness according to Breslow 2.7 mm, Clark's level IV; N, asparagine; NM, nodular melanoma; P, proline; S, serine; sc, subcutaneous; simult, simultaneous; SSM, superficial spreading melanoma; T, thymine; T1857A, change from thymine to adenine at nucleotide 1857 (according to NCBI accession number NM_004333, the start codon ATG corresponding to nucleotide positions 1–3); V, valine; V599E, substitution of valine to glutamic acid at codon 599 (according to NCBI accession number NP_004324.1); WT, wild type of protein sequence.
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| 15935100 | PMC1164406 | CC BY | 2021-01-04 16:03:04 | no | BMC Cancer. 2005 Jun 3; 5:58 | utf-8 | BMC Cancer | 2,005 | 10.1186/1471-2407-5-58 | oa_comm |
==== Front
BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-601594904510.1186/1471-2407-5-60Research ArticleDetermination of caspase-3 activation fails to predict chemosensitivity in primary acute myeloid leukemia blasts Staib Peter [email protected] Jan [email protected] Timo [email protected]öthe Timo [email protected] Clinic I for Internal Medicine, The University Hospital of Cologne, Kerpener Str. 62, 50924 Cologne, Germany2005 11 6 2005 5 60 60 25 2 2005 11 6 2005 Copyright © 2005 Staib et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Ex-vivo chemosensitivity tests that measure cell death induction may predict treatment outcome and, therefore, represent a powerful instrument for clinical decision making in cancer therapy. Such tests are, however, work intensive and, in the case of the DiSC-assay, require at least four days. Induction of apoptosis is the mode of action of anticancer drugs and should, therefore, result in the induction of caspase activation in cells targeted by anticancer therapy.
Methods
To determine, whether caspase activation can predict the chemosensitivity, we investigated enzyme activation of caspase-3, a key executioner caspase and correlated these data with chemosensitivity profiles of acute myeloid leukemia (AML) blasts.
Results
There was, however, no correlation between the ex-vivo chemosensitivity assessed by measuring the overall rates of cell death by use of the DiSC-assay and caspase-3 activation.
Conclusion
Thus, despite a significant reduction of duration of the assay from four to one day, induction of apoptosis evaluated by capase-3 activity does not seem to be a valid surrogate marker for chemosensitivity.
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Background
Cytogenetic abnormalities, age, initial leucocyte count and P-glycoprotein expression are examples of widely accepted prognostic factors in acute myeloid leukemia [1-5]. However, the prognosis of an individual patient may still not accurately be determined by these factors. The ex-vivo chemosensitivity profile may help to predict the individual response to therapy and, more importantly, to individualize the treatment and to identify new agents [6-8].
Many ex-vivo chemosensitivity assays have been investigated for over 40 years. The clonogenic assay is a long term assay but technical drawbacks and the long culturing time have limited the clinical use of the method [9,10]. Most assays used today are short term total cell kill assays, however, the methods to determine viable cells after incubation with cytotoxic agents vary greatly between direct evaluation of the cells and, in the vast majority, indirect evaluation using different surrogate markers. The differential staining cytotoxicity assay (DiSC) uses microscopic evaluation of dye exclusion in viable cells [11,12]. In the MTT assay, surviving cells reduce the methyl-thiazol-tetrazolium (MTT) into highly colored formazan which can be quantified by spectrophotometry [13-15]. Other assays have used fluorescein diacetate (FMCA) or cellular ATP content as a surrogate marker of cell viability [16,17]. All these assays mentioned above are based on essentially the same culturing procedures in terms of incubating fresh leukemic cells with cytotoxic agents over a period of four to five days. A shorter duration of assays, however, may be useful to obtain ex-vivo chemosensitivity profiles prior the onset of treatment and would allow to individualize the therapy in most patients.
It is generally accepted that cytotoxic drugs eliminate malignant cells by inducing apoptosis (programmed cell death) [18,19]. This process starts, as it was shown in hematopoietic tumor cell lines, within hours of exposure to cytotoxic drugs [20]. Thus, quantification of a surrogate marker of apoptosis might be a suitable method for a fast ex-vivo chemosensitivity assay. The appearance of phospholipids like phosphatidylserine at the cell surface is a common marker for apoptosis which can be detected by Annexin V using flow cytometry [21,22]. Apoptosis may also be quantified by changes of the mitochondrial electrochemical transmembrane potential using DiOC6 or JC-1 [21,23], by detection of DNA- respectively nuclear-fragmentation using the TdT-assay [23], gel-electrophoresis or acridine orange [21,24-26]. Further apoptotic markers are the cleaved forms of poly (ADP-ribose) polymerase (PARP) and caspase-3 which both are detectable by specific antibodies [24,25,27-29].
Most of these methods allow some grade of automation and a reasonable number of samples to be processed, which is needed for the clinical use of an ex-vivo chemosensitivity assay as well as for the identification of new agents. A development allowing more automation and larger sample sizes might be the detection of activated caspase-3, the main effector caspase in the apoptotic enzyme cascade, by using fluorogenic substrates and detection of enzyme activation in microplate readers [30]. To evaluate the usefulness and accuracy of such a procedure, we investigated the ex-vivo chemosensitivity assessed by measuring caspase-3 activation and, also, compared these data to results obtained by DiSC assay in AML blasts.
Methods
Patient samples
Bone marrow (BM) or peripheral blood (PB) were taken from adult patients with acute myeloid leukemia (AML) with informed consent during routine diagnostic BM aspiration and/or phlebotomy before treatment was started. Samples were collected in heparinized tubes from patients treated at our institution.
Cell lines
For establishing the caspase-3 assay the AML cell lines THP-1, HEL and KG-1 were used.
Drugs
Drugs were dissolved according to manufacturers' instructions and diluted in RPMI 1640 culture medium (Gibco, Paisley, UK). In order to induce cytotoxicity levels ex vivo comparable with those induced in vivo, the drugs were used at concentrations encompassing the range of clinically relevant plasma levels, as previously reported [31]. Cytarabine (Ara-cell™, cell-pharm, Hannover, Germany) was used at final concentrations in the range from 0.06 μg/ml to 5 μg/ml in the DiSC-Assay and from 0.06 to 20 μg/ml in the caspase-3 assay. Daunorubicin (Daunoblastin™, Pharmacia, Erlangen, Germany) was tested at final concentrations ranging from 0.008 μg/ml to 2 μg/ml and from 0.008 μg/ml to 5 μg/ml, mitoxantrone (Novantron™, Lederle, Münster, Germany) from 0.006 μg/ml to 0.5 μg/ml and from 0.006 μg/ml to 20 μg/ml in the DiSC-assay and the caspase-3 assay, respectively. The drugs were tested in duplicate samples at 5 different concentrations in the DiSC-Assay and in triplicate samples at six different concentrations in the caspase-3 assay.
Differential Staining Cytotoxicity (DiSC)-Assay
The DiSC-assay was performed as previously described with minor modifications [11,32-34]. After density gradient isolation 105 fresh leukemic cells were resuspended in RPMI 1640 medium (Life Technologies, Germany) containing 10% heat-inactivated fetal calf serum (Biochrom, Germany) and penicillin-streptomycin (Life Technologies, Germany). Cells were incubated either without (control samples) or with added cytotoxic drug in a total volume of 200 μl (37°C, humidified 5% CO2) for 96 hours. After the incubation 1% fast-green and 0.5% nigrosin (Sigma Aldrich Chemie, Taufkirchen, Germany), including fixed duck erythrocytes as an internal standard, were added to each tube. Cells were transferred to collagen surfaced microscope slides by cytocentrifugation, air-dried and counterstained with May-Grünwald-Giemsa stain (Sigma Aldrich Chemie). Subsequent evaluation of slides by light microscopy facilitated the determination of drug efficacy at each drug concentration compared with controls.
Caspase-3 activity assay
For measuring the activation of the caspase-3 we used the fluorometric assay TIA (Test for Induced Apoptosis) Product No. CDC_101 Evotec technologies, subsequently called Casp3-test (Evotec Analytical Systems, Erkrath, Germany). This assay employs the peptide substrate DEVD-AMC. 105 ficoll isolated mononuclear cells were incubated without (control samples) or with added cytotoxic drug in a total volume of 100 μl (37°C, humidified 5% CO2) for 16 hours. Afterwards, cell lysis was performed by adding 10 μl of lysis-solution (Evotec) and 90 μl lysis buffer (Evotec) to each well and incubating the suspension for one hour at room temperature in the dark on a shaker. For fluorometric detection of caspase-3 activity 1% (v/v) substrate DEVD covalently linked to the fluorogenic dye (7-amino-4-methyl coumarin, AMC) was added. Upon cleavage by caspase-3 free dye as an indicator for cumulated caspase-3 activity was detected using a fluorescence microplate reader (SPECTRAFluor, Tecan, Grödig/Salzburg, Austria) with a 360 nm excitation and a 465 nm emission filter for 30 to 60 minutes.
Data analysis
For the DiSC assay logistic curves were fitted to the cell count survival data; the logit of survival probability was taken to be linear with respect to the logarithm of drug concentration [8,34,35]. LC90-doses, that is, the concentration of drug to produce a 90% reduction in cell survival compared with control cells, were determined by calculating the log dose at which the fitted survival probability was equal to 0.1. As LC90-results are log normal, all LC90-values were logged (base 10) before calculation of median, ranges of concentrations and statistical tests.
Established analyzing procedures for ex-vivo chemosensitivity assays assume that the minimum and the maximum drug-effects are defined. In case of enzymes like the caspases the in-vivo maximum activity in live cells cannot be reliably obtained. Therefore, an analyzing procedure to describe the relationship between drug concentration and caspase activation had to be developed for the Casp3-test and is presented as part of the results. For comparison between DiSC assay and Casp3-test results the Spearman's regression was applied.
Results
Patient characteristics
In this study, we examined 42 AML-specimens. Patient characteristics are shown in Table 1. The diagnosis and classification of AML was confirmed by central review of bone marrow slides, at our institution patients were treated according to treatment protocols of the AML-Cooperative Group (AMLCG, Germany).
Evaluation of caspase-3 assay (Casp3-test)
Before using the Casp3-test for the assessment of the ex-vivo drug sensitivity an analyzing procedure had to be developed. With increasing drug concentrations we observed an increasing fluorescence signal following an S-shaped curve for the caspase-3 activity. This dependence of fluorescence upon the concentration of a cytotoxic drug with the appearance of a saturation behavior corresponds to kinetics described by the Michaelis-Menten equation. The base-level of fluorescence was integrated into the Michaelis-Menten equation by adding a constant "CutOff " as follows:
V = CutOff + Vmax*[S]/([S]+Km)
with V being the gradient of fluorescence, Vmax being the maximum gradient of fluorescence, [S] the drug concentration, CutOff the base-level of fluorescence and Km being the Michaelis constant describing the drug concentration at half maximum rate of reaction (Figure 1). The Km served as a measure for the cytotoxic efficacy of the chemotherapeutic agents.
For defining an optimal incubation period to allow time for all drugs studied to exert their apoptotic effect and, also, to find stable caspase-3 activity rates we performed time course experiments. The AML cell lines HEL, THP-1 and KG-1 were incubated with daunorubicin (0.03 – 5 μg/ml) and cytarabine (0.19 – 20 μg/ml). The incubation was stopped after every two hours up to 24 hours. Plotting the fluorescence signal in dependence upon incubation time, maximum values were observed at different time points corresponding to different drug concentrations. Usually, the highest drug concentrations achieved their maximum of relative fluorescence (compared to controls) after 8 to 12 hours of incubation in contrast to lower concentrations with 12 to 14 hours. Generally, higher fluorescence levels were obtained by higher drug concentrations.
Stability of Km
The Km as a marker for the cytotoxic efficacy of a specific drug was determined according to the Michaelis-equation as described above. Stable Km-values were achieved during incubation with daunorubicin within 14 hours and with cytarabine within 12 hours (Figure 2). In order to have reliably stable rates of apoptosis we chose an incubation time of 16 hours.
Ex-vivo chemosensitivity by the DiSC-assay
In 37 of 42 AML samples (88%) the DiSC-assay was successfully performed. In 5 cases the assay failed due to bacterial contamination. All chemotherapeutic drugs tested – ara-C, daunorubicin and mitoxantrone – induced cell death in a dose-dependent manner (Figure 1). The sensitivities of the AML cells expressed as LC90s showed variability between the patients from as much as 22-fold below (for ara-C) to 105-fold above (also for ara-C) the median value (Table 2). For each drug, a range of sensitivities was observed, ranging from very sensitive to very resistant [11,36]. These individual patient drug sensitivity profiles reflect the patient heterogeneity seen in the clinic [37]. If more than 90% of the tumor cells were already killed by the lowest drug concentration, the LC90-value was set equal to the corresponding lowest drug concentration [13]. This was observed in seven cases after incubation with cytarabine.
Ex-vivo chemosensitivity by the Casp3-test
The Casp3-test was successful in all 42 AML samples in terms of measurable caspase activity. But as far as the various cytotoxic drugs – ara-C, daunorubicin and mitoxantrone – are concerned apoptosis could be demonstrated in a dose-dependent manner only in a subset of 116 of 126 tests (92%) (Figure 1). The sensitivities of the AML blasts expressed as Km showed extreme variability between the patients from as much as 40-fold below (for daunorubicin) to 6,3 × 108-fold above (for ara-C) the median value (Table 2). In 14 cases Km-values were less than the lowest concentration of ara-C used in the assay, hence the Km-values was set equal to the lowest ara-C concentration tested in these cases.
Comparison between DiSC-assay and Casp3-test
Both assays were simultaneously performed on each of the 42 AML samples. Correlation between the two tests was possible for cytarabine in 33 cases, for daunorubicin in 36 cases and for mitoxantrone in 33 cases.
For each drug tested the Km-values by the Casp3-test showed an extreme variability as compared to the LC90-values by the DiSC-assay, although the median values were quite similar (Table 2). LC90-values did not correlate with corresponding Km-values of cytarabine (R2 = 0.38, p = 0.491), daunorubicin (R2 = 0.001, p = 0.964) or mitoxantrone (R2 = 0.005, p = 0.544) according to the regression after Spearman-Rho. The associations of LC90-values with Km-values of the three drugs are shown in Figure 3.
Discussion
The major goal of this study was to determine whether the ex-vivo drug resistance in fresh leukemic cells from patients with AML is reliably assessed by a short-time apoptosis-assay that measures caspase-3 activation. To this end, we compared the Casp3-test with established methods like the differential staining cytotoxicity (DiSC)-assay that measures reliably the overall rate of cell death induced by anticancer drugs [6,8,11,34,37,38]. The cytotoxic drugs cytarabine, daunorubicin and mitoxantrone, commonly used in the treatment of AML were tested in both assays.
In the Casp3-test, the activation of proteases that recognize the DEVD peptide motif (DEVD-ases), especially caspase-3 was detected by measuring a fluorescence signal generated by a fluorogenic dye upon cleavage by caspase-3. It has been repeatedly demonstrated that apoptosis usually is the dominant mode of tumor cell death promoted by chemotherapy [19,39,40], and caspase-3 is a major effector protease in this process [41-44]. Our results indicate, however, that levels of caspase-3 activation, i.e. DEVD-ases, do not correlate with sensitivity or resistance to commonly employed anticancer drugs in AML blasts. Even though apoptosis is the main pathway for drug-induced cell death caspase-3 activities fail to predict the response to chemotherapy as determined by measuring ex-vivo chemosensitivity by the DiSC-assay. There was no association between LC90-values (DiSC-assay) and corresponding Km-values (Casp3-test) of cytarabine (p = 0.491), daunorubicin (p = 0.964) or mitoxantrone (p = 0.544).
The DiSC-assay measures total cell death and, therefore, integrates apoptotic and non-apoptotic death induced by chemotherapy. Moreover, the DiSC-assay was shown to identify response to therapy and also long-term survival in various hematological neoplastic diseases [8,35]. This assay has been extensively evaluated in B-CLL, AML and, also, ALL [8,11,36-38,45]. In a large series of AML patients, we have recently shown an overall predictive accuracy for the DiSC-assay of 98.2% concerning treatment response and proofed the ex-vivo chemosensitivity evaluated by the DiSC-assay as one of the strongest prognostic factors [34]. For the DiSC-assay, it has also been demonstrated that it runs more reliable than colony-forming assays that measure clonogenic growth and that results obtained by DiSC-assay are equivalent to both, the colony-forming assay and the colorimetric MTT-assay [32,36]. Based on this background, the DiSC-assay serves as an established standard method to evaluate the individual ex-vivo chemosensitivity and, also, as an appropriate reference method to evaluate and establish new ex-vivo chemosensitivity assays. Furthermore, the DiSC-assay has also been used to study various parameters influencing the apoptotic process [46,47].
Therefore, due to the lack of correlation between the Casp3-test and the DiSC-assay it must be concluded that the Casp3-test is not equivalent to the DiSC-assay and, consequently, not appropriate for the assessment of the individual ex-vivo chemosensitivity and the identification of new agents. This observation may be due to the test-system itself, but it seems more likely that it is due to the methodology of the Casp3-test to assess chemosensitivity.
Most established ex-vivo chemosensitivity assays measure total apoptotic cell death after all drugs tested have exerted their apoptotic effect. In contrast, determination of caspase-3 activation only reflects the apoptosis at a single time. However, total apoptotic cell death is the result of all apoptotic processes which occur during the whole incubation time of 96 h in the DiSC assay allowing enough time for all drugs studied to exert their apoptotic effect. It becomes clear that the intensity of apoptosis at one time point does not necessarily represent the intensity of all apoptotic processes. Moreover, anticancer drugs may also induce caspase-independent cell death and this could be neglected when only caspase-3 activities are determined [26]. It might be argued that 16 hours of incubation with cytotoxic agents in fresh leukemic cells may be too short for the evaluation of caspase-3 activity, but in all cell line experiments Km-values were found stabilized within 14 hours. Additionally, Km-values could be calculated in 92% of the Casp3-tests done on patient material, and the median Km-values were comparable with median LC90-values of the DiSC-assay implicating an adequate incubation period.
Conclusion
In conclusion, the use of a caspase-3 activity test for the assessment of the ex-vivo chemosensitity does not appear to be suitable for the prediction of drug responses. Therefore, established tests like the DiSC-assay or the MTT-assay are still the best choice for predicting chemosensitivities of cancer cells.
Competing interests
The company Evotec Analytical Systems, Erkrath, Germany provided funding to perform this study at our institution. The authors declare that they are not related to the company Evotec Analytical Systems in any way and that they have no financial or non-financial competing interests. Furthermore, the authors declare that the description of the results in this manuscript was not influenced by the company and that no information was withheld.
Authors' contributions
PS conceived of the study, and participated in its design and coordination and drafted and revised extensively the manuscript. JT performed the caspase-3 activity tests and its interpretation and drafted the manuscript. TST performed the DiSC-assays and its interpretation. TS participated in the design of the study and performed the statistical analysis and helped to draft the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was supported by a grant from Evotec Analytical Systems, Erkrath, Germany. We thank Dana Chrobok for her skillful technical assistance. We gratefully acknowledge Volker Diehl, MD, PhD, for encouraging this work to be done at the time he was chief of the department. We are also grateful to Michael Hallek, MD, PhD, chief of the department, for helpful suggestions and editorial assistance with the manuscript.
Figures and Tables
Figure 1 Example of a typical dose-response curve received by the Casp3-test (a) and the DiSC-assay (b) in leukemic cells from a patient with AML after incubation with daunorubicin. FU = Fluorescence Units, TCS = tumor cell survival.
Figure 2 Average Km-values of daunorubicin and incubation time in the cell lines HEL, THP-1 and KG-1.
Figure 3 Distribution of the LC90- (DiSC-assay) and Km-values (Casp3-test) for the tested drugs a) cytarabine (N = 33), b) daunorubicin (N = 36), and c) mitoxantrone (N = 33).
Table 1 Patient characteristics
Patients diagnosed with AML, no 42
Age [years]: median (range) 45 (19 – 73)
Female/male ratio, no 16/26
Successful DiSC-assays, no (%) 37/42 (88%)
Successful Casp3-tests, no (%) 42/42 (100%)
Successful single drug tests, no (%) 116/126 (92%)
DiSC-assay / Casp3-test correlation, no (%) 37/42 (88%)
Correlation between single drugs, no (%) 94/126 (75%)
FAB-groups No
M1 3
M2 12
M3 4
M4 10
M5 5
M6 1
Secondary AML 7
Table 2 Median LC90- (DiSC-assay) and Km-values (Casp3-test) for cytarabine, daunorubicin and mitoxantrone (range).
Drug LC90 (μg/ml) Km (μg/ml)
Cytarabine 1.40 (0.06 – 147.44) 0.270 (0.062 - >20)
Daunorubicin 0.63 (0.2 – 1.59) 0.443 (0.011 - >5)
Mitoxantrone 0.235 (0.02 – 3.34) 0.222 (0.007 - >20)
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| 15949045 | PMC1164407 | CC BY | 2021-01-04 16:03:04 | no | BMC Cancer. 2005 Jun 11; 5:60 | utf-8 | BMC Cancer | 2,005 | 10.1186/1471-2407-5-60 | oa_comm |
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BMC Cardiovasc DisordBMC Cardiovascular Disorders1471-2261BioMed Central London 1471-2261-5-111592707710.1186/1471-2261-5-11Research ArticleAntibiotics for the primary prevention of acute rheumatic fever: a meta-analysis Robertson Katharine A [email protected] Jimmy A [email protected] Bongani M [email protected] Primary Health Care Directorate, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa2 The Cardiac Clinic, Department of Medicine, Groote Schuur Hospital and University of Cape Town, Cape Town, South Africa2005 31 5 2005 5 11 11 22 8 2004 31 5 2005 Copyright © 2005 Robertson et al; licensee BioMed Central Ltd.2005Robertson et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Rheumatic fever continues to put a significant burden on the health of low socio-economic populations in low and middle-income countries despite the near disappearance of the disease in the developed world over the past century. Antibiotics have long been thought of as an effective method for preventing the onset of acute rheumatic fever following a Group-A streptococcal (GAS) throat infection; however, their use has not been widely adopted in developing countries for the treatment of sore throats. We have used the tools of systematic review and meta-analysis to quantify the effectiveness of antibiotic treatment for sore throat, with symptoms suggestive of group A streptococcal (GAS) infection, for the primary prevention of acute rheumatic fever.
Methods
Trials were identified through a systematic search of titles and abstracts found in the Cochrane Central Register of Controlled Trials (Cochrane Library Issue 4, 2003), MEDLINE (1966–2003), EMBASE (1966–2003), and the reference lists of identified studies. The selection criteria included randomised or quasi-randomised controlled trials comparing the effectiveness of antibiotics versus no antibiotics for the prevention of rheumatic fever in patients presenting with a sore throat, with or without confirmation of GAS infection, and no history of rheumatic fever.
Results
Ten trials (n = 7665) were eligible for inclusion in this review. The methodological quality of the studies, in general, was poor. All of the included trials were conducted during the period of 1950 and 1961 and in 8 of the 10 trials the study population consisted of young adult males living on United States military bases. Fixed effects, meta-analysis revealed an overall protective effect for the use of antibiotics against acute rheumatic fever of 70% (RR = 0.32; 95% CI = 0.21–0.48). The absolute risk reduction was 1.67% with an NNT of 53. When meta-analysis was restricted to include only trials evaluating penicillin, a protective effect of 80% was found (Fixed effect RR = 0.20, 95% CI = 0.11–0.36) with an NNT of 60. The marginal cost of preventing one case of rheumatic fever by a single intramuscular injection of penicillin is approximately US$46 in South Africa.
Conclusion
Antibiotics appear to be effective in reducing the incidence of acute rheumatic fever following an episode of suspected GAS pharyngitis. This effect may be achieved at relatively low cost if a single intramuscular penicillin injection is administered.
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Background
Rheumatic fever is the most common cause of acquired heart disease in children and adults worldwide [1,2]. Acute rheumatic fever is expressed as an inflammatory reaction that involves many organs, primarily the heart, the joints, and the central nervous system [3]. The clinical manifestations of acute rheumatic fever follow a group A streptococcal (GAS) infection of the tonsillopharynx after a latent period of approximately 3 weeks. The major importance of acute rheumatic fever is its ability to cause fibrosis of heart valves, leading to crippling hemodynamics of chronic heart disease, heart failure and death. Open heart surgery may be needed to repair or replace heart valves in patients with severely damaged valves, the cost of which is exorbitant and a drain on the limited health resources of poor countries [4,5].
The global burden of disease caused by rheumatic fever currently falls disproportionately on children living in the developing world. Rheumatic heart valve disease causes 400,000 deaths annually mainly among children and young adults living in developing countries. At least 12 million people are estimated to be currently affected by rheumatic heart disease with two million patients requiring repeated hospitalisation and one million requiring, often unaffordable, heart surgery in the next 5 to 20 years [6]. In many developing countries, the incidence of acute rheumatic fever approaches or exceeds 100 per 100,000, whereas the incidence is currently estimated at less than 2 per 100,000 in the US.[1,2] This decline of rheumatic fever in the industrialised world has been partially attributed to the use of antibiotics for the treatment of GAS infections [7], but it is also believed to be the result of improved living conditions [8]. However, the recent resurgence of rheumatic fever in middle-class families in some parts of the economically developed world is a reminder that even in industrialised countries, there is no room for complacency [9,10].
The bulk of the research on the prevention of rheumatic fever dates back to the mid-20th century and was conducted primarily on adult populations living in the United States [11]. Methods for preventing rheumatic heart disease have included both primary and secondary prevention strategies. Primary prevention is achieved by disrupting the initial transmission of GAS infection or by blocking the progression of GAS infection to rheumatic fever. Secondary prevention is used following an attack of acute rheumatic fever to prevent the progression to cardiac disease. The consensus reached in the 1950's was that the most effective and efficient method for preventing rheumatic heart disease was through the primary prevention of acute rheumatic fever using antibiotics to treat the preceding GAS infection (WHO 1954) [12]. Where primary prevention failed, a secondary prevention plan also relying on antibiotic therapy was recommended for preventing the progression of cardiovascular complications [13].
Treatment of streptococcal pharyngitis with antibiotics is currently standard practice in most of the developed world. To note some exceptions, there are a few countries that now recommend no investigation or treatment of sore throat based on recent fears of increasing antibiotic resistance compounded with the findings of a recent study highlighting the negligible benefits gained from antibiotic treatment [14]. This new rationale for treating sore throat with antibiotics may not be applicable to a developing country setting where there remains a real threat of rheumatic fever. Antibiotic treatment of streptococcal pharyngitis appears to be an effective health intervention that is simple to administer; however, its benefits have not been realised in much of the developing world [1].
Controversy exists over the priority that the primary prevention of rheumatic fever deserves in the competition for scarce resources for healthcare in developing countries [1]. In this context it has been argued that secondary rather than primary prevention should be the mainstay of community-based approaches to the control of rheumatic fever and rheumatic heart disease [1]. Unfortunately, these discussions take place in an information vacuum, since the benefits of primary prevention of rheumatic fever, to the best of our knowledge, have not been quantified in a systematic fashion. This review aims to summarise the evidence on the effectiveness of antibiotics for the prevention of acute rheumatic fever, and seeks to determine to what extent the introduction of primary prevention programmes for rheumatic fever is supported by existing research.
Methods
Study search
We systematically searched the Cochrane Central Register of Controlled Trials (Cochrane Library Issue 4, 2003), MEDLINE (1966–2003), EMBASE (1966–2003), and the reference lists of identified studies. Search terms included: sore throat, pharyngitis, rheumatic, streptococcal pharyngitis, strep throat, antibiotics, and tonsillitis. We reviewed the selected titles and abstracts (when available) to identify which studies were trials. There were no language restrictions.
Inclusion criteria
We included only randomised and quasi-randomised controlled trials on a patient population presenting with a sore throat (pharyngitis) with or without confirmation of GAS infection by a throat culture and/or a rapid test, and no history of rheumatic fever. Trials that did not specify the method of randomisation and for which the authors were not available for follow-up were excluded.
Trials were required to use the Jones Criteria for the diagnosis of rheumatic fever. A positive diagnosis required the presence of 2 major criteria [carditis, migrating polyarthritis of the big joints, chorea, erythema marginatum, subcutaneous nodules] or 1 major and 2 minor criteria [fever, arthralgia without arthritis, previous history of RF or RHD, prolonged P-R interval, elevated erythrocyte sedimentation rate, positive C-reactive protein, leucocytosis] [15].
Acceptable interventions included any antibiotic versus placebo or no treatment. Acceptable trials included the incidence of a first attack of acute rheumatic fever following throat infection as an outcome. Secondary outcomes that were noted but not required included adverse events to antibiotic use, adherence to antibiotic therapy and mortality associated with the first attack of rheumatic fever.
Data extraction
Using a standardized data form, two reviewers (KR, BM) independently extracted data from each eligible trial. The methodological quality of the trials was assessed based on the method of randomisation, concealment of allocation, blinding and loss to follow-up. Additional data extracted from each trial included information on the study setting (i.e.: hospital vs. community-based trials, geographic location), on the participants (age, presenting complaint), and on the details of the interventions.
Data analysis
The results from each study were summarised using relative risk and a 95% confidence interval (CI). The chi-squared test for heterogeneity was used, along with the visual inspection of graphs to determine the level of between study variation. As the p-value associated with this test was large (>0.10), indicating low-levels of statistical heterogeneity between study results, a fixed-effect model was used to pool the data to estimate an overall effect. A subgroup analysis included only studies where penicillin was used as the intervention.
Results
Study search
Seventy seven potentially eligible studies were identified by the preliminary search. Based on their titles and abstracts (when available), 41 studies were retrieved for a thorough evaluation, performed independently by two reviewers (KAR, BMM) (Fig. 1). Discrepancies in the selection of studies were resolved through discussion. The reviewers were not blinded to the source, institution or results of the studies.
Figure 1 The QUORUM statement.
Thirty one of 41 studies that were retrieved in full-text were excluded for the following reasons: rheumatic fever not included as an outcome (11), not trials (5), no randomisation or method of randomisation not described (5), trial on secondary prevention of rheumatic fever (3), control group not used (3), retrospective study (2), patients included with a history of rheumatic fever (1), non-human subjects used (1).
Characteristics of included studies
Ten studies were included in the review (Table 1) [16-25]. The studies were all hospital-based, 8 out of 10 of which were conducted at U.S. military hospitals from 1950 to 1957 [16,18-24]. Only 3 of the studies used placebos for the control group [20,21,23]. The remainder used either no treatment or symptomatic treatment as controls. Patients in the US-based studies comprised males, aged 17 years and older [16,18-24]. One trial included children aged 3–16 [25], and another involved adults and children [17]. We sought only studies on patients with no previous history of rheumatic fever. While 3 of the 10 included studies reported some patients with a rheumatic fever history [16,19,22], the rates were below the margin of error (5.0%) and therefore negligible. All 10 studies made the attempt to limit the study participants to those with suspected GAS infection, a diagnosis that was based on the observance of exudate on the tonsils or pharynx in 9 out of 10 studies. The use of this single criterion for the diagnosis of GAS infection was supported by a study estimating that 70–90% of patients admitted to army hospitals around the same time period with streptococcal tonsillitis or pharyngitis presented with exudate on their tonsils or oropharynx [26].
Table 1 Characteristics of included trials
Study ID Randomisation and concealment of treatment allocation Participants Intervention Effect size % Randomised included in analysis
Bennike, 1951 Quasi-randomised; Inadequate concealment of treatment 349 admitted to hospital with ordinary acute tonsillitis, plegmonous tonsillitis or ulcerative tonsillitis 1. Penicillin: IM, 300,000 units/day for 6 days (adults)
2. Control – symptomatic treatment Not Estimable 88%
Brink, 1951 Quasi-randomised; No concealment of treatment 475 males, aged 17–21, admitted to U.S. military hospital with respiratory symptoms or fever with exudate on tonsils or pharyngeal mucosa 1. Procaine penicillin G: IM 300,000 units/day for 4 days
2. Aureomycin: avg. 2 g/day orally for 4 days
3. Control – no treatment RR = 0.29 [0.06,1.46] Unknown
Brock, 1953 Randomised; No concealment of treatment 349 males admitted to U.S. military hospital with exudative pharyngitis and laboratory-confirmed GAS infection 1. Procaine penicillin G: IM 600,000 units/day for 3 days
2. Control: IM saline placebo, day 1 and day 5 RR = 0.11 [0.00,2.71] Unknown
Brumfitt, 1957 Quasi-randomised; No concealment of treatment 121 males, aged 18–21, admitted to U.S. military hospital with sore throat, pyrexia and no clinical evidence of more generalized disease of which sore throat may have been coincident feature 1. Combination of procaine penicillin G: IM 600,000 units/day for 4 days and crystalline penicillin: IM 200,000 units/day for 4 days
2. Control – symptomatic treatment Not estimable Unknown
Chamovitz, 1954 Quasi-randomised; Unknown whether treatment allocation concealed 241 males admitted to U.S. military hospital with exudative tonsillitis or pharyngitis 1. DBED penicillin: IM 1,200,000 units
2. Control – IM placebo RR = 0.17 [0.01,3.41] Unknown
Denny, 1950 Quasi-randomised; No concealment of treatment 1602 males admitted to U.S. military hospital with respiratory symptoms and observed exudate on the tonsils or pharyngeal wall 1. Penicillin G: IM 200,000 units/day for 3 days of 300,000 units/day for 4 days
2. Control: symptomatic treatment RR = 0.12 [0.03,0.50] 81.8%
Denny, 1953 Randomised; Unknown whether treatment allocation concealed 207 males admitted to U.S. military hospital with suspected streptococcal infection based on presence of exudate on tonsils or pharynx and total leukocyte count exceeding 10,000 1. Crystalline procaine penicillin: IM 600,000 units/day for 5 days
2. Crystalline aureomycin: avg. 2 g/day for 5 days
3. Crystalline terramycin: avg. 2 g/day for 5 days
4. Control: oral lactose placebo for 5 days RR = 0.64 [0.06,6.92] Unknown
Houser, 1953 Quasi-randomised; No concealment of treatment 2044 males, ages 17–21, admitted to U.S. military hospital with exudative lesions on their tonsils or pharynx 1. Aureomycin: avg. 2 g/day for avg. 5 days
2. Control: no specific treatment RR = 0.63 [0.34,1.17] 88%
Siegel, 1961 Quasi-randomised; No concealment of treatment 1213 children, aged 3–16, with uncomplicated acute upper-respiratory tract disease and laboratory-confirmed GAS infection 1. Benzathine penicillin G: IM 600,000 units
2. Control: symptomatic treatment RR = 0.20 [0.01,4.14] 95%
Wannamaker, 1951 Quasi-randomised; No concealment of treatment 2340 males, aged 17–20, admitted to U.S. military hospital with respiratory symptoms and exudative lesions on the tonsils or oropharynx, or oral temp. > 100°F 1. Procaine penicillin G: IM various dosages (1,200,000 over 4 days; 600,000 units over 3 days; 600,000 single dose)
2. Control: no specific treatment RR = 0.21 [0.09,0.47] 83.3%
All 10 studies conducted appropriately timed follow-up visits at 3–4 weeks following the initial onset of symptoms given the 3-week delay in the onset of clinical signs of rheumatic fever. Adherence to therapy was not measured in any of the studies, as all of the studies included were hospital-based and antibiotics were not self-administered. Approximately half of the studies mentioned side effects as an outcome; however, most were not quantified. Symptoms mentioned were pain at the site of injection, nausea and diarrhea.
The methodological quality of the studies, in general, was poor. Nine out of the ten studies reviewed date back to the 1950's when randomised trial methods were still evolving and good guidelines for conducting trials were not yet available [16-24]. Regarding randomisation and allocation concealment, two trials used shuffled cards and were considered to be truly randomised [20,21]. The remaining studies were categorized as 'quasi-randomised' as allocation was based on methods such as Air Force Serial number, and date of hospital admission. As an "open" system of randomisation was used in all trials, allocation concealment was deemed inadequate in all cases. Three out of 10 studies were placebo-controlled [20,22,23], however, this did not ensure blinding of patients and providers as the placebo used was not identical in appearance to the experimental treatment. Four studies employed blinded assessors during follow-up for the diagnosis of rheumatic fever [16,18,19,22], 2 did not specify whether the assessor was blinded [24,25], and 1 used no blinding [17]. Five out of 10 studies reported on loss to follow-up [16,17,19,22,25]. Data from these trials on the number of patients that were randomised which could be included in the final analysis ranges from 81.8% to 95%.
Quantitative data synthesis
1. Incidence of rheumatic fever
Antibiotics versus control
All 10 studies evaluated the effectiveness of antibiotics in preventing acute rheumatic fever (n = 7665). Three thousand nine hundred and ninety six (3996) received antibiotic treatment and 3669 received either placebo or no specific treatment. As no statistical heterogeneity was present (p = 0.28) we pooled the results of the individual studies using the fixed-effect model. Meta-analysis revealed a substantial protective effect against the onset of acute rheumatic fever in patients treated with antibiotics (RR = 0.32; 95% CI = 0.21–0.48) (Fig. 2). Twenty nine of 3996 (0.73%) patients taking an antibiotic, and 89 of 3669 (2.4%) patients receiving no antibiotic developed acute rheumatic fever 1–2 months following a suspected streptococcal sore throat infection. These findings suggest that administering antibiotics to a patient with a sore throat and symptoms suggestive of GAS infection, who has no history of rheumatic fever, will reduce his or her risk of acute rheumatic fever by almost 70%. The number of patients with suspected GAS throat infection needed to treat with antibiotics to prevent 1 case of acute rheumatic fever is 53 (NNT = 53).
Figure 2 Forest plot of antibiotic trial effect sizes.
Sub-group analysis – intramuscular penicillin versus control
Nine out of ten studies compared the effect of intramuscular penicillin versus control for the prevention of acute rheumatic fever [16-21,23-25]. We found no statistical heterogeneity (p = 0.86) and conducted a fixed-effect, meta-analysis revealing an even greater protective effect against acute rheumatic fever with penicillin than all antibiotics combined (RR = 0.20, 95% CI = 0.11–0.36) (Fig. 3). Twelve of 3464 patients (0.35%) treated with penicillin, and 63 of 3238 (2.0%) patients on no antibiotic developed acute rheumatic fever 1–2 months following a suspected streptococcal sore throat infection. These results indicate an 80% reduction in risk of acute rheumatic fever for patients with sore throat and symptoms suggestive of GAS infection when treated intramuscularly with penicillin. This sub-group analysis yielded an NNT of 60.
Figure 3 Forest plot of penicillin trial sizes.
2. Adverse outcomes
Only 4 out of 10 studies reported any adverse outcomes associated with antibiotic treatment [17,18,20,23]. One study reported on pain at the site of injection [23], and the others reported on nausea, vomiting, and diarrhea following antibiotic treatments. No study reported mortality associated with an attack of acute rheumatic fever (Table 1).
Discussion
This systematic review found that antibiotic treatment of sore throat with accompanying symptoms suggestive of group A streptococcal (GAS) infection is effective in reducing the attack rate of acute rheumatic fever by 70%. Intramuscular penicillin appears to reduce the attack rate by as much as 80%. There was one fewer case of acute rheumatic fever for every 50–60 patients treated with antibiotics. These findings suggest that antibiotic treatment can be effective for preventing acute rheumatic fever in a population with suspected GAS throat infection.
The treatment of GAS pharyngitis is directed towards eradication of the bacteria from the upper respiratory tract. The infection can usually be eradicated by a single intramuscular injection of benzathine benzylpenicillin or by 10 days' treatment with oral penicillin [1]. While the use of intramuscular penicillin is supported by clinical trials, few trials have been done to test the efficacy of the oral penicillin for preventing acute rheumatic fever [27]. There is resistance to using intramuscular penicillin in some developing countries due to the perceived higher risk of anaphylaxis, the dangers associated with the potential reuse of needles and the discomfort of intramuscular injections. Concerns over safety issues have resulted in government orders prohibiting penicillin injections in hospitals and clinics [28]. Government regulations in response to some of these fears are warranted, particularly in the area of infection control through the prevention of needle reuse. However, with respect to the dangers of anaphylaxis, more than 50 years of experience with penicillin has shown that, while toxic reactions to intramuscular penicillin have been reported, severe reactions are exceedingly rare, especially in children. Therefore, when given under sterile conditions, fear regarding the use of parenteral penicillin is unwarranted [1].
The challenge for policymakers and clinicians is how best to apply these findings to a developing country setting where the risk of rheumatic fever persists. The most evident hindrance to the generalisability of these findings is the discrepancy in characteristics of the studied population and the population currently at risk for rheumatic fever. All of the included studies were conducted in a developed country setting a half century ago, and 8 out of 10 studies included only young adult males in their study population. Variables such as geographic location and age will affect the incidence of GAS infection as well as the attack rate of acute rheumatic fever within a population. These epidemiological differences across populations will in turn affect the number of cases needed to treat (NNT) with antibiotics in order to prevent one case of rheumatic fever. Unfortunately, trials similar to those included in this review have not been conducted in developing countries. However, the strong findings of this review do provide supportive evidence for the development of primary prevention programs to decrease the long-term sequelae associated with GAS pharyngitis in developing countries.
The validity of applying the NNT found in this review to pediatric populations in developing countries is questionable due to probable differences in GAS infection rates. In populations with a high incidence of GAS infection, the proportion of patients with respiratory symptoms that are infected with GAS will be higher than in a setting of low GAS infection incidence. A higher GAS incidence will yield a lower NNT, under the assumption that antibiotics provide the same protective effect in both populations. In the population studied, the proportion of suspected GAS infections that were true positives was large given the high specificity of the presence of exudate in the oropharynx area, estimated at 70–90% [26]. To improve the generalisability of these results to the present developing country setting, criteria for identifying suspected GAS would need to exhibit similar levels of specificity.
Identifying true cases of streptococcal pharyngitis is difficult in a developing country setting where laboratory confirmation of GAS infection is not readily available. Therefore there is a need to use a clinical prediction rule (CPR) for the diagnosis of GAS infection that takes into account these limitations while at the same time maximizing both sensitivity and specificity. Several CPR's have been developed and implemented worldwide for diagnosing streptococcal pharyngitis. A CPR put forth by the WHO Acute Respiratory Infection (ARI) Guideline suggests that both pharyngeal exudate and an enlarged and tender cervical node should be present [29]. An evaluation of this CPR in a developing country setting estimated the sensitivity and specificity of these criteria at 12% and 94% respectively [30]. The high specificity ensures that suspected GAS cases have a high probability of being true cases; however, the low sensitivity suggests that many true cases are not identified. It is at this point that a CPR suitable for a developed country diverges from a CPR suitable for a developing country where the risk of acute rheumatic fever is significantly higher.
In developing countries, it is more important to detect and treat all possible cases of GAS infection than to prevent antibiotic treatment for sore throats not attributable to GAS infection. Therefore an appropriate CPR for this setting will have high sensitivity, despite the subsequent tradeoffs of a lowered specificity. It was found that a revised version of the WHO CPR that included either the presence of exudate or enlarged cervical nodes exhibited a sensitivity and specificity of 84% and 40% respectively when tested in a developing country setting [30]. It should be noted that the loss of specificity will lead to an increase in the NNT. The tradeoffs that exist between a CPR's sensitivity, specificity, and the resulting NNT have implications for the cost of implementing a primary prevention program for rheumatic fever.
In the absence of accurate epidemiological data on streptococcal pharyngitis and rheumatic fever for the developing world, a formal cost-effectiveness analysis of the use of antibiotics for the primary prevention of rheumatic fever is not feasible. However, anecdotal evidence exists supporting the cost-effectiveness argument. The cost of one intramuscular injection of penicillin (1.2 million units) in South Africa is ~ R5.00. Using the NNT found in this review, one case of rheumatic fever will be prevented for every 60 patients receiving one intramuscular injection of penicillin for the treatment of suspected streptococcal pharyngitis. Therefore the marginal cost of preventing one case of rheumatic fever is R300.00, or US$46. A recent study in Sao Paulo, Brazil estimates the economic burden associated with rheumatic fever and its long-term sequelae in a low-income population to be over US$50 million. Furthermore, treatment costs for chronic rheumatic patients were estimated at US$319/patient/year [31]. Comparing these costs with the estimated costs of prevention provides support for the cost-effectiveness of intramuscular antibiotic treatment. An accurate cost-effectiveness analysis can not yet be done for oral antibiotics until more conclusive evidence on its efficacy is available.
Intuitively, primary prevention is a more desirable outcome for the individual as well as the health sector. Primary prevention decreases the burden placed on patients and health facilities by decreasing reliance on time- and resource-intensive secondary prevention programs, the success of which is entirely dependent on patient compliance. However, secondary prevention programmes are currently thought to be a more cost-effective approach to RF/RHD prevention and therefore more deserving of scarce resources available to communities in poor countries. This study highlights the lack of reliable data required to accurately assess the cost-effectiveness of a primary prevention program. While there is some scattered data on the prevalence of RHD in developing countries, additional data on the incidence of RF in these settings as well as the incidence of GAS infections is still needed. Furthermore, the preliminary cost-analysis put forth by this study suggesting that primary prevention is likely to be cost-effective, when considered in light of the favorable outcome of primary prevention, calls for additional attention and evaluation. At this point, primary prevention should not be dismissed.
It is important to note that the results of the included trials could have been distorted by either selection bias, detection bias, or both, arising from poor methodological quality. Few studies were adequately randomised, none used an appropriate method for concealing allocation, only four blinded the outcome assessors, and only five commented on subjects lost to follow-up. Empirical evidence suggests that trials with inadequate or unclear allocation concealment or lack of blinding, on average, lead to overestimation of the effects of interventions [32].
Conclusion
Acute rheumatic fever is common among children living in poor socioeconomic conditions. The findings of this review support the notion that antibiotic treatment given to cases of suspected streptococcal pharyngitis is an effective and safe option for reducing the complication of acute rheumatic fever. In other parts of the world the incidence of acute rheumatic fever is so low that the risks of antibiotic use may outweigh the potential benefits. Evidence is needed concerning the effects of antibiotic therapy for preventing rheumatic fever and rheumatic heart disease in children living in developing countries. Given the overwhelmingly positive results in the trials included in this review, placebo-controlled trials could be considered unethical. Trials comparing antibiotic types, routes of administration, and treatment regimens within high-risk groups, however, should be considered. Additionally, the collection of baseline epidemiological data on GAS infections, rheumatic fever, and rheumatic heart disease, and compliance with existing guidelines [33] is needed at the country level. Data on the ratio of symptomatic to sub-clinical cases of GAS pharyngitis is also essential for health planners to assess the level of prevention that is obtainable through primary prevention. Together, these data will enable countries to make more accurate estimates of the NNT and to assess the potential impact of both primary and secondary prevention programs on the health and economic outcomes associated with rheumatic fever.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
KAR and BMM contributed to all aspects of this study. JAV contributed to study design, data analysis and writing of the paper. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was funded in part by the Heart Foundation of South Africa, the Medical Research Council of South Africa, and the University of Cape Town Research Committee.
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| 15927077 | PMC1164408 | CC BY | 2021-01-04 16:30:07 | no | BMC Cardiovasc Disord. 2005 May 31; 5:11 | utf-8 | BMC Cardiovasc Disord | 2,005 | 10.1186/1471-2261-5-11 | oa_comm |
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BMC EcolBMC Ecology1472-6785BioMed Central London 1472-6785-5-41592152810.1186/1472-6785-5-4Research ArticleTree thinning as an option to increase herbaceous yield of an encroached semi-arid savanna in South Africa Smit Gert N [email protected] Department of Animal, Wildlife and Grassland Sciences, University of the Free State, P.O. Box 339, Bloemfontein 9300, Republic of South Africa2005 28 5 2005 5 4 4 27 1 2005 28 5 2005 Copyright © 2005 Smit; licensee BioMed Central Ltd.2005Smit; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The investigation was conducted in a savanna area covered by what was considered an undesirably dense stand of Colophospermum mopane trees, mainly because such a dense stand of trees often results in the suppression of herbaceous plants. The objectives of this study were to determine the influence of intensity of tree thinning on the dry matter yield of herbaceous plants (notably grasses) and to investigate differences in herbaceous species composition between defined subhabitats (under tree canopies, between tree canopies and where trees have been removed). Seven plots (65 × 180 m) were subjected to different intensities of tree thinning, ranging from a totally cleared plot (0 %) to plots thinned to the equivalent of 10 %, 20%, 35 %, 50% and 75 % of the leaf biomass of a control plot (100 %) with a tree density of 2711 plants ha-1. The establishment of herbaceous plants (grasses and forbs) in response to reduced competition from the woody plants was measured during three full growing seasons following the thinning treatments.
Results
The grass component reacted positively to the tree thinning in terms of total dry matter (DM) yield, but forbs were negatively influenced. Rainfall interacted with tree density and the differences between grass DM yields in thinned plots during years of below average rainfall were substantially higher than those of the control. At high tree densities, yields differed little between seasons of varying rainfall. The relation between grass DM yield and tree biomass was curvilinear, best described by the exponential regression equation. Subhabitat differentiation by C. mopane trees did provide some qualitative benefits, with certain desirable grass species showing a preference for the subhabitat under tree canopies.
Conclusion
While it can be concluded from this study that high tree densities suppress herbaceous production, the decision to clear/thin the C. mopane trees should include additional considerations. Thinning of C. mopane with the exclusive objective of increasing productivity of the grass layer would thus invariably involve a compromise situation where some trees should be left for the sake of the qualitative benefits on the herbaceous layer, soil enrichment, provision of browse and stability of the ecosystem.
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Background
In addition to browsing by domestic stock and game the direct uses of woody plants in southern African savannas include their use as firewood, rough construction timber, the production of charcoal and woodcarvings. In areas where trees are used for timber, harvest rates commonly exceed replacement rates. In other areas where woody plants are not subjected to harvesting and where cattle and game ranching are practised exclusively, an increase in woody plant abundance is common. This increase in woody plant abundance is commonly referred to as "bush encroachment" and involves the invasion of grasslands and the thickening of savanna [1].
The reasons for bush encroachment in savanna are diverse and complex. In most situations the determinants of savanna ecosystems were modified by man, either directly or indirectly. These determinants may either be primary (such as climate and soil) or secondary (such as fire and the impact of herbivores) [2-4]. The latter are of particular interest since, although they act within the constraints imposed by the primary determinants, they can often be directly modified by management. Examples are the exclusion of occasional hot, top killing brush fires, the replacement of most of the indigenous browsers and grazers by domestic (largely grazing) livestock often at extremely high stocking rates, the restriction of movement of herbivores by the erection of fences, long-term overgrazing of the herbaceous layer, notably during wet seasons, and the provision of artificial watering points [5,6].
In extensively managed semi-arid savannas, the productivity of herbaceous plants, notably the grasses, is of primary importance. The Colophospermum mopane Kirk ex J. Léonard (Kirk ex Benth) dominated savanna of South Africa, like most of the southern African savanna ecosystems, is water-limited and an increase in woody plant abundance invariably results in the suppression of herbaceous plants (e.g. [7-11]). Due to this suppression effect, the grazing capacity of large areas of the South African C. mopane savanna is reported to have declined due to bush encroachment, often to such an extent that many previously economic livestock properties are now no longer economic [6]. This is often the major reason why tree thinning or even total clearing is considered. The most commonly used methods of bush control include both mechanical and chemical measures. Arboricides with tebuthiuron as active ingredient are often used, but due to the non-selective nature of this arboricide, trees are also mechanically cut and the stumps treated with arboricides of which picloram is the most common active ingredient. Due to a general lack of adequate herbaceous biomass for fuel, fire is not used in the area.
Results of tree thinning may, however, differ between vegetation types, and is complicated by the existence of not only negative tree-grass interactions, but also positive tree-grass interactions. Due to the enrichment of soil under tree canopies [12-14], trees may have positive effects on grass growth. Positive interactions, such as the association of certain desirable grass species (notably Panicum maximum) with tree canopies, are a consequence of subhabitat differentiation (canopied and uncanopied subhabitats). Subhabitat differentiation is dependent on tree density, tree species and tree size [14,15], while interactions with soil can also play a role.
The C. mopane savanna is an important savanna vegetation type of southern Africa (South Africa, Namibia, Botswana and Mozambique) and the total area in southern Africa under C. mopane vegetation types is estimated at 555000 km2 [16]. The C. mopane trees have extensive root systems [17] and their selective removal has a profound effect on the soil water regime [18]. This will invariably influence the tree-grass competitive interaction. An understanding of the exact nature and magnitude of such influences is an important prerequisite towards an understanding of the complex biological interactions that exist in these ecosystems.
As part of a comprehensive investigation into the effect of tree thinning on the South African C. mopane savanna, the objectives of this study were: (i) to determine the influence of intensity of tree thinning on the dry matter yield of herbaceous plants (notably grasses), (ii) to establish relations between tree density and herbaceous production, and (iii) to investigate differences in herbaceous species composition between defined subhabitats (under tree canopies, between tree canopies and where trees have been removed).
Study area
The study was conducted in the Limpopo Province of South Africa on a site located at 29°12'E, 22°19'S, 560 m above sea level. The savanna vegetation is locally described by Acocks [19] as "Mopani veld" and by Low & Rebelo [20] as "Mopane Bushveld". Louw [21] made a further division of seven plant communities within the South African Mopane Bushveld, and the study area was located in, what he named the Colophospermum-Boscia community. This community covers about 60 000 ha of the Mopane Bushveld. Louw [21] described this community as a virtually pure stand of Colophospermum mopane (synonym: Hardwickia mopane), interposed with a few individuals of Boscia foetida subsp. rehmanniana and Salvadora australis (synonym: S. angustifolia var. australis). Within the study area the most important grass species are Enneapogon cenchroides, Aristida adscensionis, Brachiaria deflexa, Cenchrus ciliaris and Digitaria eriantha.
The study area, before tree thinning, was characterized by the virtual absence of herbaceous plants, accompanied by severe soil degradation in the form of surface erosion and crust formations. Crust formations are known to reduce infiltration and cause substantial losses due to rainfall runoff (e.g. [22-25]). The site was previously used as grazing for cattle, but due to a lack of adequate grazing, the cattle was removed from the area for a period of at least five years. Since then the area has mainly been grazed and browsed by an unknown number of free ranging game species.
The rainy season usually extends from October to March inclusively, but rainfall is irregularly distributed and unpredictable. Mean long-term seasonal rainfall (July-June) for the period 1966/67 to 1990/91 was 376 mm (SE ± 27.6, range: 140–620 mm). The probability of rain falling during January is greater than for other months. The area is largely frost-free and is well known for its high summer temperatures and moderate to warm winter temperatures.
The underlying rock type is mainly sandstone [21] and the soil is predominantly sandy (80% sand, 8 % silt, 12 % clay). A detailed description of the soil is given by Smit & Rethman [18].
Results
Areas of subhabitats
Three subhabitats were distinguished: between tree canopies (uncanopied – UCA), under tree canopies (canopied – CA) and where trees have been removed (removed canopy – RCA). The areas covered by the various subhabitats are presented in Table 1. The subhabitat between trees (uncanopied – UCA) predominates, also with small variation between treatments (mean of 85.8 %). Through tree thinning the subhabitat under trees canopies (canopied – CA) decreased over the gradient of decreasing tree density, up to the point of no representation in the 0% plot. In contrast, the subhabitat where trees were removed (removed canopy – RCA), increased over this gradient, not being represented in the control plot (100 % plot).
Table 1 Percentages of the total surface area covered by the various habitats and subhabitats in each of the experimental plots.
Subhabitat Experimental plot Area (%)
Between trees 0 % 87.10
,, 10 % 86.58
,, 20 % 83.99
,, 35 % 86.65
,, 50 % 87.46
,, 75 % 85.92
,, 100 % 82.92
Under trees 0 % 0.00
,, 10 % 2.85
,, 20 % 5.57
,, 35 % 5.80
,, 50 % 8.82
,, 75 % 10.85
,, 100 % 17.08
Where trees 0 % 12.90
were removed 10 % 10.57
,, 20 % 10.44
,, 35 % 7.55
,, 50 % 3.72
,, 75 % 3.23
,, 100 % 0.00
Leaf volume of the woody layer
The estimated number of Evapotranspiration Tree Equivalents (ETTE) ha-1 of the C. mopane trees over the trial period is presented in Table 2. Detailed results and a discussion of the woody layer are reported elsewhere [26-28].
Table 2 The number of EvapotranspirationTree Equivalents (ETTE) ha-1 within the various tree thinning plots and seasons.
Season Exp. plot ETTE ha-1
After thinning 10 605.5
20 1 406.1
35 1 717.4
50 3 176.2
75 3 376.7
100 5 509.9
Season 1 10 707.5
20 1 669.5
35 2 008.0
50 3 487.6
75 3 602.9
100 5 900.8
Season 2 10 809.9
20 1 885.5
35 2 136.1
50 3 503.3
75 3 769.3
100 5 962.3
Season 3 10 998.4
20 2 171.8
35 2 540.8
50 3 870.8
75 4 197.2
100 6 733.0
Dry matter yield of the herbaceous layer
The total seasonal DM yield of grasses (subhabitats combined) is presented in Figure 1 and the total seasonal DM yield of grasses within the various subhabitats is presented in Figure 2. The grass DM yields were generally low during the first, low rainfall season season 1). In the following seasons the yields were substantially higher, with marked differences between treatments. Comparison of the grass DM yields between subhabitats, revealed differences. The yields between tree canopies (UCA) (Figure 2a) were initially of the same order as under tree canopies (CA) (Figure 2b), with the yields where trees have been removed (RCA) the highest (Figure 2c). While the seasonal grass yield patterns largely followed the rainfall pattern (Figure 3), the yields of the UCA subhabitat in the totally cleared plot (0% plot) continued to improve during the third season, which received less than half the rainfall of the previous season (season 2).
Figure 1 Total seasonal DM yields of grasses as measured in die various tree thinning treatment plots during the three seasons following the tree thinning (the DM yields of the uncanopied, canopied and removed canopy subhabitats were combined).
Figure 2 Total seasonal DM yields of grasses within the defined subhabitats during the three seasons following the tree thinning: (a) between tree canopies (uncanopied), (b) under trees (canopied), and (c) where trees have been removed (removed canopy).
Figure 3 Monthly rainfall recorded at the Colophospermum mopane experimental site during the three seasons (July-June) of the trial period.
The fitting of polynomials to data of season 1 was unsuccessful, mainly due to the low rainfall and subsequent poor response of the grass layer. Polynomials were subsequently only fitted to the data for seasons 2 and 3 (Table 3). The testing of paired combinations (contrasts) [29] of these selected polynomials for significant differences on the x-axis (ETTE ha-1) showed that total grass DM yields did not differ significantly (P > 0.05) between the UCA and CA-subhabitats, during both seasons 2 and 3. In contrast, during season 2, up to 3927 ETTE ha-1, grass DM yields differed significantly (P < 0.05) between the UCA and the RCA-subhabitats, with the yields being higher in the RCA-subhabitat. These differences changed during season 3, with yields not differing significantly (P > 0.05) between the latter subhabitats over the complete ETTE gradient. The test of the contrast CA versus RCA showed that during season 2 yields differed significantly (P < 0.05) up to a density of 3817 ETTE ha-1 (0 %, 10%, 20 %, 35 % and 50 % plots), with the yields being higher in the RCA-subhabitat. Similar to the UCA/RCA contrast, yields did not differ significantly (P > 0.05) between these subhabitats during season 3.
Table 3 Polynomials with the best fit (y = total grass DM yield, x = ETTE ha-1)
Subhabitat Season after thinning Polynomial r2 P
UCA 2 quadratic: y = 948.0 - 0.278 + 0.000021x2 0.78 0.021
UCA 3 quadratic: y = 980.0 - 0.3985 + 0.000039x2 0.90 0.005
CA 2 cubic: 686.3 - 0.4284x + 0.000254x2 + 0.29E-7x3 0.99 0.003
CA 3 quadratic: 630.0 - 0.2132x + 0.000019x2 0.64 0.099
RCA 2 quadratic: 1 968.0 - 0.066x - 0.000073x2 0.95 0.005
RCA 3 quadratic: 1 047.3 - 0.3558x + 0.000038x2 0.98 0.001
Relationship between tree leaf biomass and herbaceous dry matter yield
The relationship between tree leaf biomass, expressed as Evapotranspiration Tree Equivalents (ETTE) ha-1 and total grass DM yield (all subhabitats combined) of each treatment plot was established (Figure 4). The relationships between ETTE ha-1 and grass DM yield within each of the defined subhabitats are presented in Table 4. The relations between ETTE ha-1 and the DM yield of forbs within these subhabitats are presented in Table 5.
Figure 4 Results of the regression analyses of the relations between the grass DM yield (subhabitats combined) and the Evapotranspiration Tree Equivalents (ETTE) ha-1 (shaded area shows the 95 % confidence limits): (a) season 1, (b) season 2, and (c) season 3.
Table 4 Results of the regression analyses of the relations between the DM yields of grasses within the defined subhabitats (dependent variable) and Evapotranspiration Tree Equivalents (ETTE) ha-1 (independent variable).
Subhabitat Season Regression equation r2 r n P
Between trees (UCA) 1 y = 57.188 - 0.00765x 0.282 -0.532 7 0.220 ns
2 ln y = 7.017 - 0.000510x 0.861 -0.928 7 0.003 **
3 ln y = 6.708 - 0.000579x 0.828 -0.910 7 0.004 **
Under trees (CA) 1 ln y = 5.052 - 0.000274x 0.679 -0.824 7 0.044 *
2 ln y = 6.936 - 0.000257x 0.662 -0.814 7 0.049 *
3 ln y = 6.099 - 0.000349x 0.567 -0.753 7 0.084 ns
Trees removed (RCA) 1 y = 274.648 - 0.0479x 0.358 -0.599 7 0.209 ns
2 ln y = 7.745 - 0.000284x 0.868 -0.932 7 0.007 **
3 ln y = 6.936 - 0.000466x 0.980 -0.990 7 0.002 **
Table 5 Results of the regression analyses of the relations between the DM yields of forbs within the defined subhabitats (dependent variable) and Evapotranspiration Tree Equivalents (ETTE) ha-1 (independent variable).
Subhabitat Season Regression equation r2 r n P
Between trees (UCA) 1 ln y = 2.756 + 0.000214x 0.137 0.370 7 0.414 ns
2 y = 43.396 + 0.01467x 0.272 0.521 7 0.230 ns
3 ln y = 3.911 + 0.000156x 0.126 0.355 7 0.434 ns
Under trees (CA) 1 y = - 65.59 + 0.12309x 0.675 0.822 7 0.045 *
2 y = 90.378 + 0.05286x 0.813 0.902 7 0.014 *
3 y = 139.76 + 0.04719x 0.416 0.645 7 0.167 ns
Trees removed (RCA) 1 y = 21.291 + 0.00130x 0.021 0.146 7 0.783 ns
2 y = 72.022 - 0.01599x 0.537 -0.733 7 0.098 ns
3 ln y = 0.888 + 0.000824x 0.611 0.781 7 0.066 ns
From Figure 4a there is a negative trend between ETTE ha-1 and total grass DM yield of the combined subhabitats. However, following thinning, this negative trend changed significantly with each season. It changed from a non-significant (P > 0.05) linear relation during the first (dry) season (Figure 4a) to a significant curvilinear (P < 0.05) relationship during the second and third seasons (Figure 4b &4c). The best fit to these curvilinear relations was achieved by the exponential regression equation. The gradient of the curve was steeper in the relationship established for season 3 (Figure 4c), indicating an increasing difference between grass DM yields of the totally cleared plot (0 %) and the rest of the treatments.
Examination of the grass DM yields within the respective subhabitats revealed trends similar to that already presented for the combined subhabitats. Significant (P < 0.05) negative relationships between grass yield and ETTE ha-1 are particularly eminent after the second and third seasons within the UCA and RCA-subhabitats. This negative relationship was less strongly defined in the CA-subhabitat (Table 4).
The reaction of forbs to the thinning of C. mopane differed markedly from that of the grasses (Table 5). With few exceptions, the yields of forbs were mostly positively associated with ETTE ha-1, though the relations were mostly statistically non-significant (P > 0.05). This implies that they were predominantly negatively affected by tree thinning. Some variation between subhabitats was also found, but a consistent pattern was lacking.
Grass species differences between subhabitats
The mean percentage contributions (on a dry mass basis) of the most abundant grass species to the total grass DM yield within the defined subhabitats are presented in Table 6. By non-statistical inspection it appeared as if Tragus berteronianus, Aristida species and Oropetium capensis were mostly more abundant within the UCA-subhabitat. Preferences for the CA-subhabitat were shown by Cenchrus ciliaris, Digitaria eriantha and Panicum maximum. Those with no apparent preferences were Brachiaria deflexa and Enneapogon cenchroides, being abundant in all subhabitats. No conclusion can be drawn for Urochloa mosambicensis, Bothriochloa radicans and Sporobolus ioclados due to a low representation. Preferences for the RCA-subhabitat are likely to be transient in view of the expected short-term advantage that the RCA-subhabitat offers.
Table 6 Mean percentage contribution (on a dry mass basis) of the most abundant grass species to the total grass DM yield within the defined subhabitats.
Grass species Exp. plot Mean % contribution (standard error)
Between trees (UCA) Under trees (CA) Trees removed (RCA)
Tragus berteronianus 0 % 14.70 (7.30) - 7.23 (4.55)
,, 10 % 8.30 (4.33) 4.30 (3.09) 5.73 (5.29)
,, 20 % 19.03 (12.97) 6.50 (3.26) 13.40 (6.95)
,, 35 % 28.57 (14.32) 9.03 (4.12) 7.03 (6.54)
,, 50 % 14.23 (7.23) 3.57 (2.51) 5.13 (4.84)
,, 75 % 11.00 (6.32) 3.60 (2.75) 5.90 (5.55)
,, 100 % 10.87 (8.76) 2.10 (2.10) -
Aristida species 0 % 56.80 (2.59) - 10.53 (1.43)
,, 10 % 38.23 (2.69) 12.93 (7.35) 6.23 (1.53)
,, 20 % 31.17 (4.93) 14.33 (5.80) 6.90 (3.58)
,, 35 % 8.97 (2.41) 5.23 (3.25) 8.27 (3.93)
,, 50 % 12.00 (6.89) 6.57 (3.31) 5.47 (2.39)
,, 75 % 23.37 (20.02) 4.53 (2.58) 9.37 (7.00)
,, 100 % 23.73 (7.05) 0.13 (0.13) -
Oropetium capensis 0 % 1.93 (1.41) - 0.13 (0.13)
,, 10 % 3.47 (1.09) 0.70 (0.60) 0.27 (0.22)
,, 20 % 6.33 (4.16) 2.17 (1.31) 0.03 (0.03)
,, 35 % 9.37 (4.27) 3.67 (1.95) 0.17 (0.17)
,, 50 % 14.17 (3.68) 3.90 (1.57) 0.10 (0.10)
,, 75 % 35.60 (15.69) 5.97 (0.85) 0.00 (0.00)
,, 100 % 21.83 (2.28) 4.93 (2.17) -
Cenchrus ciliaris 0 % 1.67 (1.67) - 2.10 (1.24)
,, 10 % 0.00 (0.00) 0.00 (0.00) 2.97 (1.49)
,, 20 % 0.00 (0.00) 7.63 (3.71) 1.20 (1.20)
,, 35 % 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)
,, 50 % 0.00 (0.00) 4.33 (4.33) 0.00 (0.00)
,, 75 % 0.00 (0.00) 10.77 (9.21) 0.50 (0.50)
,, 100 % 0.00 (0.00) 19.63 (11.67) -
Digitaria eriantha 0 % 0.00 (0.00) - 1.87 (1.13)
,, 10 % 1.20 (1.20) 6.00 (3.81) 2.00 (0.91)
,, 20 % 0.00 (0.00) 6.50 (1.50) 8.17 (3.13)
,, 35 % 0.00 (0.00) 2.10 (2.10) 2.80 (0.90)
,, 50 % 0.00 (0.00) 17.60 (10.49) 8.80 (4.28)
,, 75 % 0.00 (0.00) 10.10 (3.26) 9.93 (8.52)
,, 100 % 2.37 (2.37) 40.10 (15.54) -
Panicum maximum 0 % 0.00 (0.00) - 6.53 (6.53)
,, 10 % 0.00 (0.00) 12.60 (12.60) 0.00 (0.00)
,, 20 % 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)
,, 35 % 0.00 (0.00) 7.43 (7.43) 2.60 (2.60)
,, 50 % 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)
,, 75 % 0.00 (0.00) 3.57 (3.57) 3.83 (3.83)
,, 100 % 0.00 (0.00) 0.00 (0.00) -
Brachiaraia deflexa 0 % 4.93 (3.79) - 15.40 (11.72)
,, 10 % 6.40 (3.13) 21.33 (11.18) 18.77 (15.40)
,, 20 % 1.17 (0.69) 15.67 (10.05) 11.87 (7.89)
,, 35 % 12.10 (6.14) 26.57 (13.32) 30.40 (19.93)
,, 50 % 25.77 (12.91) 36.60 (20.58) 35.77 (15.44)
,, 75 % 21.57 (12.27) 41.00 (21.95) 30.23 (13.99)
,, 100 % 10.60 (6.74) 26.40 (19.59) -
Enneapogon cenchroides 0 % 16.57 (8.67) - 47.10 (13.78)
,, 10 % 36.63 (2.47) 38.83 (13.97) 51.63 (21.41)
,, 20 % 37.57 (13.30) 42.40 (9.12) 54.20 (17.82)
,, 35 % 12.03 (6.03) 29.20 (17.05) 39.53 (16.10)
,, 50 % 26.03 (12.07) 18.77 (12.37) 42.83 (15.25)
,, 75 % 6.23 (3.83) 20.57 (12.13) 36.30 (5.18)
,, 100 % 0.60 (0.60) 6.30 (5.33) -
Bothriochloa radicans 0 % 0.00 (0.00) - 0.00 (0.00)
,, 10 % 0.00 (0.00) 0.00 (0.00) 9.13 (9.13)
,, 20 % 4.43 (4.43) 4.83 (4.25) 0.00 (0.00)
,, 35 % 7.37 (2.35) 14.40 (9.46) 9.23 (3.38)
,, 50 % 4.17 (2.77) 0.00 (0.00) 0.00 (0.00)
,, 75 % 2.30 (2.30) 0.00 (0.00) 1.00 (1.00)
,, 100 % 19.97 (10.38) 0.50 (0.50) -
Sporobolus ioclados 0 % 0.00 (0.00) - 6.63 (6.63)
,, 10 % 5.83 (5.83) 1.97 (1.82) 3.30 (2.10)
,, 20 % 0.00 (0.00) 0.00 (0.00) 1.67 (1.67)
,, 35 % 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)
,, 50 % 1.33 (1.33) 0.00 (0.00) 1.97 (1.97)
,, 75 % 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)
,, 100 % 0.00 (0.00) 0.00 (0.00) -
Urochloa mosambicensis 0 % 3.40 (1.76) - 2.40 (1.88)
,, 10 % 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)
,, 20 % 0.00 (0.00) 0.00 (0.00) 2.60 (2.60)
,, 35 % 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)
,, 50 % 2.30 (2.30) 0.00 (0.00) 0.00 (0.00)
,, 75 % 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)
,, 100 % 0.00 (0.00) 0.00 (0.00) -
Discussion
The botanical composition and productivity of any mature stand of vegetation is largely determined by competition [30]. The roots of woody plants are fundamental in their competitive interactions with herbaceous plants and other woody plants. Roots determine the spatial distribution of water and nutrient uptake and can cause an increase or a decrease in resource availability [31]. This aspect was clearly illustrated within the study area [28] where it was demonstrated that the total root biomass of C. mopane ranged from 9760 kg ha-1 to 29790 kg ha-1 (mean: 17354 kg ha-1). Of these a mean of 19 % was in the 0–1.0 mm diameter class, and 20.3 %, 16.2 % and 44.5 % in the >1.0–5.0 mm, >5.0–10.0 mm and >10 mm diameter classes respectively. A mean of 66.1 % of all fine roots (<5.0 mm) was found within the first 400 mm of the soil [28].
A subsequent study [18] presented evidence that the roots of the C. mopane trees are able to utilise soil water at a matric potential lower than that of grasses (ψ < -1500 kPa). This feature, combined with high rainwater runoff losses due to a lack of a herbaceous cover, resulted in a dramatic reduction in the amount of plant available water with an increase in tree density. This enables the C. mopane trees to compete successfully with herbaceous plants and to prevent their establishment at high tree densities.
In view of this knowledge the observed increase in grass DM yield after the thinning of the C. mopane trees was expected. Indeed competitive interactions between the woody and herbaceous components of savannas, involving mainly available soil water as the primary determinant of production, have been reported world-wide (Australia: [32-35]; North America: [36-40]; southern and east Africa: [8,9,11,12,14]). While the existence of negative competition interactions between woody and herbaceous plants are thus nothing new, the results of this study is of particular significance, which relates to the magnitude and scale of the competition interaction.
The suppressive effect of the C. mopane trees on the grass DM yield at high tree densities is severe and for this reason the thinning of the C. mopane trees resulted in significant and desirable increases in grass DM yields (Figures 1 and 2). It is also worthy to note that during a wet year, like the second season, the grass DM yield differed substantially (1211%) between the extreme ends of the competition gradient (0 % versus 100 % plots), but the difference enlarged even further (2778 %) during a dry season (season 3). This substantiates the general assumption that the consequences of bush encroachment is at its worst during dry periods [9].
The gradual establishment of the strong negative relation between grass DM yield and ETTE ha-1 is clearly illustrated in Figure 4. As bare soil became colonised by grasses in the plots with a low tree density, runoff of rainfall was increasingly reduced, increasing the amount of soil water available to the establishing grasses [18]. This resulted in increased differences in grass DM yield between plots at the extreme ends of the ETTE gradient. This phenomenon is clearly illustrated in Figure 1 where the grass DM yield in all the plots followed the pattern of seasonal rainfall, except in the totally cleared plot (0%) which continued to improve during the third season, while receiving less than half of the rainfall of the previous wet season (second season). At the other end of the gradient, grass DM yields differed little between years of below and above average rainfall (75 % and 100 % plots). This is typical of a human induced drought situation and not a climatic drought.
The negative curvilinear relationship between tree density and grass DM yield evident on the experimental site as a whole, as well as within the individual subhabitats, corresponds to those described for some other savanna vegetation types (e.g. [7-9], [32-34]). This relation implies that the highest grass DM yield is obtained where all the C. mopane trees are removed. Since grass yields under tree canopies were not significantly higher than between tree canopies, and the high yields where trees were removed are likely only temporary, it would appear that no advantageous tree-grass interactions, evident in several other savanna vegetation types (e.g. [12-15], [41-44]) occur in this vegetation type. This, at least, applies to the total grass DM yield and not to possible differences in grass species composistion.
While subhabitat differentiation did not present any advantage in respect of total grass DM yield, differences with regard to grass species composition between subhabitats were, however, present (Table 6). On evaluating these species differences, it is clear that the CA-subhabitat is important to the presence of desirable perennial grass species like Cenchrus ciliaris, Digitaria eriantha and Panicum maximum. These are also the species with the highest nutritional characteristics. Though these species did not constitute a large proportion of the grass species composition, they may play an important role in the total nutrition of grazing herbivores should they increase under improving management conditions.
A possible explanation for the negative reaction of forbs to tree thinning, lies in the seemingly inability of forbs to compete with establishing grasses. Thus, in those plots at the low end of the ETTE gradient, forbs were being replaced by strongly competitive grasses. The decrease of forbs can therefore be considered as a secondary consequence of the removal of C. mopane trees. However, it can be expected that the different forb species will react differently to competition from grasses, as well as to subhabitat changes. Thus, a proper understanding of the dynamics of forbs would necessitate an evaluation on a species basis.
The results of this study must be viewed in relation to different hypotheses of tree-grass dynamics, especially in semi-arid environments. This will invariably have an influence on the decision of the desirability to thin or clear the C. mopane trees for the purpose of increasing the herbaceous yield.
The terms "equilibrium" and "non-equilibrium" as used in rangelands, are points of strong debate among scientists. The central aspect of this debate is the definition of the degree to which climate or consumers (herbivores) influence vegetation. One view is that consumers reach densities that degrade environments from a previous condition of equilibrium and the other view is that the dynamics of pastoral systems are non-equilibrial and primarily dictated by variability in rainfall [45].
Higgins et al. [46] suggested a non-equilibrium mechanism of coexistence for savanna ecosystems. According to their model, grasses and trees coexist for a wide range of environmental conditions, and exhibit long periods of slow decline in adult tree numbers interspersed with relatively infrequent recruitment events. Recruitment is controlled by rainfall (which limits seedling establishment) and fire (which prevents recruitment into adult size classes). On the other hand, Illius and O'Connor [47] argued that the view that herbivory has little impact on climatically variable ecosystems is unjustified. They proposed an alternative model in which it is assumed that despite the apparent lack of an equilibrium, animal numbers are regulated in a density-dependent manner by the limited forage available in key resource areas which are utilized in the dry season. Their model asserts that strong equilibrial forces exist over a limited part of the system, with the animal population virtually uncoupled from resources elsewhere in the system.
While these arguments mainly relate to the causes and mechanisms according to which the woody plants increase (bush encroachment), the results of this study clearly showed that once the C. mopane has established, the suppression of the herbaceous layer was such that rainfall had very little effect on annual herbaceous yields in plots with high tree densities. Rainfall only played a significant role on herbaceous yields in plots where tree densities were reduced. Furthermore, the tree densities remained very stable at high tree densities with no indication, yet, of a natural process of restoration from its current encroached state.
Conclusion
From this study it can be concluded that the grass component of the herbaceous layer, in terms of total DM yield, reacted positively to the tree thinning treatments, but forbs were negatively influenced. It is also evident that rainfall played an important role by interacting with tree density in influencing grass DM yields. Comparatively, the grass DM yields in thinned plots were substantially higher than those of the control plot during years of below average rainfall, while at high tree densities yields differed little between seasons of varying rainfall.
At high tree densities the suppressive effect of the C. mopane trees approach complete suppression of the grass layer. The observed curvilinear relationship between grass DM yield and ETTE ha-1, best described by the exponential regression equation, implies that the highest grass DM yields will be achieved when all the C. mopane trees are removed.
The question may then be asked if total tree clearing is the recommended option for land managers who have to deal with this problem in a practical manner. Based on some observed qualitative benefits of subhabitat differentiation by the C. mopane trees, with certain desirable grass species that showed a preference for the CA-subhabitat, the answer is not an unconditional yes. It is assumed that these desirable grass species would probably be lost with the complete removal of the C. mopane trees. From the literature it is also known that during practical tree thinning operations, re-encroachment is a common problem [28]. Through selective tree thinning, the development of a structured savanna with large trees is encouraged, and these large trees are able to suppress the establishment of new seedlings [5]. Total removal of all the C. mopane trees is therefore expected to be conducive to the rapid re-encroachment of the cleared area. Thinning of C. mopane with the exclusive objective of increasing productivity of the grass layer would thus invariably involve a compromise situation where some trees should be left for the sake of the qualitative benefits on the herbaceous layer, soil enrichment, provision of browse and stability of the ecosystem.
While the benefits of tree thinning (not total clearing) in terms of increased herbaceous yield was demonstrated in this study, the issue of cost poses a substantial limitation on the practical implementation of bush control measures in the C. mopane savanna vegetation. An economical evaluation of different chemical and mechanical bush control measures was beyond the scope of this study, but hopefully this study will provide essential quantitative botanical data for a thorough economical evaluation.
Methods
Trial layout
The study area consisted of seven, 1.17 ha plots (180 m × 65 m), thinned to differing tree densities. The plots were located next to each other on a homogeneous area of 8.2 ha. Treatments were allocated randomly to the plots. The control plot was left undisturbed (referred to as the 100 % plot), and the others thinned to the approximate equivalents of 75%, 50%, 35 %, 20 %, 10 % and 0 % (total clearing) of the tree biomass of that of the 100 % plot. The control plot was characterized by a dense stand of C. mopane with herbaceous plants almost completely absent.
The occurrence of dwarf growth forms of C. mopane is known to exist. In the Kruger National Park, all C. mopane growing on soils derived from basic material i.e. basalt, diabase/dolorite and gabbro are multi-stemmed shrubs with a mean height of 1–2 m, while C. mopane growing on sandy soils are usually single-stemmed and up to 5 m tall [48]. The C. mopane trees of the study area at the onset of the study had a mean height of 2.47 m (SE ± 0.052), with a mean canopy diameter of 1.68 m (SE ± 0.064). A large percentage of them was multi-stemmed. No specific information on the ages of the trees is available, but according to local inhabitants this specific dense stand of C. mopane trees was in existence for a number of years, though some older members can remember a time when the area was sparsely covered with trees.
Trees were randomly marked for removal during the thinning process. This ensured a fairly even spread of the remaining trees without favouring a particular tree size. The resultant thinned plots resembled the structure of naturally occurring open stands of C. mopane. During thinning, trees were sawn off at ground level and removed from the plot. The stumps were sprayed with a 1 % concentration of picloram and triclopyr (Tordon Super) mixed in diesel, thus ensuring that the sawn trees were killed without affecting the remaining plants. The study area was fenced to exclude grazing or browsing animals. The tree thinning was completed during the winter of 1989 and the tree densities (trees ha-1) were as follows: 100 % (control) plot – 2711; 75 % plot – 1978; 50 % plot – 1233; 35% plot – 744; 20 % plot – 589; 10 % plot – 300 and 0 % – 0 trees ha-1. The response of the herbaceous layer was studied during the three growing seasons following tree thinning.
Rainfall
Daily rainfall data were recorded as the mean of four standard rain gauges (127 mm diameter), placed at each of the four corners of the experimental area (Figure 3).
Quantification of the woody layer
The purpose of the survey of the woody layer was primarily aimed at obtaining some quantitative data of the leaf biomass of the remaining C. mopane trees for purposes of establishing the relationship between the above ground woody and herbaceous biomass.
At the end of each growing season, normally April or May, the canopy of all rooted live C. mopane trees encountered in fixed transects (5 m × 180 m) located in the middle of each of the experimental plots, was measured. The measurements consisted of the following [49,50]: (i) maximum tree height, (ii) height where the maximum canopy diameter occurs, (iii) height of first leaves or potential leaf bearing stems, (iv) maximum canopy diameter, and (v) base diameter of the foliage at the height of the first leaves. The canopy volume of the trees, regardless of their shape or size, was calculated from these dimension measurements by using the volume formulas of an ellipsoid, a right circular cone, a frustum of right circular cone or a right circular cylinder. Depending on the shape of the tree, any one of these volume formulas may be used, or more likely two of them in combination. A comprehensive description of the procedure is given by Smit [50].
Leaf volume estimates (cm3) were calculated using the BECVOL-model (Biomass Estimates from Canopy Volume) [26,51], which is based on the quantitative description technique proposed by Smit [49,50]. It includes regression equations, developed from harvested trees, which relate the spatial canopy volume (independent variable) to the actual leaf volume (dependant variable): ln y = -4.34074 + 0.7601x, r = 0.963, P < 0.001. Spatial tree canopy volume (x) is transformed to its normal logarithmic value, while y represents the estimated leaf volume (cm3). The number of Evapotranspiration Tree Equivalents (ETTE) ha-1 was subsequently calculated from the leaf volume estimates (1ETTE = mean leaf volume of a 1.5 m tall single-stemmed tree = 500 cm3 leaf volume) [49]. Since the ETTE-values is based on estimates of actual leaf biomass it is considered a more accurate measure of potential competition of woody plants compared to simple density data (plants ha-1).
Quantification of the herbaceous layer
Three subhabitats were distinguished: between tree canopies (uncanopied – UCA), under tree canopies (canopied – CA) and where trees have been removed (removed canopy – RCA). The C. mopane trees do not have wide spreading canopies. Closed canopies [15] are thus largely absent. The various subhabitats were consequently not considered to be purely a function of the area overspanned by the tree canopies, but also of the soil. Large scale loss of topsoil, partially retained under the trees, has led to distinctive elevated soil surface patterns. The CA and RCA subhabitats are subsequently often smaller in diameter than the immediate overstory canopy spread. Due to the relatively close proximity of the trees, as well as an extensive horizontal spread of the roots of C. mopane [17], the uncanopied subhabitat fell within the root zone of the trees, even at the lowest tree density.
Areas covered by the various subhabitats were determined for each of the experimental plots. Subhabitat areas, which were mostly circular in shape, were determined from two diameter measurements rectangular to each other. The area of either a fitting circle or ellipsoid was calculated [52]. Only the areas of the CA and RCA subhabitats were measured. For each experimental plot the area of the UCA subhabitat was calculated from subtracting the combined areas of the two measured subhabitats from the total area of each experimental plot (1.17 ha).
Above-ground dry matter (DM) yield of herbaceous plants within the seven tree density plots was determined at the end of each growing season, normally April or May. A harvest technique [53,54] was employed, which provided estimates of net primary production [55,56] less possible dry matter loss due to grass mortality. Losses due to grazing during the growing season were prevented by the fencing of the study area. Controlled grazing by cattle during the dormant season annually, ensured that carry-over from one season to another was low.
Grasses (species basis) and forbs (non-species basis) were harvested in quadrates (0.25 m2), randomly placed in each of the subhabitats. A total of 60 quadrates per experimental plot were harvested, 20 randomly allocated per subhabitat. In those plots where only 2 of the 3 defined subhabitats were represented (0 % and 100 % plots), 30 quadrates were harvested on each of the 2 represented subhabitats. Rooted herbaceous plants within each quadrate were clipped to stubble height using hand clippers. Stubble height varied from 0.1–3.0 cm, depending whether the species was tufted or not. The clipped material was dried to a constant mass (70°C) and weighed.
Data analyses
In testing for treatment effects, care was taken to avoid the use of pseudo-replications [57]. Relations between tree leaf biomass (dry basis) and the DM yield of herbaceous plants (grasses and forbs) were established using regression analyses [58,59]. For the determination of differences in trend of grass DM yield between habitats and subhabitats, the total grass DM yield within the various habitats and subhabitats of the experimental plots after each successive season (x-axis), was subjected to the fitting of polynomials. Polynomials (linear, quadratic or cubic) with the best fit were selected and paired combinations of these selected polynomials were subsequently tested for contrasts on the x-axis (tree density) using the procedures of Groeneveld [29].
List of abbreviations
BECVOL – Biomass Estimates from Canopy Volume
CA – Canopied (under tree canopies)
DM – Dry mass
ETTE – Evapotranspiration Tree Equivalents (1 ETTE = mean leaf volume of a 1.5 m single-stemmed tree = 500 cm3 leaf volume)
RCA – Removed Canopy (where trees were removed)
UCA – Uncanopied (between tree canopies)
Authors' contributions
All aspects of the study were conducted by the author with assistance as indicated under Acknowledgements.
Acknowledgements
The field assistance of A. le Roux and the late J.S. Swart is gratefully acknowledged. Mrs M.F. Smith is thanked for her help with the statistical analysis.
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| 15921528 | PMC1164409 | CC BY | 2021-01-04 16:29:15 | no | BMC Ecol. 2005 May 28; 5:4 | utf-8 | BMC Ecol | 2,005 | 10.1186/1472-6785-5-4 | oa_comm |
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BMC Fam PractBMC Family Practice1471-2296BioMed Central London 1471-2296-6-231594638210.1186/1471-2296-6-23Research ArticleFollow-up care by patient's own general practitioner after contact with out-of-hours care. A descriptive study van Uden Caro JT [email protected] Paul J [email protected] Sjoerd O [email protected] Andre JHA [email protected] Geertjan [email protected] Schayck Onno CP [email protected] Harry FJM [email protected] Department of Integrated Care, Research Institute Caphri, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, the Netherlands2 Department of General Practice, Research Institute Caphri, Maastricht University, P.O. Box 616, 6200 MD Maastricht, the Netherlands3 Department of Health Organization Policy and Economics, Research Institute Caphri, Maastricht University, P.O. Box 616, 6200 MD Maastricht, the Netherlands4 Department of Respiratory Diseases, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, the Netherlands2005 9 6 2005 6 23 23 30 12 2004 9 6 2005 Copyright © 2005 van Uden et al; licensee BioMed Central Ltd.2005van Uden et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Little is known about the care process after patients have contacted a GP cooperative for out-of-hours care. The objective of this study was to determine the proportion of patients who seek follow-up care after contact with a GP cooperative for out-of-hours care, and to gain insight into factors that are related to this follow-up care.
Methods
A total of 2805 patients who contacted a GP cooperative for out-of-hours care were sent a questionnaire. They were asked whether they had attended their own GP within a week after their contact with the cooperative, and for what reason. To investigate whether other variables are related to follow-up care, a logistic regression analysis was applied. Variables that entered in this analysis were patient characteristics (age, gender, etc.) and patient opinion on correctness of diagnosis, urgency and severity of the medical complaint.
Results
The response rate was 42%. In total, 48% of the patients received follow-up care from their own GP. Only 20% were referred or advised to attend their own GP. Others attended because their medical condition worsened or because they were concerned about their complaint. Variables that predicted follow-up care were the patient's opinion on the correctness of the diagnosis, patient's health insurance, and severity of the medical problem.
Conclusion
Almost half of all patients in this study who contacted the GP cooperative for out-of-hours care attended their own GP during office hours within a week, for the same medical complaint. The most important factor that predicted follow-up care from the patient's own GP after an out-of-hours contact was the patient's degree of confidence in the diagnosis established at the GP cooperative. Despite the limited generalisability, this study is a first step in providing insight into the dimension of follow-up care after a patient has contacted the GP cooperative for out-of-hours primary care.
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Background
During the last decade, out-of-hours primary care in the Netherlands has been reorganized from practice-based services to large-scale general practitioner (GP) cooperatives [1]. Currently, over 90% of the Dutch population is covered by more than 120 GP cooperatives for out-of-hours primary care. The initiative for this reorganization has come mainly from the medical profession itself. Research has shown that similar reorganisations had beneficial effects in other countries like the UK and Denmark; GPs' satisfaction with out-of-hours services increased and the number of hours the GP has to be on call dropped substantially [2]. Also patients seemed to be fairly satisfied with out-of-hours primary care delivered by GP cooperatives. However, patients seemed to be less satisfied when receiving telephone advice only [3-5].
GP cooperatives, being a new type of organisation in Dutch health care, aim to enhance the efficiency of current care provision. Besides the increased satisfaction of GPs, improved efficiency of the organisation may well be possible. Research into utilisation of out-of-hours services and patient flow can generate relevant insight into functioning of out-of-hours care organisations. Insight into how many patients utilise out-of-hours services, what type of consultation they receive, and which care process follows their contact with the GP cooperative can supply information about the efficiency of the out-of-hours care organisation. Utilisation of out-of-hours services and the type of consultations patients receive have been regularly investigated [2]. However, little is known about the care process after patients have contacted the GP cooperative for out-of-hours care. Only a few studies have included analyses on demand of follow-up care at the patient's own GP's practice, but showed wide variability in numbers and out-of-hours care settings. McKinley et al. [6] found that 54% of all patients who received telephone advice only during out-of-hours (provided by patients' own GP or by commercial deputising services) attended their own GP during office hours with the same problem within two weeks after their out-of-hours contact. For patients who received a home visit this proportion was 45%. Two studies on GPs working at a hospital's emergency department reported that 22 to 26% of the patients went to their own GP within three months after their contact with the GP at the emergency departmen [7,8]. Neither one of these studies, which used patient reports, give insight into how many of these patients were advised to see their own GP for follow-up care, or attended at their own initiative. A study by Shipman et al. [9] showed that GPs working out-of-hours in a practice-based setting referred about 17% of all patients to the patient's own GP the next day. This leaves unknown how many attended at their own initiative.
The purpose of this study is to determine the dimension of follow-up care after contact with a GP cooperative for out-of-hours care, and to gain insight into factors that are related to this follow-up care.
Methods
The study was conducted in the province of Limburg in the south of the Netherlands. In this province there are seven GP cooperatives operational, which cover a population of about 1.1 million (total population of the Netherlands is approximately 16 million). With respect to out-of-hours primary care, the province is organisationally divided in five regions. Two of these regions each have two cooperatives (NL and ML), one region (OZL) has one GP cooperative with two satellite centres, and in the other two regions (WM and MH) only one GP cooperative is operational. In the year 2002, a total of 307,346 patient contacts with these five organisations were registered. On average, 39% of these contacts were handled with telephone advice only, 51% consisted of a consultation at the GP cooperative, and 10% consisted of home visits. From March to June 2003, a sample of 2805 patients from these five GP cooperative organisations were sent a questionnaire within three weeks after they had been in contact with the GP cooperative. Sampling was performed per out-of-hours care organisation. With respect to patients who received telephone advice only and those who attended the GP cooperative, a computer program selected each fourth patient contact with the out-of-hours primary care organisation backwards from the moment of sampling. Since the number of home visits is limited, all 150 patients, who were visited by a GP from the cooperative prior to the moment of sampling, received a questionnaire. Per region 450 questionnaires were sent out; 150 to patients who received only telephone advice, 150 to patients who visited the GP cooperative, and 150 to patients who received a home visit. Because of parallel research, more questionnaires were sent out in one of the regions (WM): 1005 questionnaires equally distributed among the three types of patient contact with the GP cooperative. Three to four weeks after the questionnaire had been sent, a reminder was sent by mail to patients who had not returned the questionnaire, with the exception of the WM area. This study was part of a larger study on patient satisfaction with out-of-hours primary care [10]. The study was approved by the institutional medical ethics board of the University Hospital Maastricht.
Patients were asked to report whether they had attended their own GP within a week after their contact with the GP cooperative for out-of-hours care for the same medical complaint. They were also asked about their reasons for this attendance.
To investigate whether other variables could predict follow-up care, we also collected information on patient's age and gender, patient's education level (low, medium, high), and health insurances. Health insurance was used as a measure of the patient's socio-economic status: people with an income below a certain amount (some 60% of the population) are compulsorily insured under a public scheme (the Health Insurance Fund). Everyone else has to take out private insurance. Other variables that were collected included: whether the patient thought the diagnosis made by the GP of the cooperative was correct, urgency and severity of the medical complaint (as judged by the patient), patient's concern about his medical condition, whether the patient received the type of consultation (telephone advice, consultation at the GP cooperative, home visit) he or she expected, and the patient's opinion on performance of the GP cooperative on a 10-point scale (1 = very poor and 10 = very good).
To gain insight in how well our study sample represents the study base we collected data on patient gender, age and health insurance. This was done during a four-week period in May and June in 2002 at all GP cooperatives involved in this study.
Statistics
Descriptive statistics were applied to gain insight into the extent and patients' reasons of follow-up care after contact with the GP cooperative. We performed logistic regression analysis to determine any other factors related to follow-up care. All other variables except for the patient's reason for attendance were entered in the analysis. Variables that did not significantly contribute (P > 0.10) to the predictive model were excluded by backward deletion. Only patients, who were not advised or referred to attend their own GP, were included in the logistic regression analysis.
Results
Seventy-two of the 2805 questionnaires were excluded, either because they could not be delivered (patient had moved or had given a false address), the patient had died, or the patient was sent more than one questionnaire (in case of multiple contacts). Eventually the response was 42.4% (1160/2733). Of this group, 834 patients reported to have been helped by the GP or the doctor's assistant of the GP cooperative and did not receive care by a medical specialist at the hospital's emergency department (see figure 1). In total, 47.7% (398/834) of these patients reported to have attended their own GP within a week after their contact with the GP cooperative for the same medical problem. 19.9% (166/834) attended their own GP on advice of the GP or doctor's assistant of the GP cooperative. About one-third of all patients not referred or advised to attend their own GP, still went to see their own GP within a week after their contact with the cooperative at their own initiative.
Figure 1 Flow chart of patient follow-up care after contact with the GP cooperative for out-of-hours care. Grey boxes represent data on which the logistic regression has been performed. (TA = telephone advice; CC = consultation at the GP cooperative; HV = home visit; A&E = Accident and Emergency department)
Patient characteristics of the study sample with respect to the total group are very similar to the responders characteristics, except for age (Table 1). It is known that people who receive home visits are generally older, compared to those patients receiving telephone advice or attending the GP cooperative. Therefore, we also analysed age distribution per type of consultation and found no relevant difference between study base and responders (Table 2).
Table 1 Overview of patient characteristics of the study base and responders.
Study base* (n = 28,535) Responders (n = 1160)
n (%) n (%)
Gender
Male 13,234 (46.4%) 484 (45.5%)
Female 15,288 (53.6%) 579 (54.5%)
Total 28,522 (100%) 1063 (100%)
missing 13 (0.05%) 97 (8.4%)
Age#
0–20 7469 (30.0%) 282 (25.1%)
21–40 6548 (26.3%) 203 (18.1%)
40–60 5203 (20.9%) 242 (21.5%)
>60 5677 (22.8%) 396 (35.3%)
Total 24,897 (100%) 1123 (100%)
missing 15 (0.06%) 37 (3.2%)
Insurance
Public 21,875 (78.1%) 865 (76.2%)
Private 6,960 (21.9%) 270 (23.8%)
Total 27,625 (100%) 1135 (100%)
missing 910 (3.2%) 25 (2.2%)
* Based on patient contacts during a four-week period (May – June) in 2002
# Based on 24,897 contacts, excluding one region (WM).
Table 2 Age categories of patients contacting the GP cooperative for out-of-hours and age categories of responders in this study.
Telephone advice Consultation at the cooperative Home visits
Study base* (n = 5838) Responders (n = 366) Study base* (n = 4863) Responders (n = 392) Study base* (n = 1305) Responders (n = 402)
n (%) n (%) n (%) n (%) n (%) n (%)
Age
0–20 years 1786 (30.6%) 127 (35.5%) 1789 (36.8%) 146 (39.0%) 48 (3.7%) 9 (2.3%)
21–40 years 1587 (27.2%) 96 (26.8%) 1332 (27.4%) 81 (21.7%) 81 (6.1%) 26 (6.6%)
40–60 years 1226 (21.0%) 67 (18.7%) 1099 (22.6%) 82 (21.9%) 231 (17.7%) 93 (23.8%)
>60 years 1237 (21.2%) 68 (19.0%) 642 (13.2%) 65 (17.4%) 944 (72.4%) 263 (67.3%)
Total 5836 (100%) 358 (100%) 4862 (100%) 374 (100%) 1304 (100%) 391 (100%)
missing 2 (0.03%) 8 (2.0%) 1 (0.02%) 18 (4.6%) 1 (0.08%) 11 (2.7%)
* Based on patient contacts with two cooperatives in the province of Limburg during a four-week period (May – June) in 2002.
Overall
Of those patients who were not advised or referred to see their own GP within a week for the same problem but attended their own GP anyway, many reported that they had contacted their own GP because of worsening of their medical condition (23%) or because they were worried about their complaint (17%) (see Table 3). In only five percent of the cases, patients reported that wrong advice or treatment was the reason for them to attend their own GP after their contact with the GP cooperative.
Table 3 Reasons given by patients for seeking follow-up care with their own general practitioner after their contact with the GP cooperative for out-of-hours care.
Telephone advice Consultation at the GP cooperative Home visits Total
n (%) n (%) n (%) n (%)
Referred or advised by GP 62 (40.0) 60 (46.5) 44 (38.6) 166 (41.7)
Worsening of complaint 38 (24.5) 30 (23.3) 23 (20.2) 91 (22.9)
Wrong advice or treatment 11 (7.1) 6 (4.7) 3 (2.6) 20 (5.0)
Worried 26 (16.8) 15 (11.6) 26 (22.8) 67 (16.8)
Other reasons 18 (11.6) 18 (14.0) 18 (15.8) 54 (13.6)
Total 155 (100.0) 129 (100.0) 114 (100.0) 398 (100.0)
Besides these reasons, we identified four other variables to predict follow-up care: the patient's opinion on the correctness of the diagnosis, the patient's health insurance, patient satisfaction, and the severity of the medical problem as judged by the patient (Table 5). The model with these four variables was more effective in predicting those who did not attend their own GP within a week: 94.0% of non-attenders and 32.8% of attenders were correctly predicted, with an overall success rate of 73.1%. The overall variance in attendance accounted for was 13% (Cox and Snell test R2 = 0.13).
Table 5 Variables related to not-advised follow-up care with the patient's own GP after contact with the GP cooperative as dependent variable (1 = yes, 0 = no). Total group.
TOTAL GROUP B S.E. Wald df Sig. Odds ratio Exp(B) (90% C.I.)
INITIAL MODEL*
Diagnosis -1.481 0.329 20.200 1 0.000 0.228 (0.132–0.391)
Expectation -0.440 0.232 3.586 1 0.058 0.644 (0.439–0.944)
Health insurance 0.579 0.292 3.942 1 0.047 1.784 (1.104–2.882)
Gender -0.123 0.214 0.326 1 0.568 0.885 (0.622–1.259)
Age 0.001 0.005 0.020 1 0.889 1.001 (0.993–1.008)
Patient satisfaction 0.112 0.225 0.248 1 0.619 1.118 (0.773–1.619)
Performance score -0.024 0.107 0.049 1 0.824 0.977 (0.819–1.164)
Severity 0.486 0.303 2.567 1 0.109 1.625 (0.987–2.676)
Urgency -0.272 0.291 0.875 1 0.349 0.762 (0.472–1.229)
Worried 0.399 0.332 1.441 1 0.230 1.491 (0.863–2.576)
Education Low 1.142 2 0.565
Middle -0.249 0.253 0.964 1 0.326 0.780 (0.514–1.183)
High -0.177 0.302 0.345 1 0.557 0.838 (0.510–1.376)
Constant -0.134 1.372 0.010 1 0.922 0.874
FINAL MODEL#
Diagnosis a -1.758 0.289 37.078 1 0.000 0.172 (0.107–0.277)
Health insurance b 0.422 0.232 3.320 1 0.068 1.525 (1.042–2.232)
Patient satisfaction c 0.218 0.121 3.236 1 0.072 1.243 (1.019–1.517)
Severity d 0.554 0.222 6.202 1 0.013 1.740 (1.207–2.510)
Constant -0.403 0.519 0.602 1 0.438 0.669
*31.3% (209/668) missing
#13.2% (88/668) missing
a Diagnosis: 1 = right. 0 = wrong.
b Insurance: 1 = public insurance. 0 = private insurance.
c Patient satisfaction (five point scale): 1 representing highly satisfied en 5 very dissatisfied.
d Severity: 1= severe; 0 = not severe.
Telephone advice
With respect to patients who received telephone advice only, worsening of the complaint and worry about the medical condition were the most frequently reported reasons for patients to attend their own GP without being advised or referred (Table 3). In addition to these reasons, we identified the same three variables to predict follow-up care as for the total sample: correctness of the diagnosis, the severity of the medical problem as judged by the patient, and the patient's health insurance (Table 6). The model with these three variables was also more effective in predicting those who did not attend their own GP within a week: 95.0% of non-attenders and 47.1% of attenders were correctly predicted, with an overall success rate of 74.8%. The variance in attendance accounted for overall was 26% (Cox and Snell test R2 = 0.26).
Table 6 Variables related to not-advised follow-up care with the patient's own GP after contact with the GP cooperative as dependent variable (1 = yes. 0 = no). Telephone advice
B S.E. Wald df Sig. Odds ratio Exp(B) (90% C.I.)
INITIAL MODEL*
Diagnosis -2.471 0.617 16.036 1 0.000 0.085 (0.031–0.233)
Expectation -0.012 0.407 0.001 1 0.977 0.988 (0.506–1.929)
Health insurance 1.015 0.453 5.030 1 0.025 2.760 (1.311–5.810)
Gender -0.571 0.388 2.165 1 0.141 0.565 (0.298–1.070)
Age -0.006 0.009 0.410 1 0.522 0.994 (0.980–1.009)
Patient satisfaction -0.353 0.374 0.887 1 0.346 0.703 (0.380–1.301)
Performance score -0.286 0.176 2.637 1 0.104 0.752 (0.563–1.004)
Severity 0.614 0.465 1.743 1 0.187 1.848 (0.860–3.970)
Urgency -0.189 0.506 0.140 1 0.708 0.827 (0.360–1.902)
Worried -0.170 0.531 0.103 1 0.748 0.844 (0.352–2.019)
Education Low 1.724 2 0.422
Middle -0.567 0.451 1.585 1 0.208 0.567 (0.270–1.190)
High 0.036 0.473 0.006 1 0.939 1.037 (0.476–2.258)
Constant 4.101 2.355 3.031 1 0.082 60.372
FINAL MODEL#
Diagnosis a -2.749 0.478 33.081 1 0.000 0.064 (0.029–0.140)
Health Insurance b 0.860 0.388 4.905 1 0.027 2.363 (1.248–4.476)
Severity c 0.578 0.349 2.734 1 0.098 1.782 (1.003–3.166)
Constant 0.863 0.573 2.266 1 0.132 2.369
* 26.5% (62/234) missing
# 12.0% (28/234) missing
a Diagnosis: 1 = right. 0 = wrong.
b Insurance: 1 = public insurance. 0 = private insurance.
c Severity: 1 = severe; 0 = not severe.
Consultation at the GP cooperative
With respect to patients who went to the GP cooperative for consultation, again, worsening of the complaint and worry about the patient's medical condition were reported as most important reasons to attend the patient's own GP, without being advised or referred. Besides these reasons, we identified two variables to predict follow-up care: correctness of the diagnosis, and the patient's concern about his or her medical problem (Table 7). The model with these two variables was more effective in predicting those who did not attend their own GP within a week: 96.4% of non-attenders and 25.0% of attenders were correctly predicted, with an overall success rate of 76.8%. However, the overall variance in attendance accounted for was small (Cox and Snell test R2 = 0.11).
Table 7 Variables related to not-advised follow-up care with the patient's own GP after contact with the GP cooperative as dependent variable (1 = yes. 0 = no). Consultation at the GP cooperative.
CONSULTATION AT GP COOPERATIVE B S.E. Wald df Sig. Odds ratio Exp(B) (90% C.I.)
INITIAL MODEL*
Diagnosis -1.934 0.662 8.525 1 0.004 0.145 (0.049–0.430)
Expectation 0.502 0.660 0.577 1 0.447 1.652 (0.557–4.894)
Health insurance -0.038 0.555 0.005 1 0.945 0.962 (0.386–2.397)
Gender -0.079 0.418 0.036 1 0.850 0.924 (0.464–1.839)
Age 0.003 0.010 0.106 1 0.745 1.003 (0.987–1.020)
Patient satisfaction 0.211 0.486 0.188 1 0.664 1.235 (0.555–2.745)
Performance score 0.035 0.239 0.021 1 0.884 1.035 (0.699–1.534)
Severity 0.549 0.549 0.999 1 0.318 1.732 (0.701–4.274)
Urgency -0.531 0.513 1.072 1 0.300 0.588 (0.253–1.367)
Worried 1.014 0.689 2.165 1 0.141 2.757 (0.887–8.566)
Education Low 0.341 2 0.843
Middle 0.244 0.510 0.229 1 0.632 1.277 (0.551–2.957)
High -0.130 0.565 0.053 1 0.818 0.878 (0.347–2.223)
Constant -1.564 3.021 0.268 1 0.605 0.209
FINAL MODEL#
Diagnosis a -2.075 0.484 18.359 1 0.000 0.126 (0.057–0.279)
Worried b 1.073 0.520 4.266 1 0.039 2.925 (1.244–6.876)
Constant -0.083 0.651 0.016 1 0.899 0.920
*33.6% (84/250) missing
# 6.8% (17/250) missing
a Diagnosis: 1 = right. 0 = wrong.
b Worried: 1 = worried about own medical complaint. 0 = not worried.
Home visits
Similar to patients who received telephone advice only or visited the GP cooperative for consultation, patients who received a home visit reported that worry about their medical problem and worsening of the complaint were the main reasons to contact their own GP within a week after their contact with the GP cooperative. Only 3 patients (2.7%) said to have attended their own GP within a week because they believed to have received incorrect advice or treatment by the home visiting GP of the GP cooperative.
We also investigated other variables and their relationship with follow-up care, but none of the variables entered in the logistic regression analysis was able to predict whether the patient did or did not attend his or her own GP within one week after they had been visited by the GP of the cooperative at home (Table 8).
Table 8 Variables related to not-advised follow-up care with the patient's own GP after contact with the GP cooperative as dependent variable (1 = yes. 0 = no). Home visits.
HOME VISITS B S.E. Wald df Sig. Odds ratio Exp(B) (90% C.I.)
INITIAL MODEL*
Diagnosis 0.468 0.752 0.388 1 0.533 1.597 (0.464–5.503)
Expectation -1.134 0.631 3.228 1 0.072 0.322 (0.114–0.909)
Health insurance 0.507 0.749 0.458 1 0.499 1.661 (0.484–5.695)
Gender 0.550 0.440 1.567 1 0.211 1.734 (0.841–3.572)
Age -0.001 0.012 0.003 1 0.959 0.999 (0.980–1.019)
Patient satisfaction 0.990 0.508 3.801 1 0.051 2.691 (1.167–6.202)
Performance score 0.491 0.252 3.801 1 0.051 1.634 (1.080–2.473)
Severity 0.881 1.228 0.515 1 0.473 2.414 (0.320–18.198)
Urgency 1.007 0.944 1.139 1 0.286 2.738 (0.580–12.932)
Worried 0.320 0.813 0.155 1 0.694 1.377 (0.362–5.240)
Education Low 3.522 2 0.172
Middle -0.161 0.455 0.125 1 0.724 0.851 (0.402–1.801)
High -1.868 0.999 3.496 1 0.062 0.154 (0.030–0.799)
Constant -9.070 3.334 7.402 1 0.007 0.000
FINAL MODEL
- - - - - - -
* = 34.2% (63/184) missing
Discussion
This study showed that almost half of all respondents received follow-up care at their own GP's practice, within a week, for the same medical complaint for which they had contacted the GP cooperative. Although a substantial number of these patients (40%) were referred or advised by the cooperative's GPs or doctor's assistants to do so, about 60% of these patients attended their own GP at their own initiative.
According to the Statistics Netherlands Database over the last four years, about 27% of all patients require follow-up care after they have seen their GP for a medical complaint [11]. The fact that in this study substantially more patients received follow-up care can be explained by several factors. First, the medical complaints presented during out-of-hours may be more severe compared to during office hours, and therefore require follow-up care more often. Second, the GP cooperative focuses mainly on medical complaints that cannot wait until the next day; all other non-urgent disorders are often referred to the patient's own GP the next day. Third, patients may not feel fully confident or are not fully satisfied with the way their complaint has been taken care of and want to check this with their own GP. Since we have not compared the situation during office hours with outside office hours, it remains unclear which of these factors contributes the most to the difference in numbers of follow-up care.
In addition to the fact that patients have reported to attend their own GP at their own initiative mainly because their medical condition worsened or that they were worried, we found that generally three other variables were related to follow-up care. The most important variable was whether the patient believed that the correct diagnosis had been made by the cooperative's GP or doctor's assistant. However, this variable might have been biased when the patient's own GP made another diagnosis than either the cooperative's GP or doctor's assistant, since patients may have more confidence in their own GP than in an unknown GP or doctor's assistant. We also found that health insurance was a predictor of follow-up care without referral. This may be explained by the fact that privately insured patients may not be fully reimbursed for these consultations. This implies some kind of financial incentive. In addition, research has shown that patients with lower socio-economic status more frequently attend their GP [12], which is in line with our findings.
For those who received a home visit, no model could be established that predicts follow-up care at the patient's own GP cooperative. Regarding patients who received telephone advice only, or attended the GP cooperative, we found that the correctness of the diagnosis as judged by the patient was a strong predictor for follow-up care. However, many patients who received home visits will already have a known diagnosis, which may give this variable limited predictive value in this patient category. Furthermore, for patients who received a home visit the patient's own GP may often take the initiative to visit the patient, possibly because of the severity of the complaint or co-morbidity, instead of the patient taking the initiative to contact the GP. Patients receiving home visits often suffer from more severe conditions and are significantly older than those helped by telephone advice and those who visited the GP cooperative. Also these two factors may give some explanation for not finding a model to predict re-attendance, because it is known that elderly people more frequently contact the GP and that the GP routinely visits the patient to check on his or her condition.
An important limitation of the study is that the response rate to the questionnaire was only 42.4%. Therefore, care should be taken with generalising these results to all patient contacts with GP cooperatives. It could be that the number of patients seeking follow-up care in this study has been overestimated or underestimated. However, the proportion of patients who went to their own GP for the same complaint was similar to that reported in the literature [6]. In addition, the number of patients who attended their own GP for the same medical complaint on advice of the cooperative's GP or doctor's assistant reported in this study (19%), was fairly similar to that reported by Shipman et al. [9] (17%).
This study did not provide insight into appropriateness of follow-up care, but is merely a first step in revealing the extent of follow-up care after contact with a GP cooperative. Therefore, it remains unclear whether the proportion of patients who seek follow-up care by their own GP is appropriate or represents inefficient care. The latter meaning that too many patients require follow-up care because they believed that care at the GP cooperative was insufficient. Future research is warranted to confirm our study findings and to investigate the appropriateness of follow-up care.
Conclusion
This study is a first step in providing insight into the dimension of follow-up care after a patient has contacted the GP cooperative for out-of-hours primary care. We showed that about half of all respondents who contacted the GP cooperative attended their own GP for the same medical problem within a week. Only a minority of the patients was referred or advised to do so. Most cited reasons were worsening of and worry about the complaint. With respect to those that attended their own GP for the same problem on their own initiative, the perception that the correct diagnosis had been made at the GP cooperative was a strong predictor of non-attendance.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
CU participated in the design of the study, performed the statistical analysis, and drafted the manuscript. PZ, SH, AA, GW, CS, and HC participated in the design of the study, supervised the project, and provided critical edits to this manuscript.
Table 4 Characteristics of patients not advised for follow-up care with their own general practitioner.
Telephone advice (n = 234) Consultation at the GP cooperative (n = 250) Home visits (n = 184) Total (n = 668)
n (%) n (%) n (%) n (%)
Gender
Male 92 (40.9) 98 (48.5) 87 (49.2) 277 (45.9)
Female 133 (59.1) 104 (51.5) 90 (50.8) 327 (54.1)
Missing 3.8% 19.2% 2.7% 9.6%
Age (years) 30.7 (SD 24.2) 29.0 (SD 23.8) 63.5 (SD 19.1) 39.2 (SD 27.3)
Missing 2.1% 6.4% 2.7% 3.9%
Education level
Low 55 (25.2) 49 (21.0) 79 (48.8) 183 (29.9)
Average 111 (50.9) 125 (53.6) 60 (37.0) 296 (48.3)
High 52 (23.9) 59 (25.3) 23 (14.2) 134 (21.9)
Missing 6.8% 6.8% 12.0% 8.2%
Health insurance
Public 165 (71.1) 182 (74.3) 151 (83.4) 498 (75.7)
Private 67 (28.9) 63 (25.7) 30 (16.6) 160 (24.3)
Missing 0.9% 2.0% 1.6% 1.5%
Received type of consultation as expected
Yes 84 (38.0) 206 (84.8) 151 (85.8) 441 (68.9)
No 137 (62.0) 37 (15.2) 25 (14.2) 199 (31.1)
Missing 5.6% 2.8% 4.3% 4.2%
Patient's perceived severity of complaint
Not severe 87 (37.8) 85 (34.7) 9 (5.1) 181 (27.8)
Severe 143 (62.2) 160 (65.3) 168 (94.9) 471 (72.2)
Missing 1.7% 2.0% 3.8% 2.4%
Patient's perceived urgency of complaint
Not urgent 80 (35.4) 62 (25.4) 13 (7.4) 155 (24.0)
Urgent 146 (64.6) 182 (74.6) 162 (92.6) 490 (76.0)
Missing 3.4% 2.4% 4.9% 3.4%
Patient's worry about complaint
Not worried 36 (15.5) 43 (17.7) 16 (9.1) 95 (14.6)
Worried 196 (84.5) 200 (82.3) 159 (90.9) 555 (85.4)
Missing 0.9% 2.8% 4.9% 2.7%
Patient's perceived correctness of diagnosis
Not correct 47 (22.4) 25 (10.5) 18 (12.0) 90 (15.0)
Correct 163 (77.6) 214 (89.5) 132 (88.0) 509 (85.0)
Missing 10.3% 4.4% 18.5% 10.3%
Patient satisfaction 2.43 (SD 0.96) 2.03 (SD 0.70) 1.99 (SD 0.87) 2.16 (SD 0.87)
Missing 0.9% 0% 1.1% 0.6%
Performance score 6.7 (SD 2.0) 7.7 (SD 1.4) 7.6 (SD 1.8) 7.3 (SD 1.8)
Missing 5.6% 2.8% 4.3% 4.2%
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| 15946382 | PMC1164410 | CC BY | 2021-01-04 16:29:13 | no | BMC Fam Pract. 2005 Jun 9; 6:23 | utf-8 | BMC Fam Pract | 2,005 | 10.1186/1471-2296-6-23 | oa_comm |
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BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-5-191594148510.1186/1471-230X-5-19Case ReportStability of colon stem cell methylation after neo-adjuvant therapy in a patient with attenuated familial adenomatous polyposis Kim Jung Yeon [email protected] Robert W [email protected] Darryl [email protected] Departments of Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA2 Colorectal Surgery, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA2005 7 6 2005 5 19 19 8 2 2005 7 6 2005 Copyright © 2005 Kim et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Methylation at certain human CpG rich sequences increases with age. The mechanisms underlying such age-related changes are unclear, but methylation may accumulate slowly in a clock-like manner from birth and record lifetime numbers of stem cell divisions. Alternatively, methylation may fluctuate in response to environmental stimuli. The relative stability of methylation patterns may be inferred through serial observations of the same colon.
Case presentation
A 22 year-old male with attenuated familial adenomatous polyposis received neo-adjuvant chemotherapy and radiation prior to surgery for rectal adenocarcinoma. Colon crypt methylation patterns before and after neo-adjuvant therapy (62 days apart) were essentially identical with respect to percent methylation and diversity. Consistent with previous studies, methylation patterns recorded no evidence for enhanced colon crypt stem cell survival with a germline mutation (codon 215) proximal to the mutation cluster region of APC.
Conclusion
The inability of neo-adjuvant therapy to significantly alter crypt methylation patterns suggests stem cells are relatively protected from transient environmental changes. Age-related methylation appears to primarily reflect epigenetic errors in stem cells that slowly accumulate in a clock-like manner from birth. Therefore, life-long human stem cell histories are potentially written within and may be read from somatic cell epigenomes.
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Background
Recent studies reveal methylation of certain CpG rich sequences increases with age in normal human colon [1,2]. These CpG rich sequences, like most CpG islands [3], are unmethylated at birth and appear to somatically accumulate cytosine methylation over many years. The etiology of such age-related methylation is uncertain, but one possible mechanism is random somatic errors during DNA replication [4]. By this mechanism, methylation would inherently increase with cell division because the CpG rich sequences are initially unmethylated. Consistent with random replication errors, methylation patterns (the 5' to 3' order of methylation) differ between cells within the same colon [4] and overall levels increase with age [1,2].
Random epigenetic errors potentially records cell histories because methylation exhibits somatic inheritance [3]. Daughter cells generally inherit the same methylation pattern as their parents, but rarely replication errors occur which are subsequently passed to progeny (Figure 1). Somatic errors can only accumulate within stem cells because non-stem cells and their errors are lost within days from normal crypt cell differentiation and migration [4,5]. Therefore, CpG rich sequences, like sequences used to reconstruct species evolution [6], potentially function as somatic epigenetic molecular clocks that can be used to reconstruct somatic cell evolution. Preliminary studies are consistent with this hypothesis because seemingly random human colon crypt methylation patterns appear to record life-long stem cell fates within crypt niches [4].
Although crypt methylation patterns from multiple different aged colons are consistent with a clock-like accumulation of epigenetic errors, environmental factors may also alter methylation. Age-related methylation levels differ between individuals of the same age [1,2,4], which may represent life-long biological differences between individuals, stochastic differences, or transient responses to environmental changes. It may be possible to distinguish between these alternatives through serial examinations of the same colon. A somatic molecular epigenetic clock predicts the amount of additional methylation is a function of the time between examinations. In contrast, if methylation is more labile, patterns may quickly change after a severe environmental insult. Here we sample methylation 62 days apart in the same colon before and after cytotoxic neo-adjuvant chemotherapy and radiation for a rectal adenocarcinoma. Methylation patterns were essentially unchanged, suggesting crypt stem cell epigenomes are relatively protected from transient environmental changes.
Case presentation
A 22 year-old male presented with anemia. Colonoscopy revealed eight hyperplastic to adenomatous polyps ranging in size from 0.6 to 3.0 cm scattered through the colon, and a four cm, locally invasive, ulcerative rectal adenocarcinoma five cm from the anus. The clinical impression was attenuated familial adenomatous polyposis (AFAP), with a germline heterozygous truncating APC mutation at codon 215 (CAG to TAG; Gln to stop). The first sample for methylation analysis (Day 1) was a biopsy of normal appearing sigmoid colon.
The patient received neo-adjuvant chemotherapy consisting of three, two week cycles of oxaliplatin and 5-fluorouracil, with the last cycle ending two weeks before colectomy. The patient exhibited Grade 3 toxicity with mucositis, stomatitis, diarrhea, and parasthesia. He also received radiation therapy to the pelvis (2,500 centigrays over five days), ending 19 days before colectomy. The specimens obtained for analysis were outside of the primary radiation field.
Colectomy revealed approximately 50 scattered small hyperplastic to adenomatous polyps (0.1 to 0.3 cm), and a 0.7 cm ulcerative lesion at the site of the rectal adenocarcinoma containing a few residual neoplastic glands with superficial invasion of the muscularis propria. Lymph nodes were negative for metastatic carcinoma. The second specimen for analysis (Day 62) was taken from a normal appearing region of the sigmoid colon.
Methylation analysis
Individual crypts were isolated from the first (a colonoscopic biopsy) and last (a 1–2 cm2 patch of mucosa) specimens using an EDTA-containing solution as previously described [4]. DNA, extracted from individual crypts placed in microfuge tubes, was bisulphite treated (to convert C's to U's, whereas methylated-C's are unchanged), and amplified by PCR at two different CpG rich regions called CSX (nine different crypts) and BGN (eight of the same crypts). PCR products were cloned into bacterial vectors and eight clones representing individual PCR products were sequenced from each crypt [4].
Crypt patterns (5' to 3' order of methylation) before and after neo-adjuvant therapy were complex and seemingly random, consistent with stochastic epigenetic errors (Figure 2). The CSX (cardiac specific homeobox) sequence or "tag" is autosomal (5q34, also called Mint23 [7]) and has eight CpG sites located in the 3' untranslated region and 256 possible patterns ([2]8 possible 5' to 3' orders of methylation). The BGN (biglycan) tag is on the X-chromosome (Xq28, one allele per male cell) and has nine CpG sites located just distal to the promoter and 512 ([2]9) possible patterns. These tags are not expressed in the colon and therefore any methylation is unlikely to confer selection [4]. Crypt methylation patterns are summarized by two statistics: percent methylation (proportion of methylated CpG sites) and diversity or numbers of unique tags per eight sampled from each crypt.
Methylation tag values differed between crypts sampled before and after neo-adjuvant therapy, but these differences were not significant and did not follow a trend (Figure 2). Average CSX tag values were lower and BGN tags values were higher after neo-adjuvant therapy. A model [4] in which random methylation errors start to accumulate from birth is consistent with the lack of significant tag differences between serial specimens and the observed crypt variations. The model can be simulated on a computer (using previously published human colon crypt parameters [4]) to estimate average tag values and intervals that include 95% of simulated individual crypt outcomes (Figure 3). Different crypts within the same colon may have different methylation patterns because errors are stochastic and independent between cells and CpG sites. Methylation patterns before and after neo-adjuvant therapy are predicted to be similar because additional errors acquired in the 62 days between the samples are relatively insignificant compared to the 22 years since birth. Observed crypt tag values were generally consistent with the predictions of the model (Figure 3).
In addition to numbers of divisions since birth, methylation patterns (Figure 2) may also record stem cell dynamics. For example, crypt diversity or unique tags per crypt appears to reflect crypt stem cell numbers [4]. This AFAP patient appeared to have normal numbers of crypt stem cells because his crypt diversity (3.22 and 2.33 average unique CSX crypt tags) was consistent with a normal colon (Figure 3). In contrast, significantly greater crypt diversity has been observed in some familial adenomatous polyposis (FAP) patients, with an average of 3.9 unique CSX tags per FAP crypt compared to 3.0 with non-FAP crypts, which translates to about 4-fold more FAP crypt stem cells [8]. Greater numbers of crypt stem cells correlate with germline APC mutations around the mutation cluster region (MCR) that remove some but not all β-catenin binding repeats [8]. Consistent with this observation, this patient had a truncating germline mutation (Codon 215, CAG (Gln) to TAG (stop)) that removed all β-catenin binding repeats.
Conclusion
The ability to extract historical information from normal appearing tissue is limited. However, recent advances in molecular phylogeny illustrate that genomes automatically record ancestry and time [6]. Although sequences are usually faithfully copied, errors rarely occur, which are also passed from generation to generation (Figure 1). Sequence comparisons can be used to reconstruct species evolution that occurred thousands to millions of years ago. The greater the differences between sequences, the greater the likely time since they shared a common ancestor.
Sequences comparisons depend on relatively constant error rates (a molecular clock hypothesis [6]) because time is proportional to the number of sequence differences. Although it is possible that mutation rates vary over time from environmental changes, species histories inferred from sequences are generally consistent with relatively constant mutation rates over millions of years [6]. In theory, molecular phylogeny can also be applied to somatic cell evolution because somatic cell genomes are also passed from cell to cell. Although somatic cell sequence comparisons are impractical because mutation rates are low relative to human lifetimes, epigenetic errors also potentially record ancestry because methylation exhibits somatic inheritance [3]. Epigenetic replication fidelity is less than sequence fidelity because age-related methylation is readily detected in the human colon [1,2]. Colon crypt methylation patterns have been used to infer normal human crypt niche dynamics [4], and that most directly adjacent adult crypts are not closely related because their methylation patterns differ [9]. Stem cell survival can also be inferred from methylation pattern diversity (number of unique tags). For example, increased crypt diversity and enhanced crypt stem cell survival is observed in some FAP patients with truncating germline mutations that remove some but not B-catenin binding repeats [7]. In contrast and as with this patient, germline APC mutations in the N-terminus of APC have not been associated with enhanced stem cell survival [7], and are associated with fewer polyps and AFAP [10].
A general problem with the use of sequences to reconstruct ancestry is the stochastic nature of most errors. It is difficult to prove stochastic sequence errors actually accumulate in a clock-like manner, and error rates may potentially fluctuate in response to environmental changes. Highly variable error rates would obscure ancestral information because many errors may accumulate in relatively short periods.
Although it is difficult to test a molecular clock hypothesis for species evolution, it is possible to test a somatic epigenetic molecular clock through serial examinations of the same individual. This Case Report experimentally tests the hypothesis that age-related methylation predominantly reflect stem cell errors that start to slowly accumulate from birth. Consistent with this hypothesis, methylation patterns were not significantly different after 62 days despite intervening neo-adjuvant therapy. Neo-adjuvant chemotherapy targets rapidly dividing cells and normal colon is relatively unaffected, although diarrhea (Grade 3 toxicity) may and did occur in this patient. However, the therapy did not appear to alter stem cell methylation patterns, suggesting stem cells are protected from this type of therapy. Therefore, age-related methylation appeared to be relatively stable and primarily a function of the time or number of stem cell divisions since birth. Further serial examinations with more patients and longer intervals may provide additional evidence supporting or refuting this hypothesis.
In summary, age-related methylation appears to slowly accumulate in a clock-like manner in stem cells from birth. The stochastic nature of somatic epigenetic errors requires an algorithm for interpretation, which essentially translates the sophisticated methods used to extract ancestry from species genomes to reconstruct somatic cell histories. The unseen fates of human crypt stem cells may be potentially read from anyone because methylation automatically records their autobiographies.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Dr. Kim performed most of the experiments and helped write the manuscript. Dr. Beart provided the specimens and clinical information. Dr. Shibata analysed the data and wrote the manuscript
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We thank the assistance of the patient who cooperated with this study. Written consent was obtained from the patient for publication of this study. We thank the technical assistance of Mario Campuzano. This work was supported by a grant from the National Institutes of Health (DK61140).
Figures and Tables
Figure 1 Somatic cell inheritance of methylation patterns. The tags used in this study are unmethylated at birth (unmethylated CpG sites represented by open circles, with filled circles representing methylated sites). Generally the same methylation pattern is faithfully copied and passed on to daughter cells. However, random epigenetic errors may occur, and these errors are also subsequently inherited. Methylation patterns (5' to 3' order of methylation) potentially encode numbers of divisions since birth and ancestral relationships between cells. Of note, new errors in non-stem cells will be lost during normal crypt differentiation, migration and death.
Figure 2 Crypt methylation before and neo-adjuvant therapy. There are eight tags per crypt with each tag arranged in a 5' to 3' horizontal order of CpG sites. CSX (eight CpG sites) and BGN (nine CpG sites) tags were sampled from eight of the same crypts. Crypt methylation patterns are summarized by percent methylation and numbers of unique tags per crypt. Methylation patterns were not significantly different before and after neo-adjuvant therapy, with averages summarize in the Table. The seemingly random methylation patterns are consistent with a model [4] in which stochastic methylation errors start to accumulate from birth.
Figure 3 Crypt methylation tags are consistent with a clock-like stochastic accumulation of methylation errors from birth. Such a model using previously published crypt parameters [4] can be simulated on a computer to yield expected individual (dotted lines include 95% of simulated crypt outcomes) and average (solid grey line) crypt values. Little difference is expected between the initial (black X 's are individual crypt values and black triangles are average values) and final specimens (red bars are individual crypt values and red circles are average values) because by this model, methylation errors slowly accumulate from birth, and few additional changes are expected to accumulate during the 62 days between specimens.
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| 15941485 | PMC1164411 | CC BY | 2021-01-04 16:03:27 | no | BMC Gastroenterol. 2005 Jun 7; 5:19 | utf-8 | BMC Gastroenterol | 2,005 | 10.1186/1471-230X-5-19 | oa_comm |
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-641587635410.1186/1471-2164-6-64Research ArticleGene expression profile of cervical tissue compared to exfoliated cells: Impact on biomarker discovery Steinau Martin [email protected] Daisy R [email protected] Mangalathu S [email protected] Suzanne D [email protected] Mack T [email protected] Elizabeth R [email protected] Centers for Disease Control and Prevention, Atlanta, GA, USA2 University of Michigan, Ann Arbor, MI, USA2005 5 5 2005 6 64 64 2 3 2005 5 5 2005 Copyright © 2005 Steinau et al; licensee BioMed Central Ltd.2005Steinau et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Exfoliated cervical cells are used in cytology-based cancer screening and may also be a source for molecular biomarkers indicative of neoplastic changes in the underlying tissue. However, because of keratinization and terminal differentiation it is not clear that these cells have an mRNA profile representative of cervical tissue, and that the profile can distinguish the lesions targeted for early detection.
Results
We used whole genome microarrays (25,353 unique genes) to compare the transcription profiles from seven samples of normal exfoliated cells and one cervical tissue. We detected 10,158 genes in exfoliated cells, 14,544 in the tissue and 7320 genes in both samples. For both sample types the genes grouped into the same major gene ontology (GO) categories in the same order, with exfoliated cells, having on average 20% fewer genes in each category. We also compared microarray results of samples from women with cervical intraepithelial neoplasia grade 3 (CIN3, n = 15) to those from age and race matched women without significant abnormalities (CIN1, CIN0; n = 15). We used three microarray-adapted statistical packages to identify differential gene expression. The six genes identified in common were two to four fold upregulated in CIN3 samples. One of these genes, the ubiquitin-conjugating enzyme E2 variant 1, participates in the degradation of p53 through interaction with the oncogenic HPV E6 protein.
Conclusion
The findings encourage further exploration of gene expression using exfoliated cells to identify and validate applicable biomarkers. We conclude that the gene expression profile of exfoliated cervical cells partially represents that of tissue and is complex enough to provide potential differentiation between disease and non-disease.
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Background
Early cancer detection requires noninvasive sampling is for general screening populations. Exfoliated cervical cells have been used for cytologic screening of cervical cancer. These accessible cells could also be ideal for molecular screening based on gene expression if their mRNA can be isolated and is representative of the expression profile of the underlying tissue. We have previously shown that satisfactory RNA can be isolated from pap smear material in amounts sufficient for microarray analysis [1]. However since exfoliated cells are keratinized and terminally differentiated, it remains to be demonstrated that they have an active mRNA profile that adequately represents the molecular signature of cervical tissue. While one group reported success using exfoliated oral cells as a source of gene expression biomarkers [2], others have not obtained satisfactory results [3].
This study addresses the representation of gene expression profiles in exfoliated cells and full thickness epithelium. To further explore the possibility of using exfoliated cells for molecular screening, we compared the gene expression in samples with cervical intraepithelial neoplasia grade 3 (CIN3) to those without or only low grade lesions (CIN0, CIN1).
Results
Gene expression profiles in cervical exfoliated cells and tissue
The MWG A, B and C human 30 k arrays include probes evaluating 25,353 different genes of the transcribed human genome. Of these genes, 14,544 (57%) were detected in uterine cervix tissue and 10,158 (40%) in exfoliated cells (Figure 1). Of those detected in tissue, 7,320 (50.3%) were also detected in exfoliated cells. Genes detected in each sample type grouped into the same major GO categories in the same order of abundance (Figure 2), although the numbers in each category were on average 20% less in exfoliated cells (range 4.4 – 27.3%). The exception is the GO category, regulation of gene expression, epigenetics, in which the few genes that were detected were only found in exfoliated cells.
Figure 1 Number of genes expressed above cutoff in cervical tissue and exfoliated cells.
Figure 2 GOs of biological processes in cervical exfoliated cells and full thickness epithelium. While expressed genes represent major categories in the same order, a proportion of each (4.4 to 27.3 %) is not present in exfoliated cells.
EASE analysis of the 7,224 genes detected only in cervical tissue found the biologic themes of structural cell components, ribosomal function and structure, ion transport and regulation to be enriched relative to their representation on the arrays (Table 1). Biologic themes enriched in genes detected only in exfoliated cells were mainly differentiation processes like neurogenesis, morphogenesis, oncogenesis, organogenesis and development (Table 2).
Table 1 Biological themes enriched in genes detected only in tissue
Gene Category # of Genes EASE score†
Molecular Function Structural molecule activity 228 2.27E-07
Structural constituent of ribosome 78 4.21E-05
Extracellular matrix structural constituent 38 4.12E-04
Monovalent cation \:proton antiporter activity 7 3.90E-03
Sodium \:hydrogen antiporter activity 7 3.90E-03
Transcriptional repressor activity 18 1.31E-02
Voltage-gated sodium channel activity 9 1.58E-02
Cation \:cation antiporter activity 8 2.24E-02
Cellular Component Large ribosomal subunit 23 1.06E-03
Ribosome 90 2.20E-03
Cytosolic ribosome 24 6.14E-03
Cytosolic large ribosome subunit 16 9.87E-03
Cellular component unknown 160 1.22E-02
Collagen 15 1.36E-02
Ribonucleoprotein complex 127 1.52E-02
Biological process unknown 252 7.91E-04
†Cut-off <0.025
Table 2 Biological themes enriched in genes detected only in exfoliated cells
Gene Category # of Genes EASE score†
Biological process Neurogenesis 62 6.46E-04
Development 205 1.38E-03
Behavior 18 6.30E-03
Morphogenesis 122 1.04E-02
Regulation of cell adhesion 7 1.10E-02
Oncogenesis 14 1.38E-02
Cellular process 614 1.82E-02
Organogenesis 108 1.82E-02
* Chromatin remodeling complex 10 6.73E-03
Cell fraction 91 2.44E-02
** Protein tyrosine/serine/threonine phosphatase activity 91 3.23E-03
†Cut-off <0.025; *Cellular Component; **Molecular Function
Gene expression comparison of CIN3 samples with CIN0 and CIN1 controls
Of the 5461 genes on the A arrays that were included in this analysis, the average expression of 95 were at least 2-fold greater whereas 10 were at least 2-fold lower in CIN3 compared to CIN 0/CIN 1. Based on CV, the CIN 3 class showed more heterogeneity than the CIN 0 class (Figure 3) or the combined CIN 0/CIN 1 controls (data not shown).
Figure 3 Plot of CV differences between CIN3 and CIN0 samples. CVs were calculated from normalized, log2 transformed sARM values for each gene. If variance in gene expression was random in both groups, approximately half the genes would be more variable in CIN 3 (positive) and half in CIN 0 (negative). The vertical dashed line divides the total number of genes in half (at gene 2730), the solid line marks where were the CVs are equal (at gene 3364). The increased number of genes with greater CV in the CIN 3 group indicates greater heterogeneity of expression profiles in this disease group compared with the control (CIN 0) group.
The univariate parametric p-values of the 20 most significant genes that discriminate between the CIN3 and the CIN 0/CIN 1 groups selected in the BRB Array Tools two-sample T-test ranged between 0.00077 – 0.0067316. None had a false discovery rate (FDR) of less than 10%. The GO class comparison yielded 11 categories significant at the nominal 0.005 level of permutation tests. These were helicase activity, replication fork, DNA-directed DNA polymerase activity, regulation of viral life cycle, peroxidase activity, DNA replication and endoplasmatic reticulum membrane, or a closely related subcategory (Table 3).
Table 3 GO categories with higher than expected numbers of genes differentially regulated in CIN3(p < 0.005).
GO category GO description LS Permut. p-value KS Permut. p-value
0004376 Helicase activity 0.00129 0.00718
0001724 RNA helicase activity 0.00196 0.01317
0030894 Replisome 0.00811 1e-04
0005657 Replication fork 0.01098 7e-04
0003887 DNA-directed DNA polymerase activity 0.01822 0.00253
0050792 Regulation of viral life cycle 0.03079 0.00364
0004601 Peroxidase activity 0.03864 0.00382
0016684 Oxidoreductase activity 0.03864 0.00382
0006260 DNA replication 0.06296 0.00403
0006270 DNA replication initiation 0.07700 0.00066
0004789 Endoplasmic reticulum membrane 0.09838 0.00409
SAM yielded a trimmed list of 14 genes with a median FDR of 35.7. Nine of these genes were also in the top list generated with BRB Tools. Among the first 21 genes that Focus identified as agreeing best with the hypothesized pattern change, 12 were seen in BRB Array Tools top list and 9 by SAM. Table 4 shows the six genes identified by all three methods. All were upregulated in CIN3 by an average fold change of 2.3 (2.0 – 3.3).
Table 4 Genes with statistically significant upregulated expression in CIN3. Six genes were indicated by all three analysis methods (BRB Array-Tools, SAM, Focus). Fold changes are shown as calculated by SAM.
GenBank ID Gene Name Fold Change
NM_021988 ubiquitin-conjugating enzyme E2 variant 1 2.0
NM_018307 ras homolog gene family, member T1 2.1
NM_024719 growth hormone regulated TBC protein 1 2.3
NM_007083 nudix type motif 6 2.1
NM_022841 hypothetical protein FLJ12994 3.3
NM_016175 truncated calcium binding protein 2.0
Discussion
Exfoliated cells from normal squamous epithelium are derived from the terminally differentiated superficial layers and may have a more restricted representation of the underlying tissue than those derived from a neoplastic epithelium where differentiation is reduced or lost completely. Therefore results from the normal samples should provide a conservative estimate of the degree of similarities between cells and tissue. Grouping the detected genes by broad ontology categories, the cervical tissue and cervical exfoliated samples showed a similar distribution, however exfoliated cells had fewer numbers of genes in each category. The genes in common between tissue and exfoliated cell profiles represented 50% of the total genes detected in tissue and 72% of the total detected in the cells. Therefore, while much of the tissue gene profile is included in the exfoliated cells, this representation is only partial.
The 7224 genes that were not detected in exfoliated cells could be expected to be involved in proliferation of the basal epithelium. Even though the GOs of these genes did not indicate a direct involvement of these genes in cell division they did include basic components of cell structure and function. Interestingly, 6 of the 15 most significant GOs were related to ribosome activity implying that the ribosomal complex is not renewed after initial establishment in the basal cells.
It is interesting to note that 2,838 genes found in the exfoliated samples were not detected in the tissue. One explanation may be the presence of inflammatory or other cells such as endometrium that are not present in tissue. With keratinocyte differentiation the profile becomes more specialized as some genes are down regulated [4]. Therefore, another explanation may be that the restricted RNA profile allows genes below the cutoff in tissue to exceed the threshold for detection in exfoliated cells. This is supported by the fact that average signal intensity (sARM) of these genes was only one third of these expressed in both specimens. The observation that the GOs of these genes were relate to advanced differentiation processes favors the latter explanation.
Since we used only one cervical tissue sample we undoubtedly underestimated the true biologic variability introduced by age, hormonal status and other environmental factors. In addition, requiring detection in 6 of 7 exfoliated samples limits the transcriptome to those genes that could reliably and reproducibly be detected by microarrays. Additional study, including cervical tissues and matched exfoliated samples from a spectrum of disease states would be required to fully define the extent to which tissue and exfoliated cell profiles overlap, nonetheless we conclude that exfoliated cells are worthy of further investigation as a source of molecular markers for screening.
To begin biomarker discovery we conducted a pilot microarray study of exfoliated cells from women with CIN3 to those with no disease (CIN0) or CIN 1 to determine if differential gene expression could be identified and evaluate the degree of variation within disease groups to determine the number of samples that would be required to stabilize selection of differentially expressed genes. Not surprisingly, the gene expression profiles of the two groups were very similar. Based on cytology, less than 10% of the sampled cells are neoplastic, so the dilution effect on abnormal transcript profiles could be considerable. A "field effect", that is extension of molecular changes to an area larger than the histologically identifiable lesion, has been demonstrated in other cancers including head and neck [5], colon [6] and bladder [7]. There is some evidence that this may occur in the cervix [8], but the extent to which this occurs is not clear.
Using three different statistical approaches to identify differentially expressed genes resulted in lists of candidate genes with little overlap and relatively high estimates of false discovery rates. These inconsistencies reflect the relatively small differences between disease classes compared to within-class variation. We intentionally represented a wide range of age and race in this pilot so as not to over simplify the within-class variation thereby maximizing the specificity of the identified candidate biomarkers. Interestingly, despite matching age and race between the disease groups, the within-class variation was greater for CIN 3 than for no disease or combined CIN 0 and CIN 1. This suggests that cytologically identical CIN3 lesions may represent different molecular pathways to oncogenesis.
There were 6 genes that were identified by all three analysis methods. Given the central role of HPV in cervical carcinogenesis it is interesting that one of these, the ubiquitin-conjugating enzyme E2 variant 1, participates in the degradation of p53 through interaction with HPV E6 protein [9]. Similarly, it is interesting to note an over-representation of genes in the GO "viral life cycle" in CIN3. While none of the others have been previously implicated in cervical carcinogenesis this is not too surprising, as their role may be restricted to premalignant lesions or to host response.
Conclusion
The primary goal of this pilot study was to explore the possibility of using exfoliated cells for genomic biomarker discovery. We conclude that RNA from these cells can indeed be applied to genomic studies. Exfoliated cells display an expression profile that reflects the tissue albeit with limited complexity. In addition, the characteristic expression differences between CIN 3 and control samples (CIN 1 and no disease) are small and future studies need to be designed to address these factors.
Methods
Study population
The study population consisted of women enrolled into an ongoing study of cervical neoplasia in high-risk urban women [10]. Participants were recruited from non-pregnant, HIV-negative women, aged 18–69 years, attending colposcopy clinics at urban public hospitals in Atlanta, Georgia and Detroit, Michigan. Specimens used in this study were from participants enrolled between December 2000 and December 2002. Cervical disease status was determined based on the summary results of cytology, colposcopy and biopsy examination. We selected 15 women with high grade lesions (CIN3) as cases and age (± 4 years) and race matched women without or with only low grade lesions as controls (7 CIN0 and 8 CIN1).
Sample collection and RNA extraction
After visualization of the cervix, ecto- and endocervical cells were collected using a CytoBroom (Cytyc Corporation, Malborough, MA) and dislodged into PreservCyt collection media (Cytyc Corporation). If a cytology diagnosis was required, the collection device was used to prepare a conventional Pap smear and then placed into the PreservCyt collection media. Samples were transported to the laboratory at ambient temperature and stored at 4°C until processed. Within two weeks of sample collection, total nucleic acids (TNA) were extracted from 14 ml of each 20 ml PreservCyt sample using modifications of the MasterPure Complete DNA and RNA Purification kit (Epicentre, Madison, WI) as previously described (Habis et al 2004). The TNA extract was resuspended in 50 μL TE buffer with 50 units of RNasin (Promega Corporation, Madison, WI) and stored at -70°C until use. Total RNA derived from normal uterine cervix tissue (age 48, unknown ethnicity) was purchased (Stratagene®, La Jolla, CA). Quality of all samples was visually evaluated by gel electrophoresis and quantitation was assessed by densitometric measurement (FluorChem® Digital Imaging System, Alpha Innotech, Inc., San Leandro, CA) of the ribosomal bands, with comparison to a standard 28S and 18S control marker.
Microarray assays
We used MWG Human 30 k Arrays (A/ B/ C) (MWG Biotech, Ebersberg, Germany). Each array was hybridized with cDNA prepared from 500 ng total RNA. Conditions for labeling and hybridization were as described elsewhere [11]. Briefly, samples were pretreated with DNase I and cDNA was prepared and labeled with biotin-11-dUTP (Enzo, Farmingdale, NY) using SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) with oligo dT and random primers. The automated Discovery™ System (Ventana Medical Systems, Tucson, AZ) was used to hybridize slides for 8 hours at 42°C, and detect hybridization with anti-biotin Gold Resonance Light Scattering (RLS) Particles (Invitrogen). Slides were scanned with the GSD-501™ RLS scanner (Invitrogen) and 16-Bit Tiff images were subsequently quantified with Array Vision™ Software 8.0 (Imaging Research, St Catherines, ON, Canada). We used sARM values (artifact removed density minus the background density) of each feature for statistical analysis and a signal to noise ratio (S/N = sARM, divided by the SD of the background density) of 1.5 as the cut-off for detection of gene expression.
Comparison of gene expression in cervical exfoliated cells and uterine cervix tissue
We used the results of the 7 samples of exfoliated cells from women with no abnormalities (CIN0) to characterize the profile of exfoliated cells and of the uterine cervix tissue RNA assayed in duplicate to characterize the tissue profile. A gene was included in the profile if detected in >85% of the exfoliated samples (6 of 7) or in both replicates of the tissue sample. We used the web based database for annotation, visualization and integrated discovery (DAVID) [12] for functional annotation and ontology of the detected genes, and the expression analysis systematic explorer (EASE) for identification of enriched biological themes within the gene lists as reflected by an EASE score of < 0.025 [13].
Differential gene expression in CIN0/CIN1 and CIN3 samples
Expression data were derived from the results of all 30 exfoliated samples hybridized to MWG A arrays. Features were restricted to those with a S/N above 1.5 detected in at least 12 of the 15 samples (80%) of either class (CIN0/CIN1 or CIN3). 5461 genes that passed this filter were subjected to further statistical analysis.
We calculated the mean and coefficient of variation (CV) of the log2 transformed median centered sARM for each gene within the CIN 3, CIN 0 and CIN 1/CIN 0 groups. We used the CV in expression of each gene as a measure of homogeneity. For each gene we calculated the difference between the CV of the CIN 3 group minus that of the other groups and plotted these values in descending order to visualize discrepancies in the variation of genes expression between the classes.
To identify expression differences between the two groups we used the following three different microarray-adapted statistical software packages:
(1) BRB Array Tools 3.2 : Log2 transformed sARM values were normalized over the median of each array and subjected to a two-sample T-test with a random variance model and 1000 permutations. We considered the top 20 genes with the lowest univariate parametric p-values as differentially expressed. Multivariate permutation tests were applied to estimate the proportion of false discoveries in the discovery list. The indicated genes were assigned to gene ontology (GO) categories. Additionally, all GO categories that included at least 5 genes represented on the microarray were analyzed to identify biological themes overrepresented in genes differentially expressed. A GO category was selected if its corresponding LS or KS permutation p-value was below the threshold of 0.005 [14].
(2) Significant analysis of microarrays (SAM) 1.21 : sARM values were log2 transformed and centered to the median of each array. Normalized data were analyzed in an unpaired, two-class model with gene specific t-tests and 200 permutations to estimate false discovery rate (FDR) from multiple testing. Genes were scored based on change in expression relative to the standard deviation of the repeated measurements in order to identify those with differential expression [15].
(3) Focus 5.1 : Raw sARM data were normalized to a modified Z-transformation and tested for the hypothesis, gene expression in CIN3 samples is upregulated over controls. The applied contrast coefficient of 1.0 scored genes directly according to the average intensity difference between the two classes [16]. Genes agreeing best with the hypothesized pattern change were trimmed to a list of 20 with the highest interest scores and at least a 2-fold change.
Authors' contributions
MS performed the microarray experiments, statistical analysis and bioinformatics. DRL organized sample collection and processing. MSR, SDV and MTR participated in aspects of the study design and contributed to the discussion and interpretations of the results. ERU initiated the project participated in its design and coordination and helped drafting the manuscript.
All authors read and approved the final manuscript.
Acknowledgements
The authors thank Angelique Habis for contributions to microarray technique, Ira R. Horowitz, Lisa C. Flowers and David Kmak for clinical evaluations, Talaat Tadros, George Birdsong and Mujtaba Husain for contributing to pathology review, and Ainsley Nicholson for insights into the analysis of variance within classes.
Supported in part by NCI's Early Detection Research Network (EDRN) Interagency Agreement Y1-CN-0101-01.
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| 15876354 | PMC1164412 | CC BY | 2021-01-04 16:39:53 | no | BMC Genomics. 2005 May 5; 6:64 | utf-8 | BMC Genomics | 2,005 | 10.1186/1471-2164-6-64 | oa_comm |
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BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-5-371590450510.1186/1471-2334-5-37Research ArticleClinical outcomes in typhoid fever: adverse impact of infection with nalidixic acid-resistant Salmonella typhi Kadhiravan Tamilarasu [email protected] Naveet [email protected] Arti [email protected] SK [email protected] K [email protected] Anoop [email protected] Department of Medicine, All India Institute of Medical Sciences, New Delhi, India2 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India3 Department of Paediatrics, All India Institute of Medical Sciences, New Delhi, India2005 18 5 2005 5 37 37 23 10 2004 18 5 2005 Copyright © 2005 Kadhiravan et al; licensee BioMed Central Ltd.2005Kadhiravan et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Widespread use of fluoroquinolones has resulted in emergence of Salmonella typhi strains with decreased susceptibility to fluoroquinolones. These strains are identifiable by their nalidixic acid-resistance. We studied the impact of infection with nalidixic acid-resistant S. typhi (NARST) on clinical outcomes in patients with bacteriologically-confirmed typhoid fever.
Methods
Clinical and laboratory features, fever clearance time and complications were prospectively studied in patients with blood culture-proven typhoid fever, treated at a tertiary care hospital in north India, during the period from November 2001 to October 2003. Susceptibility to amoxycillin, co-trimoxazole, chloramphenicol, ciprofloxacin and ceftriaxone were tested by disc diffusion method. Minimum inhibitory concentrations (MIC) of ciprofloxacin and ceftriaxone were determined by E-test method.
Results
During a two-year period, 60 patients (age [mean ± SD]: 15 ± 9 years; males: 40 [67%]) were studied. All isolates were sensitive to ciprofloxacin and ceftriaxone by disc diffusion and MIC breakpoints. However, 11 patients had clinical failure of fluoroquinolone therapy. Infections with NARST isolates (47 [78%]) were significantly associated with longer duration of fever at presentation (median [IQR] 10 [7-15] vs. 4 [3-6] days; P = 0.000), higher frequency of hepatomegaly (57% vs. 15%; P = 0.021), higher levels of aspartate aminotransferase (121 [66–235] vs. 73 [44–119] IU/L; P = 0.033), and increased MIC of ciprofloxacin (0.37 ± 0.21 vs. 0.17 ± 0.14 μg/mL; P = 0.005), as compared to infections with nalidixic acid-susceptible isolates. All 11 patients with complications were infected with NARST isolates. Total duration of illness was significantly longer in patients who developed complications than in patients who did not (22 [14.8–32] vs. 12 [9.3–20.3] days; P = 0.011). Duration of prior antibiotic intake had a strong positive correlation with the duration of fever at presentation (r = 0.61; P = 0.000) as well as the total duration of illness (r = 0.53; P = 0.000).
Conclusion
Typhoid fever caused by NARST infection is associated with poor clinical outcomes, probably due to delay in initiating appropriate antibiotic therapy. Fluoroquinolone breakpoints for S. typhi need to be redefined and fluoroquinolones should no longer be used as first-line therapy, if the prevalence of NARST is high.
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Background
Typhoid fever is a common illness in developing countries like India [1] and is a potential threat to developed nations, in an era of increasing air travel and global operations [2]. In the absence of appropriate chemotherapy, typhoid fever was often a fatal illness and introduction of effective antibiotic therapy in 1950s led to a sharp decline in the rates of complications and mortality due to typhoid fever [3]. However, in early 1990s multidrug-resistant strains of Salmonella enterica serotype typhi (MDR-ST) that were resistant to all the three first-line drugs then in use, namely chloramphenicol, amoxycillin and co-trimoxazole emerged, and sooner MDR-ST became endemic in many areas of Asia, including India [4]. This change in pattern of susceptibility was reflected even in places far away, such as the United Kingdom [5] and the United States of America [6]. Fluoroquinolones are very effective against MDR-ST, achieving fever clearance in less than four days with cure rates exceeding 96%, and are currently the first-line drug for the treatment of typhoid fever [7].
However, towards the end of the last decade, it was observed that fever took longer time than before to clear, and at times surprisingly failed to respond to ciprofloxacin therapy [8-10]. These isolates had comparatively higher minimal inhibitory concentrations (MIC) of fluoroquinolones, although they were susceptible to fluoroquinolones by conventional disc diffusion testing and recommended MIC breakpoints [8-10]. Nevertheless, such strains of S. typhi are resistant to nalidixic acid and it was noted that clinical response to fluoroquinolones in patients infected with nalidixic acid-resistant S. typhi (NARST) was inferior to the response in those infected with nalidixic acid-sensitive S. typhi (NASST) strains [11]. However, it is not clear whether fluoroquinolones can still be used as first-line drug for the treatment of typhoid fever, and if used whether this has any adverse impact on clinical outcomes other than treatment failure such as development of complications and morbidity assessed in terms of total duration of illness. In this scenario, the present study was undertaken to evaluate the impact of infection with NARST on clinical outcomes in patients with typhoid fever.
Methods
Study population
This study was conducted at the All India Institute of Medical Sciences (A.I.I.M.S.) hospital, New Delhi, India. This is a tertiary level medical centre located in north India, serving predominantly to low and middle-income groups of the population. All consecutive patients including children with blood culture-proven typhoid fever, admitted to the A.I.I.M.S. hospital during the period November 2001 through October 2003, and those treated as outpatients during this period and were available for at least one follow-up visit, were prospectively included in the study. Patients were evaluated as per a pre-designed instrument, regarding the demographic details, presenting symptoms, physical and laboratory findings and complications. Response to treatment was assessed in terms of fever clearance time.
Microbiological methods
All isolates from blood culture were identified by standard biochemical tests and confirmed by slide agglutination test using specific Salmonella antiserum (Murex Diagnostics Ltd., UK). Antibiotic susceptibility of the isolates was determined by disc diffusion method using 5 μg disc of ciprofloxacin and 30 μg disc of nalidixic acid (HiMedia Laboratories Ltd., India), as per the National Committee for Clinical Laboratory Standards (NCCLS) guidelines and interpretive criteria [12]. The isolates were also tested for susceptibility to chloramphenicol (30 μg), amoxycillin (10 μg), co-trimoxazole (1.25/23.75 μg) and ceftriaxone (30 μg) (HiMedia Laboratories Ltd., India) by disc diffusion method [12]. Minimum inhibitory concentrations (MIC) of ciprofloxacin and ceftriaxone were determined by the E-test method, using commercially available strips (AB Biodisk, Sweden), as per manufacturer's specifications.
Definitions
A case of typhoid fever was defined as one that presented with a febrile illness, whose blood culture yielded S. typhi. Susceptibility break points to various antibiotics tested, were taken as per NCCLS definitions [12]. Anaemia was defined as haemoglobin less than 100 g/L. Leucopenia was defined as total leucocyte count less than 4 × 109 cells/L and thrombocytopenia as platelet count less than 100 × 109platelets/L. Leucocytosis was defined as total leucocyte count more than 11 × 109 cells/L. Liver enzymes were considered as elevated if the levels were more than two times the upper limit of normal (cut-off of 100 IU/L for both alanine aminotransferase and aspartate aminotransferase; 560 IU/L for alkaline phosphatase). Fever clearance time was defined as the time from the start of appropriate antibiotic therapy to the first instance, when oral temperature fell to 37.5°C and remained below that level continuously for 48 hours. Clinical failure of fluoroquinolone therapy was defined as continuing fever, even after five days of continuous treatment with a fluoroquinolone.
Treatment of typhoid fever
The choice of antibiotic therapy was at the discretion of the treating physician. The practice in general, was to prescribe oral ciprofloxacin (adults: 500 mg b.i.d.; children: 15 mg/kg/day) or ofloxacin (adults: 400 mg b.i.d.; children: 15 mg/kg/day) or cefixime (20 mg/kg/day) for outpatient treatment and to admit the patient for in-patient care, if felt by the treating physician to be not responding to therapy or acutely ill. Hospitalised patients received parenteral antibiotic therapy, either ceftriaxone (adults: 2 g b.i.d.; children: 75 mg/kg/day) or a fluoroquinolone or a combination regimen, as decided by the treating physician.
Statistical analysis
All statistical analyses were done using a software package (SPSS for Windows, Version 10.0.1, SPSS Inc., Chicago, IL). Continuous variables are presented as mean ± standard deviation (SD) or as median with interquartile range (IQR). Categorical variables are expressed as proportions. Independent sample t test or Mann-Whitney U test as appropriate was applied for the comparison of continuous variables between two groups and categorical variables were compared by chi-squared test or Fisher's exact test. Correlation between two continuous variables such as total duration of illness and duration of prior antibiotic intake was tested by Pearson's product moment correlation. Comparison of continuous variables between more than two groups was done by Kruskal-Wallis test. P-value less than 0.05 was considered statistically significant. All tests were two-sided.
Results
Over a period of two years, 60 patients (hospitalised: 49, outpatients: 11) with blood culture-proven typhoid fever were included in the study. In patients who had relapse, only the first episode was considered in the analysis. Mean age of the patients was 15 ± 9 years (range: 6 months-39 years). Overall, males were predominantly affected (40 patients, 67%). The predilection for male gender was seen in all age groups (in decades), except in infants and preschool children (0–5 years) where the gender distribution was nearly equal (n = 13; 7 males). The frequency of various symptoms and physical findings at presentation is shown in Table 1.
Table 1 Clinical features at presentation, in a study of 60 patients with blood culture-proven typhoid fever
Characteristic Frequency, overall (n = 60) NARST (n = 47) NASST (n = 13) MDR-ST (n = 22) NonMDR-ST (n = 38)
Age, years * 15 ± 9 13 ± 9 † 23 ± 8 † 14 ± 9 16 ± 10
Gender (male/female) 40/20 32/15 8/5 15/7 25/13
Fever, days ‡ 8 (4.8–14) 10 (7–15) † 4 (3–6) † 9 (7–14.3) 7 (3.8–14)
Chills 39 (65) 30 (64) 9 (69) 15 (68) 24 (63)
Anorexia 58 (97) 46 (98) 12 (92) 22 (100) 36 (95)
Abdominal pain 23 (38) 18 (38) 5 (38) 10 (45) 13 (34)
Vomiting 33 (55) 27 (57) 6 (46) 14 (64) 19 (50)
Diarrhoea 27 (45) 22 (47) 5 (38) 11 (50) 16 (42)
Constipation 7 (12) 6 (13) 1 (8) 2 (9) 5 (13)
Intestinal bleeding 1 (2) 1 (2) -- -- 1 (3)
Headache 24 (40) 19 (40) 5 (38) 6 (27) 18 (47)
Altered sensorium 3 (5) 3 (6) -- 2 (9) 1 (3)
Cough 21 (35) 18 (38) 3 (23) 10 (45) 11 (29)
Relative bradycardia 6 (10) 4 (9) 2 (15) 1 (5) 5 (13)
Jaundice 3 (5) 3 (6) -- -- 3 (8)
Hepatomegaly § 29 (49) 27 (57) ∥ 2 (15) ∥ 11 (50) 18 (47)
Splenomegaly § 30 (51) 25 (53) 5 (38) 14 (64) 16 (42)
NARST = nalidixic acid-resistant Salmonella typhi
NASST = nalidixic acid-sensitive Salmonella typhi
MDR-ST = multidrug-resistant Salmonella typhi
-- no patients in this category
All data presented as number (%) of patients, except when indicated
* data presented as mean ± SD
† P-value < 0.05 for NARST vs. NASST; compared by independent t test or Mann-Whitney U test
‡ duration of fever at presentation; presented as median (IQR)
§ one patient with underlying chronic myeloid leukaemia was not included
∥ P-value < 0.05 for NARST vs. NASST; compared by Fisher's exact test
All 60 isolates were sensitive to ciprofloxacin by disc diffusion testing and the MIC values of ciprofloxacin for all the isolates were within the susceptible range (0.016–1 μg/mL) as per current NCCLS definitions (susceptible if, MIC ≤ 1 μg/mL). Forty seven isolates (78%) were resistant to nalidixic acid (NARST) and the frequency of resistance to other antibiotics namely, chloramphenicol, amoxycillin and co-trimoxazole was 26 (43%), 28 (47%) and 30 (50%) respectively. About a third of isolates were MDR-ST (22 isolates, 37%) and no isolate was resistant to ceftriaxone (MIC range: 0.016–0.064 μg/mL; susceptible if, MIC ≤ 8 μg/mL).
When compared with NASST, the mean MIC of ciprofloxacin was significantly higher for NARST isolates (Table 2; P = 0.005). However, the distribution of MIC values of ciprofloxacin around the mean, in the two groups was wide and there was considerable overlap. When dichotomised using the median value of MIC (0.25 μg/mL), isolates with MIC values above the median were significantly more likely to be resistant to nalidixic acid (NARST) than were isolates with MIC values below the median (P = 0.013; Figure 1). As a surrogate marker, nalidixic acid-resistance was 82% sensitive and 100% specific for identifying isolates with MIC of ciprofloxacin ≥ 0.125 μg/mL.
Table 2 Antibiotic susceptibility and clinical outcomes in a study of 60 patients with blood culture-proven typhoid fever
Variable All patients (n = 60) NARST (n = 47) NASST (n = 13) MDR-ST (n = 22) NonMDR-ST (n = 38)
MIC ciprofloxacin, μg/mL 0.33 ± 0.21 0.37 ± 0.21* 0.17 ± 0.14* 0.37 ± 0.26 0.30 ± 0.18
MIC ceftriaxone, μg/mL 0.036 ± 0.016 0.038 ± 0.018 0.031 ± 0.01 0.033 ± 0.017 0.039 ± 0.016
Fever clearance time, days 5.1 ± 3.5 5.5 ± 3.8 3.7 ± 1.3 4.9 ± 2.7 5.3 ± 3.9
Total duration of illness, days 14.3(9.8–21) 16.3(11.4–24)* 9.6(6.8–10)* 14.5(9.3–21.4) 14.3(9.9–22)
Complications † 11 (18) 11 (23) -- 2 (9) 9 (24)
NARST = nalidixic acid-resistant Salmonella typhi
NASST = nalidixic acid-sensitive Salmonella typhi
MDR-ST = multidrug-resistant Salmonella typhi
MIC = minimum inhibitory concentration
-- no patients in this category
* P-value < 0.05 for NARST vs. NASST; compared by independent t test or Mann-Whitney U test
† includes encephalopathy, hepatitis, lower gastrointestinal bleeding, meningitis and myocarditis. Presented as number (%) of patients; all other data given as mean ± SD or median (IQR)
Figure 1 Histogram depicting the distribution of minimum inhibitory concentration (MIC) of ciprofloxacin of 60 Salmonella typhi isolates and the proportion of nalidixic acid-resistant (NARST) as well as nalidixic acid-susceptible (NASST) isolates in each column
About half of the patients (47%) presented in the first week of illness. The duration of fever at presentation was significantly longer in patients with NARST isolates (Table 1; P = 0.000) as was the total duration of illness (Table 2; P = 0.000), than in patients with NASST isolates. The frequency of various laboratory abnormalities, according to the susceptibility of the isolate is shown in Table 3. Antibiotic susceptibility pattern had no significant association with any of the physical findings except for hepatomegaly (P = 0.021), which was more frequent in patients with NARST isolates (Table 1). Elevation of hepatic enzymes was a common feature (27 patients, 45%) and in four patients the elevation was marked (> 5 times the upper limit of normal), among which three patients had concomitant hyperbilirubinaemia. Patients with NARST isolates had significantly higher levels of aspartate aminotransferase than patients with NASST isolates (121 [66–235] vs. 73 [44–119] IU/L; P = 0.033). Though the mean levels of alanine aminotransferase and alkaline phosphatase were higher in patients with NARST isolates, these differences were not statistically significant (data not shown).
Table 3 Haematological and biochemical findings in a study of 60 patients with blood culture-proven typhoid fever
Variable Frequency, overall (n = 60) NARST (n = 47) NASST (n = 13) MDR-ST (n = 22) NonMDR-ST (n = 38)
Anaemia * 19 (32) 15 (32) 4 (31) 6 (27) 13 (34)
Leucopenia * 10 (17) 8 (17) 2 (15) 4 (18) 6 (16)
Leucocytosis * 6 (10) 5 (11) 1 (8) 2 (9) 4 (11)
Thrombocytopenia * 8 (13) 6 (13) 2 (15) 3 (14) 5 (13)
AST > 2 × ULN 27 (45) 23 (49) 4 (31) 9 (41) 18 (47)
AST > 5 × ULN 8 (13) 8 (17) -- 3 (14) 5 (13)
ALT > 2 × ULN 18 (30) 16 (34) 2 (15) 6 (27) 12 (32)
ALT > 5 × ULN 4 (7) 4 (9) -- 1 (5) 3 (8)
NARST = nalidixic acid-resistant Salmonella typhi
NASST = nalidixic acid-sensitive Salmonella typhi
MDR-ST = multidrug-resistant Salmonella typhi
AST = alanine aminotransferase
ALT = aspartate aminotransferase
ULN = upper limit of normal
MIC = minimum inhibitory concentration
-- no patients in this category
* defined by cut-off values as given in the text
All data presented as number (%) of patients
Twenty six patients (43%) had received prior oral antibiotic therapy as outpatients, for a variable duration (2–17 days) before presentation: of which 11 patients (42%) had received fluoroquinolones for at least five days duration and thus were deemed to have clinical failure of fluoroquinolone therapy (others: cefixime – 7 patients [2–15 days]; ciprofloxacin – 4 patients [2–4 days]; amoxycillin – 3 patients [2–4 days]; azithromycin – 1 patient [2 days]). Among these 11 patients, three patients remained febrile even after 10 days of fluoroquinolone therapy. However, all the 11 isolates from patients with fluoroquinolone-failure were sensitive to ciprofloxacin in vitro, according to current NCCLS breakpoints (mean MIC: 0.34 ± 0.11 μg/mL; range: 0.125–0.5 μg/mL). Of the 11 instances of clinical failure of fluoroquinolone, in 10 patients the isolates were NARST.
Fever clearance times were assessable in 51 patients (5 patients were lost to follow-up and in the other 4 patients defervescence was confounded by co-existing conditions). The initial antibiotic regimens were: ceftriaxone alone (35 patients) or in combination with another antibiotic (6 patients) and ofloxacin alone (7 patients) or in combination (3 patients) (Figure 2). Overall, the mean fever clearance time was 5 ± 3.5 days (range: 1–18.5 days). Clearance of fever took seven days or more in 10 patients (20%). Fever clearance time had no significant association with age, gender, antibiotic susceptibility pattern, MIC of ciprofloxacin, MIC of ceftriaxone, or the antibiotic regimen used for treatment. Overall, 11 patients (18%) developed complications, which included encephalopathy (4 patients), meningitis (1 patient), hepatitis characterised by marked elevation of transaminases (4 patients), myocarditis and massive lower gastrointestinal bleeding (1 patient each). All 11 patients who developed complications had NARST isolates. However, this apparent difference between NARST and NASST in the development of complications was not statistically significant (P = 0.1).
Figure 2 Antibiotic therapy and fever clearance time in a study of 60 patients with blood culture-proven typhoid fever. Numbers in parentheses represent the number of patients * 26 patients had received some antibiotic for variable duration before presentation, of which 11 patients remained febrile, even after taking fluoroquinolones for ≥ 5 days † fever clearance time, presented as mean ± SD (days) ‡ other antibiotics given in combination with ceftriaxone were: ofloxacin (3 patients), amikacin (2 patients) and azithromycin (1 patient) § miscellaneous regimens were: cefixime + amoxycillin (1 patient), ceftriaxone + ofloxacin + metronidazole (1 patient) and ceftriaxone + ciprofloxacin + gentamicin (1 patient) ∥ antibiotics added were: amikacin (3 patients) and ofloxacin (2 patients) ¶ switched over to azithromycin (1 patient) and ofloxacin (1 patient)
Total duration of illness was significantly longer in patients who developed complications than in patients who did not (22 [14.8–32] vs. 12 [9.3–20.3] days; P = 0.011). This difference became non-significant (P = 0.087) after adjusting for the duration of prior antibiotic intake, suggesting that the difference in the duration of prior antibiotic intake contributed to the apparent difference in the total duration of illness. The duration of fever at presentation was significantly longer in patients who had received prior antibiotic therapy than in patients who had not (12 [7–22.5] vs. 7 [3-10]; P = 0.009) and there was a strong positive correlation between duration of prior antibiotic intake and duration of fever at presentation (r = 0.61; P = 0.000) as well as total duration of illness (r = 0.53; P = 0.000). When patients with NARST isolates were subdivided on the basis of presence or absence of complications, it became apparent that even among patients with NARST isolates, the duration of fever at presentation was comparatively longer in those with complications than those who had NARST isolates but no complications, exhibiting a linear trend (Figure 3; P = 0.001). No significant differences were observed when patients with MDR-ST isolates were compared with the rest, with respect to any of the parameters studied, including rate of complications and total duration of illness (Tables 1, 2 and 3).
Figure 3 Duration of fever at presentation, according to nalidixic acid-susceptibility status and presence of complications. Data presented as mean ± SE NASST = nalidixic acid-sensitive Salmonella typhi NARST = nalidixic acid-resistant Salmonella typhi
Discussion
The present study brings out the high frequency of nalidixic acid-resistance among S. typhi isolates from New Delhi, India. Moreover, this study reconfirms the occurrence of ciprofloxacin-susceptible but nalidixic acid-resistant S. typhi isolates and their relation to clinical failure of fluoroquinolone therapy. The frequency of nalidixic acid-resistance as found in the present study (78%) is high in comparison with earlier studies from India, in which it was in the range of 60–67% [13,14]. However, it should be highlighted that these studies were temporally separated, hindering direct comparison between them. Being a hospital-based study, NARST strains might have been inadvertently overrepresented in the study population, since patients infected with NASST are more likely to be successfully treated in the community setting with fluoroquinolones. A community-based study is needed to estimate the actual proportion of infections caused by NARST. An earlier community-based prospective study conducted in New Delhi had found an alarmingly high rate of ciprofloxacin failure (9 out of 63 patients, 14%) as early as 1995–96 [1]. In light of the consistent increase in the proportion of NARST isolates over the last decade, as suggested by data collected at our Institute [15], the high frequency of NARST strains as found in the present study seems to be a true phenomenon rather than due to selection bias. As documented in an earlier study conducted in south India [16], this increase in the frequency of NARST strains is associated with a consistent increase in the MIC levels of ciprofloxacin for S. typhi isolates [15].
Interesting is the fact that as per the current NCCLS breakpoints, these NARST isolates with higher MIC of ciprofloxacin would still be classified as being ciprofloxacin-susceptible. Notwithstanding, this subtle increase in the MIC of ciprofloxacin had an adverse impact on the clinical response to fluoroquinolone therapy: in the present study, all patients with clinical failure of fluoroquinolone therapy had isolates with MIC of ciprofloxacin ≤ 1 μg/mL. Hence, it is increasingly being felt that the common fluoroquinolone breakpoints for Enterobacteriaceae as set by the NCCLS, are to be reevaluated in the case of Salmonella species [13,17,18]. Currently, the NCCLS advises that testing of extraintestinal Salmonella isolates for nalidixic acid-resistance may be considered and that fluoroquinolone-susceptible strains of Salmonella that test resistant to nalidixic acid may be associated with clinical failure or delayed response in fluoroquinolone-treated patients with extraintestinal salmonellosis [19]. In consonance with earlier studies [13,20], the sensitivity of nalidixic acid-resistance as a surrogate marker for identifying isolates with increased levels of MIC of ciprofloxacin was found to be good in the present study. Thus, testing for nalidixic acid-resistance could be useful as a screening test for further determination of MIC levels of ciprofloxacin.
Since fluoroquinolones exhibit concentration-dependent killing, using higher doses of ciprofloxacin (750 mg b.i.d.) might compensate for this reduced susceptibility [7,21]. Area under the curve (AUC) for serum concentrations attained with this dose is 19.2 ± 1.1 μg.h/mL [22]. This extrapolates to an AUC/MIC ratio of about 77 in the case of half of the isolates encountered in the present study, which is well below the cut-off of > 250 that is required for rapid bactericidal action [23]. Thus using higher doses of ciprofloxacin is also likely to prove ineffective in this population.
Higher frequency of hepatomegaly with greater elevation of hepatic enzymes and a trend for increased incidence of complications in patients with NARST isolates, all suggest that NARST is associated with severe clinical illness. Though the present study did not find a statistically significant association between NARST and the rate of complications, current findings strongly suggest that prior intake of antibiotics not capable of achieving fever clearance is associated with the development of complications. Thus it is reasonable to presume that the use of ciprofloxacin in populations where NARST is widely prevalent would delay the initiation of appropriate antibiotic therapy and thereby would lead to an excess of complications. The current observations are consistent with this hypothesis, though not confirmatory.
Earlier studies had reported that infection caused by MDR-ST was associated with more severe illness than non-MDR-ST infection [24,25]. Noteworthy is the fact that in both these studies antibiotic used as first-line therapy was ampicillin/amoxycillin or chloramphenicol, to which MDR-ST is inherently resistant. Contrary to this, the present study and another study [26] using ciprofloxacin as first-line therapy found no significant differences between MDR-ST and non-MDR-ST infections. This analogy offers ancillary evidence in support of the hypothesis that using fluoroquinolones as first-line therapy in settings where NARST is prevalent, would result in poor outcomes. The other possibility is that development of drug resistance and virulence of the organism might be genetically linked. Blood bacterial counts were reported to be substantially higher in infections caused by MDR-ST than in infections caused by drug-susceptible S. typhi [27]. A similar phenomenon is possible in the case of NARST also, accounting for poor outcomes. But the present study was not aimed at evaluating this possibility and this merits further study.
Conclusion
In this population, nalidixic acid-resistance is very common among isolates of S. typhi and is associated with reduced susceptibility to fluoroquinolones in vitro. Clinically this translates into frequent failure of fluoroquinolone therapy. However, this is not reflected in current NCCLS breakpoints and hence the fluoroquinolone breakpoints need to be redefined for S. typhi. Moreover, use of fluoroquinolones as first-line therapy for typhoid fever delays initiation of appropriate antibiotic in this setting and is likely to result in poor outcomes. For these reasons, fluoroquinolones should no longer be used as the first-line therapy, in populations where nalidixic acid-resistance is common among isolates of S. typhi.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
TK collected the clinical data, analysed them and drafted the manuscript. NW designed the study, contributed significantly to interpretation of data and revised the draft for important intellectual content. AK participated in the design of the study, coordinated susceptibility testing and revised the draft critically for important intellectual content. SKK participated in the design of the study, contributed significantly to analysis and interpretation of data and revised the draft for important intellectual content. KR carried out microbiological studies and contributed significantly in drafting the article. AM conceived of the study, participated in the design of the study and revised the draft for important intellectual content. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We thank all Residents of the Departments of Medicine and Paediatrics and the laboratory staff of Bacteriology Laboratory, Department of Microbiology, All India Institute of Medical Sciences, for their kind help and cooperation.
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| 15904505 | PMC1164413 | CC BY | 2021-01-04 16:28:15 | no | BMC Infect Dis. 2005 May 18; 5:37 | utf-8 | BMC Infect Dis | 2,005 | 10.1186/1471-2334-5-37 | oa_comm |
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BMC Med GenetBMC Medical Genetics1471-2350BioMed Central London 1471-2350-6-231592152010.1186/1471-2350-6-23Research ArticlePolymorphisms of methylenetetrahydrofolate reductase (MTHFR) and susceptibility to pediatric acute lymphoblastic leukemia in a German study population Schnakenberg Eckart [email protected] Andrea [email protected] Gunnar [email protected] Klaus [email protected] Kathrin [email protected] Brigitte [email protected] Holger A [email protected] Karl H [email protected] Martin [email protected] Martin [email protected] Institute for Pharmacogenetic and Genetic Disposition, Langenhagen, Germany2 Children's Hospital, Pediatric Hematology and Oncology, Hannover Medical School, Germany3 Institute of Cell and Molecular Pathology, Hannover Medical School, Germany4 Department of Transfusion Medicine, Hannover Medical School, Germany2005 27 5 2005 6 23 23 16 11 2004 27 5 2005 Copyright © 2005 Schnakenberg et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Methylenetetrahydrofolate reductase (MTHFR) has a major impact on the regulation of the folic acid pathway due to conversion of 5,10-methylenetetrahydrofolate (methylene-THF) to 5-methyl-THF. Two common polymorphisms (677C>T and 1298A>C) in the gene coding for MTHFR have been shown to reduce MTHFR enzyme activity and were associated with the susceptibility to different disorders, including vascular disease, neural tube defects and lymphoid malignancies. Studies on the role of these polymorphisms in the susceptibility to acute lymphoblastic leukemia (ALL) led to discrepant results.
Methods
We retrospectively evaluated the association of the MTHFR 677C>T and 1298A>C polymorphisms with pediatric ALL by genotyping a study sample of 443 ALL patients consecutively enrolled onto the German multicenter trial ALL-BFM 2000 and 379 healthy controls. We calculated odds ratios of MTHFR genotypes based on the MTHFR 677C>T and 1298A>C polymorphisms to examine if one or both of these polymorphisms are associated with pediatric ALL.
Results
No significant associations between specific MTHFR variants or combinations of variants and risk of ALL were observed neither in the total patient group nor in analyses stratified by gender, age at diagnosis, DNA index, immunophenotype, or TEL/AML1 rearrangement.
Conclusion
Our findings suggest that the MTHFR 677C>T and 1298A>C gene variants do not have a major influence on the susceptibility to pediatric ALL in the German population.
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Background
Methylenetetrahydrofolate reductase (MTHFR) has a major impact on the regulation of the folic acid pathway due to the conversion of 5,10-methylenetetrahydrofolate (methylene-THF) to 5-methyl-THF. Two common non-synonymous coding region polymorphisms (677C>T and 1298A>C) in the MTHFR gene were shown to confer reduced enzyme activity in in vitro assays leading to a decreased pool of methyl-THF and the MTHFR variant 677C>T was associated with an increased risk of hyperhomocysteinemia, particularly in folate-deficient states [1-4]. With regard to the MTHFR 677C>T polymorphism, results from in vitro assays showed a decrease in enzyme activity to 65% for the heterozygous and to 30% for the homozygous state of the 677T variant [5]. For the 1298A>C polymorphism, enzyme activity in vitro is decreased in homozygous variants and, to a lesser extent, in heterozygotes compared with those homozygous for the wild-type allele [6,7]. It is supposed that variants (677TT/1298AA or 677CC/1298CC) of MTHFR increase the level of methylene-THF leading to a subsequent reduction of uracil in DNA. The reduction of uracil decreases the amount of misincorporations of uracil instead of thymidine into DNA. This may protect DNA from double-strand breaks and, consequently, from chromosomal alterations [1,8]. Furthermore,it is assumed that DNA methylation via MTHFR is an important epigenetic feature that modulates DNA methylation status through interaction with folate status [1,9]. Consequently, MTHFR variants are discussed to influence disease processes and several studies in the literature reported on reduced MTHFR enzyme activity and the susceptibility to different disorders, including vascular disease, neural tube defects and lymphoid malignancies [1,2,5,7,8,10-13]. Lymphoid malignancies arise as a consequence of point mutations, chromosomal rearrangements (e.g., chromosomal translocations), and epigenetic alterations in hematopoietic cells making the variant MTHFR an interesting candidate gene for studies on leukemogenesis [14]. However, especially with regard to susceptibility to acute lymphoblastic leukemia (ALL), previous studies led to discrepant results. Although both variants 677T and 1298C have been reported to decrease susceptibility to ALL in children and adults [8,10,11,13], other investigators did not support such associations [15,16]. Moreover, the protective effect of MTHFR variants was present only in children before folic acid supplement in pregnancy has been recommended [17]. These results suggest a role for gene-environment interactions in the association of MTHFR with ALL.
In the present study, we analyzed the association of MTHFR variants with pediatric ALL in a German study sample including 443 ALL patients and 379 healthy controls.
Methods
Patients and controls
The present study used data and specimens derived from patients of the ongoing ALL-BFM 2000 trial that is conducted by the Berlin-Frankfurt-Münster (BFM) study group and enrolls pediatric patients from 1 year up to 18 years of age with a diagnosis of ALL from 84 different treatment centers in Germany, Austria, and Switzerland [18]. From July 1999 until the end of February 2001, 497 patients of up to 18 years of age were enrolled onto the clinical trial after informed consent was obtained from the parents or legal guardians. The diagnosis was established in our central reference laboratory by morphological FAB criteria and cytochemistry when at least 25% lymphoblasts were present in the bone marrow, or when blasts were present in the peripheral blood. The assessments of immunophenotype, cellular DNA content, and positivity for TEL/AML1, BCR/ABL, and MLL/AF4 fusion genes were done as described previously [19,20]. Patients were included into the present study, when biological material was available at the study center leading to a study population of 443 patients (89.7% of the entire study population) who with regard to clinical characteristics did not significantly differ from the entire study population of 497 patients. Controls (n = 379) consisted of blood samples from healthy blood donors (aged between 18–68 years), with no history of malignant neoplastic disease. They were derived through the Department of Transfusion Medicine, Hannover Medical School, Hannover, Germany, and the Clinic Center of Bremen, Bremen, Germany. Individuals included in the present study were of Caucasian descent. The study was approved by the local ethics committee.
Genotyping
DNA was extracted from peripheral blood or bone marrow samples by using the QIAamp DNA Blood Midi Kit (Qiagen GmbH, Hilden, Germany). Depending on availability, either tumor material or remission samples were used. Analyses of MTHFR variants were performed on a LightCycler® instrument (Roche Diagnostics, Mannheim Germany) using a commercial real-time assay (Artus Biotech, Hamburg, Germany) according to the manufacturer's instructions. Both variants MTHFR 677C>T and 1298A>C were analyzed by simultaneous amplification in a single real-time PCR run. Melting curve analyses were performed for both variants after PCR amplification. No differences in the distribution of MTHFR variants were observed between tumor and remission samples (data not shown).
Statistical analysis
The association of MTHFR variants with risk of disease was examined by use of unconditional logistic regression analysis to calculate odds ratios (OR) and their 95% confidence intervals (CI). P values of <0.05 were considered statistically significant. The MTHFR variant was used as a categorical variable in these analyses. The expected frequency of MTHFR variants in controls was analyzed by the Hardy-Weinberg equilibrium test. The SPSS statistical package (SPSS Inc., Chicago, IL) was used for computerized calculations.
Results and Discussion
Table 1 shows the distribution of clinical characteristics in the study sample of 443 childhood ALL patients from ALL-BFM 2000. Distribution of MTHFR variants within patients and controls are summarized in Table 2. In our control population of 379 healthy individuals the frequencies of MTHFR variants 677C>T and 1298A>C were in Hardy-Weinberg equilibrium (677: χ2 = 1.81, 1298: χ2 = 0.05; P > 0.05) and demonstrated strong linkage disequilibrium (P < 0.001). None of the haplotypes was observed more frequently in the ALL group (Tab. 2). MTHFR variants that were previously reported to confer a reduced risk of ALL were similarly distributed in the patient and control group. The frequency of the variant MTHFR 677T was 0.314 in controls and 0.333 in children with ALL. Ogino et al. reported a frequency of 0.320 in a meta-analysis including 5389 individuals [21]. There was also no significant different distribution of the MTHFR 1298C variant between our patient and control groups. The 1298C variant occurs with a frequency of 0.367 in controls and 0.314 in patients with ALL. Ogino et al. reported 0.308 in their meta-analysis [21]. The calculated OR and their respective 95% CI are also shown in Table 2. No significant associations between specific MTHFR variants or combinations of MTHFR variants and risk of ALL were observed neither in the total patient group nor in analyses stratified by gender, age at diagnosis, DNA index, immunophenotype, or TEL/AML1 rearrangement (stratified analyses not shown; strata as shown in Table 1).
With regard to ALL, our results are in contrast to previous findings and do not support the assumption that higher methylene-THF levels due to MTHFR variants lead to a decreased risk of ALL in our study population [8,10,11,13]. There are different explanations for these diverging results. The present study included pediatric patients from Germany. The above mentioned studies included adult patients from United Kingdom [8] and Italy [13] but also pediatric patients from United Kingdom [10] or Brazil [11]. These latter two studies on the MTHFR 677C>T and 1298A>C polymorphisms in childhood ALL included patients diagnosed between 1992–1998 and 1991–2000, respectively, while our study included pediatric ALL patients diagnosed after July 1999. Thus, probably a large proportion of patients analyzed in the studies by Wiemels et al. [10] and Franco et al. [11] were born before folic acid supplementation in pregnancy was recommended. In a Canadian study, Krajinovic et al. reported that the protective effect of MTHFR variants was accentuated and present only in children born before 1996 [17]. This may explain the diverging results with regard to pediatric ALL and is further supported by a recent study of Balta and colleagues from Turkey, where in 142 pediatric ALL patients diagnosed between February 2000 and February 2002, no significant association between the MTHFR 677C>T polymorphism and ALL was detected [16]. However, the impact of folate metabolism on the risk of ALL may also vary from population to population because of additional gene-environment and gene-gene interactions. For example, in our study population we cannot control for differences in folate status as we have not collected information on dietary intake. Thus, a potentially existing association of MTHFR variants and ALL may be masked by the inability to control for the individual folate status in our study population. Furthermore, we cannot exclude potential gene-gene interactions. As several polymorphic key enzymes are involved in folate metabolism, a more comprehensive approach would clearly yield more precise estimates of the association of folate metabolism with risk of ALL. As an additional explanation for diverging results, specifically in subgroups with chromosomal translocations, the potential importance of parental variants and folate status with respect to an in utero pathogenesis of ALL (e.g., MLL/AF4 and TEL/AML1 fusion gene-positive ALL) should be considered [22]. Lastly, our results may simply be due to chance and small patient numbers. However, with regard to sample size, our study exceeds most other studies reported in the literature, so far. In conclusion, our findings suggest that the MTFHR gene variants analyzed here do not have a major influence on the susceptibility to pediatric ALL in the German population. However, potential effects in rare specific ALL subgroups cannot be excluded.
Conclusion
We retrospectively evaluated the association of the MTHFR 677C>T and 1298A>C polymorphisms with childhood ALL by analyzing a study sample of 443 ALL patients consecutively enrolled onto the German multicenter trial ALL-BFM 2000 and 379 healthy controls. No significant associations between specific MTHFR variants or combinations of MTHFR variants and risk of ALL were observed neither in the total patient group nor in analyses stratified by gender, age at diagnosis, DNA index, immunophenotype, or TEL/AML1 rearrangement. Our findings suggest that the MTHFR 677C>T and 1298A>C gene variants do not have a major influence on the susceptibility to pediatric ALL in the German population.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All authors were responsible for the concept of the study. ES and MSt coordinated the study. All authors were involved in sample collection, DNA preparation, genotyping and interpretation of the analyses. MSt and ES did the statistical analyses, drew-up the tables and prepared the manuscript with advice from the other authors. MSch is the chairman of the ALL-BFM 2000 study.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We thank all study participants and Thomas Laue and Dr. Tim Greve for providing kits for MTHFR genotyping.
Figures and Tables
Table 1 Clinical characteristics of patients from trial ALL-BFM 2000
Patients ALL-BFM 2000 (n = 443)
Number of subjects (%)
Gender
Male 268 (60.5)
Female 175 (39.5)
Age at diagnosis (years)
< 1 -
1-< 6 264 (59.6)
6-< 10 80 (18.1)
≥ 10 99 (22.3)
DNA indexa
< 1.16 258 (58.2)
≥ 1.16 48 (10.8)
no result 137 (30.9)
TEL/AML1 pos. 90 (20.3)
neg. 313 (70.7)
unknown 40 (9.0)
BCR/ABL pos. 9 (2.0)
neg. 412 (93.0)
unknown 22 (5.0)
MLL/AF4 pos. 1 (0.2)
neg. 394 (88.9)
unknown 48 (9.9)
Immunophenotype
Precursor B 344 (77.7)
T 81 (18.3)
Bipenotypic 4 (0.9)
Unknown 14 (3.2)
aRatio of DNA content of leukemic G0/G1 cells to normal diploid lymphocytes.
Table 2 Distribution of MTHFR variants in cases and controls and their association with childhood acute lymphoblastic leukemia in patients from trial ALL-BFM 2000
MTHFR Cases (n = 443) Number (%) Controls (n = 379) Number (%) Odds ratio (95% CI) P
677 CC/1298 AA 49 (11.1) 45 (11.9) 1.00a
677 CT/1298 AA 101 (22.8) 67 (17.7) 1.38 (0.83–2.30) 0.21
677 TT/1298 AA 44 (9.9) 41 (10.8) 0.99 (0.55–1.77) 0.96
677 CC/1298 AC 101 (22.8) 87 (23.0) 1.07 (0.65–1.75) 0.80
677 CT/1298 AC 100 (22.6) 85 (22.4) 1.08 (0.66–1.78) 0.76
677 TT/1298 AC 3 (0.7) 2 (0.5) 1.38 (0.22–8.63) 0.73
677 CC/1298 CC 45 (10.2) 52 (13.7) 0.79 (0.45–1.40) 0.43
677 CT/1298 CC n.d. n.d. - -
677 TT/1298 CC n.d. n.d. - -
n.d., not detected; CI, confidence interval; areference category
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| 15921520 | PMC1164414 | CC BY | 2021-01-04 16:03:32 | no | BMC Med Genet. 2005 May 27; 6:23 | utf-8 | BMC Med Genet | 2,005 | 10.1186/1471-2350-6-23 | oa_comm |
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BMC Med GenetBMC Medical Genetics1471-2350BioMed Central London 1471-2350-6-251594387410.1186/1471-2350-6-25Case ReportType I Gaucher disease with exophthalmos and pulmonary arteriovenous malformation Chen Chun-An [email protected] Nelson LS [email protected] Yin-Hsiu [email protected] Wei-Min [email protected] Jou-Kou [email protected] Wuh-Liang [email protected] Departments of Pediatrics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan2 Medical Genetics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan3 Department of Chemical Pathology, The Chinese University of Hong Kong, Hong Kong2005 9 6 2005 6 25 25 17 11 2004 9 6 2005 Copyright © 2005 Chen et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Gaucher disease type I, the non-neuropathic type, usually presents in adulthood with hepatosplenomegaly. We report here an adult with type I Gaucher disease presented with unusual and severe clinical manifestations.
Case presentation
Hepatosplenomegaly, bone crisis and fractures occurred at early childhood, and splenectomy was performed at the age of 5. Exophthalmos with increase in retrobulbar space was noted when the patient was 30. Cerezyme infusion started at the age of 32; but unfortunately, pulmonary arteriovenous malformation with dyspnea and hypoxemia was found two years later. Gene analysis revealed V375L/L444P mutations in the β-glucocerebrosidase gene.
Conclusion
Although both eye and lung diseases have been associated with Gaucher disease, this is the first reported demonstration of exophthalmos and pulmonary arteriovenous malformation in the same patient. This case may therefore present an extremely severe and unusual form of type I Gaucher disease.
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Backgound
Gaucher disease is a lysosomal storage disorder caused by a recessively inherited deficiency of glucocerebrosidase activity, which causes an accumulation of sphingolipid glucosylceramide in cells of the reticulo-endothelial systems [1]. The "Gaucher cells" can be found in the spleen, liver, bone, and central nerve system in affected individuals, causing hepatosplenomegaly, anemia, thrombocytopenia and skeletal diseases [1]. Three clinical forms of the disease have been described, based on the absence (type I) or the presence (types II and III) of neurological involvement in addition to the visceral findings [1].
Pulmonary involvement, with Gaucher cell infiltration of the alveolar or interstitial spaces [2], may be more common than previously thought, but clinically significant lung disease is still rare [3]. Pulmonary arteriovenous shunting has been implicated as the etiology of hypoxemia in patients with long-standing liver disease related to Gaucher disease [4,5]. Pulmonary arteriovenous malformation (AVM) in Gaucher disease, however, has not been reported in the English literature. Ocular manifestations of type I Gaucher disease include infiltration of the retina, conjunctiva and uvea with visual loss [6-8]. However, exophthalmos related to Gaucher disease has also not been described. Here we report an adult with Gaucher disease complicated with these two unusual manifestations, exophthalmos and pulmonary AVM.
Case presentation
This 34-year-old woman was a case of Gaucher disease type I with initial presentation of hepatosplenomegaly and severe bone diseases including fractures of the lower extremities and bone crisis at childhood. She was the second of a pair of twins. Her twin sister and another younger sister are both affected. There is no consanguinity in the family. She underwent splenectomy at age 5 years due to persistent thrombocytopenia, but the diagnosis of Gaucher disease was established only after a bone marrow examination at age 11 years. Her leukocytes β-glucocerebrosidase activity checked at age 26 years was 1.56 nmol/mg/h (normal: >28.42 nmol/mg/h). At that time, she had severe bone deformities, hepatomegaly, anemia, clubbing fingers and toes, but pink lips. There was no audible heart murmur, and her breathing sound was clear. Her liver size was measured 5 cm below subcostal margin at right mid-clavicular line, and neither spider angiomata nor superficial vein engorgement was found. Hemogram revealed a platelet count of 103 × 109/L and hemoglobin level of 9.6 mg/dL. DNA analysis of this patient and her two affected siblings all revealed a V375L/L444P genotype of the β-glucocerebrosidase gene.
The patient complained of bilateral orbital pain with gradual protrusion of eyes since 30 years old. Thyroid function tests, including T3, T4, thyroid-stimulating hormone and free T4, showed normal results. Ophthalmologic examinations failed to reveal any specific findings related to the exophthalmos. Magnetic resonance imaging (MRI) of the eyes revealed increases in retrobulbar space with fat-like density and mild hypertrophy of extra-ocular muscles (Fig. 1A). She received enzyme replacement therapy (ERT) with imiglucerase (Cerezyme) at a dose of 60 U/kg every two weeks since 32 years old. Her exophthalmos progressed slightly during the first year of ERT, and then started to regress. Exposure conjunctivitis still bothered her currently.
The patient suffered from intermittent dyspnea at age 34 years. She became cyanotic and had an oxygen saturation of 85% as measured by pulse oximeter. Arterial blood gas analysis revealed a pH of 7.41, carbon dioxide tension of 27.9 mmHg, and oxygen tension of 61.2 mmHg in room air. A grade III/VI bruit was audible over the right lower chest, and a chest x-ray revealed prominent pulmonary conus and increased infiltration over the right lower lung field (Fig. 1B). A high-resolution computed tomography (HRCT) of the chest suspected an AVM with engorged right inferior pulmonary artery and its draining veins (Fig. 1C); but no focal lesions were found in the lung parenchyma. Right pulmonary artery angiogram showed several dilated and tortuous vessels from the right pulmonary artery, which directly connected to vessels draining into the right pulmonary vein over the right lower lobe of the lung (Fig. 1D). Early appearance of contrast medium in the right pulmonary vein indicated the presence of pulmonary arteriovenous malformation. The patient's pulmonary artery pressures were 48/19 mmHg (mean, 33 mmHg). Embolization was achieved with 6 coils, and resulted in a rise of oxygen saturation to 96%, although residual shunt was still present. Unfortunately, two months later, dyspnea and cyanosis recurred, and her oxygen saturation dropped to 85% in room air. A pulmonary function test at that time revealed moderately severe restrictive lung disease.
Discussion
This 34-year-old patient presented symptoms and signs of exophthalmos and pulmonary AVM, which are unusual for Gaucher disease. She had normal mentality with no neurological symptoms such as ophthalmoplegia, therefore the disease could be classified as type I. However, she had severe thrombocytopenia requiring splenectomy and had had repeated fracture requiring prolonged immobilization. In a recent report from the Gaucher Registry, fracture was present in only 15% of all patients [9]. The patient's twin sister received splenectomy at young age and has bone disease similar to hers. Therefore, these sisters have an illness toward the severe end of the type I Gaucher disease.
Ocular manifestations in Gaucher disease are very rare, and the tissues reported to be involved are retina, conjunctive, and uvea [6-8]. In the MRI study, the increased retrobulbar spaces are filled with tissues with fat density, suggesting that the exophthalmos is caused by Gaucher cell infiltration. The clinical course also supports this hypothesis, since the patient's eyes started to retract after ERT.
Gaucher cells can infiltrate the alveolar spaces or the interlobular and intralobular septa, leading to air space and interstitial disease [2]. Gaucher cells can also plug in the pulmonary capillary vessels and cause pulmonary hypertension [10]. However, the chest x-ray and HRCT of the index patient revealed normal lung parenchyma, not suggesting direct alveolar or interstitial infiltration of Gaucher cells. It is also known that Gaucher disease can cause hepatic dysfunction which may induce abnormal dilatation of the intrapulmonary capillaries or the so-called hepatopulmonary syndrome [4,5]. It is possible that pulmonary AVM was the consequence of an abnormal progression of the hepatopulmonary syndrome, however, there was no overt evidence of severe hepatic dysfunction in the patient.
ERT has had a great impact on the outcome of Gaucher disease [11]. On the other hand, the effect of ERT on pulmonary hypertension remains to be established [12-15]. The patient had mild pulmonary hypertension. The pulmonary hypertension might not be related to AVM, since the latter condition usually causes low pulmonary artery pressure [16]. It has been reported that pulmonary hypertension may be triggered or aggravated by ERT [17]. It is possible that the pulmonary AVM may have existed but only caused symptoms when ERT changed intrapulmonary hemodynamics after clearance of Gaucher cells.
Through a registry of 1698 patients reported in 2000, the allele frequency of N370S was 53%, and that of L444P was 18% [9]. The L444P mutation is more common in Asians [19,20], and has been detected in Taiwanese patients with both type I and II Gaucher disease [20,21]. The prevalence of L444P and the absence of the N370S mutation may explain the more severe phenotype in Gaucher disease in Asians. The V375L mutation has been classified as a mild mutation [22], which might explain the V375L/L444P genotype in type I Gaucher disease. However, although the twin sister of the indexed person had bone disease of similar severity, she didn't have eye or lung problem. One the contrary, their younger sister has less skeletal involvemen, but had dyspnea and cyanosis, which responded to ERT. There surely are non-allelic or epigenetic factors influencing the phenotypes.
Conclusion
Although both eye and lung diseases have been associated with Gaucher disease, this is the first reported demonstration of exophthalmos and pulmonary AVM in the same patient. This case may therefore present an extremely severe and unusual form of type I Gaucher disease. Different responses of these lesions to ERT would probably be attributed to different pathogenesis and natural course in the organ involvement in Gaucher disease.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
CC prepared the manuscript of this case report. YC and WH conducted long-term follow-up and prescribed ERT for the patients. NT and WZ carried out the gene mutation analysis. JW performed coil embolization of the pulmonary AVM. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Figures and Tables
Figure 1 Type I Gaucher disease with exophthalmos and pulmonary arteriovenous malformation. (A) MRI of the head shows increases in retrobulbar space and hypertrophy of extra-ocular muscles. (B) Chest X ray reveals prominent pulmonary conus and increased infiltration over right lower lung field. (C) High-resolution computed tomography of chest reveals engorged right inferior pulmonary artery and its draining veins. (D) Pulmonary angiogram demonstrates right pulmonary arteriovenous malformation.
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| 15943874 | PMC1164415 | CC BY | 2021-01-04 16:03:32 | no | BMC Med Genet. 2005 Jun 9; 6:25 | utf-8 | BMC Med Genet | 2,005 | 10.1186/1471-2350-6-25 | oa_comm |
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BMC Med EducBMC Medical Education1472-6920BioMed Central London 1472-6920-5-191593264810.1186/1472-6920-5-19Research ArticleWhich young physicians are satisfied with their work? A prospective nationwide study in Norway Finset Kristine Benedictow [email protected] Tore [email protected] Erlend [email protected] Reidar [email protected] Oivind [email protected] Per [email protected] Department of Behavioural Sciences in Medicine, Institute of Basic Medical Sciences, Medicine, University of Oslo, Postbox 1111 Blindern, NO-0317 Oslo, Norway2005 2 6 2005 5 19 19 31 1 2005 2 6 2005 Copyright © 2005 Finset et al; licensee BioMed Central Ltd.2005Finset et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Few studies have investigated personality and medical school variables in regard to job satisfaction after graduation. It is of great importance to investigate these factors because this information may be used in the recruitment/admittance process to medical schools, and possibly to improve medical education.
Methods
We conducted a nationwide prospective 10-year follow-up study of medical students at all medical schools in Norway. They were approached three times during their medical training: at very beginning (T1), in the middle (T2), in the last year of medical school (T3), and then four years after graduation (T4). There were 210 participants who responded on all four occasions. Job satisfaction was measured with the Job Satisfaction Scale, which was used as the outcome variable. In addition to conducting multiple regression analysis for the total sample, we also conducted similar analyses separately for men and women.
Results
Among the demographic and personality variables, 'having a father who is a physician' and 'interpersonal functioning (being withdrawn)' were significantly associated with job satisfaction at T4. Among the medical school variables, 'well-being with peers', 'identification with the doctor's role at the end of curriculum', 'perceived medical school stress', and 'perceived clinical skills' were significantly associated with job satisfaction. In the multiple regression analysis only 'father as a physician' and 'perceived clinical skills' yielded an independent influence on the outcome variable in separate analyses within sub-groups of male and female students, 'perceived clinical skills' differentiated among woman only, while 'well-being with peers' differentiated only among men.
Conclusion
The main finding of this study is that the young physicians who are the most satisfied in their work are those whose fathers are physicians and those who have a high level of perceived clinical skills at the end of medical school. There are also differences in regard to predictors of job satisfaction among men and women. These findings indicate that medical schools should invest substantial effort in clinical skills training, and this seems to be especially important among female students.
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Background
Physicians' job satisfaction is important because it may affect patient satisfaction [1] and patient adherence [2] and may be inversely associated with level of stress as well as burnout [3,4]. Most studies have emphasized the importance of several aspects of the current work situation that contribute to the job satisfaction of physicians [5]. Both sense of autonomy [6] and adequate time with patients [7] have been shown to be of particular importance. The current work situation will possibly explain most of the variations in job satisfaction among young physicians. On the other hand some researchers found that differences in current work situation for pre-registered house officers did not predict whether or not they were rated as good or bad [8], on this basis the researchers propose that there are personality characteristics by the doctors themselves that make the difference. Nevertheless, are there also other factors that are important determinants of subsequent job satisfaction that could be identified during the time spent at medical school? Which medical students will truly be satisfied working as physicians? If we could identify the characteristics of such students, we might have information that could be used in the recruitment/admittance process of medical schools, and could also possibly improve our medical education. One study has reported a relationship between learning style in medical school and approach to work several years later [3]. To our knowledge, however, there are few other prospective studies that have addressed these important questions. Therefore, we find it of great value to further investigate these aspects of physician job satisfaction.
There are several factors that could be possible predictors of young physicians' job satisfaction. Age and sex may be important. Previous cross-sectional studies have, however, reported conflicting results [9-11]. Coming from a family of physicians may imply a high motivation and realistic expectations regarding working as a physician. In a previous study from this longitudinal project, having a father who was a physician predicted level of ambition in medical students [12]. Regarding the importance of personality traits, one study found that extraversion had a positive effect and neuroticism a negative effect on job satisfaction [13]. This is not surprising, given that extroversion and neuroticism are well known to affect satisfaction and well-being in general [14]. Interpersonal functioning as a personality trait is important for mastering relationships with patients and colleagues, and could therefore influence later job satisfaction. In addition, self esteem and the tendency to react with nervous symptoms could also be of importance.
Concerning medical school variables, one would expect that students who later will be satisfied with their work as physicians have reached a higher level of identification with the doctor's role [15], and have attained a high level of confidence in their own clinical skills by the end of medical school. In a review article on the performance-satisfaction relationship, Judge, Thoresen, Bono & Patton [16] propose the possibility that performance precede satisfaction. It is therefore of interest to further investigate this relationship.
Students with a high level of perceived medical school stress and/or those who are interacting poorly with student colleagues might be at risk of experiencing the same difficulties when they are working as physicians.
Based on these considerations, we have used data from a prospective longitudinal study of a nationwide sample of Norwegian medical students that were followed from the first month of university, examined again after three years, again at the end of the curriculum and finally four years later, to identify predictors of job satisfaction after graduation.
We hypothesized that the following variables would influence job satisfaction four years after the end of medical school:
1) Demographic factors: sex, age and father being a physician
2) Personality factors and nervous distress: personality traits, self esteem interpersonal problems and nervous symptoms.
3) Medical school factors: type of medical school, perceived clinical skills, well being with peers, perceived medical school stress and identification with the doctor's role at the end of the curriculum.
Methods
Sample
Students entering medical school at the four Norwegian universities in 1993 (n = 421) were invited to participate. Data were collected in the first month of medical school (T1), in the middle of the third year (T2), at the end of the sixth year (T3) and four years after graduating (T4). The response rates at the four different collections are shown in table 1. At T2, not all of the students whom were invited to participate at T1 were available, most obviously due to drop-out from school. Therefore, the population of students varied from the one assessment time to the next, making it difficult to estimate the real response rate. All the way, those responding on all four occasions was 210 individuals (50% of the original population with N = 421), 59% of whom were women; this constituted the sample to be investigated. Thus, we would estimate the "true" response rate to be closer to 60% than to 50%.
Table 1 Respons rate at the four different occasions
Point of time Response rate Total response rate in percent Response rate of original sample
1993 – T1 374 88% -
1996 – T2 287 68% 77%
1999 – T3 238 56% 64%
2003 – T4 210 50% 56%
Mean age at T1 was 21.6 years (SD 2.6) with no significant sex differences. Students with foreign citizenship accounted for 2.5% of the sample. Education of the parents at college level or higher was 77% for the fathers and 74% for the mothers, and 14.8% (n = 31) of the sample had a mother and/or a father who was a physician. There were no participants who only had mother as a physician; either both parents or father only had this occupation. There were no significant differences between the response and non-response group at T4 regarding age or citizenship. The non-response rate was somewhat higher for men than for women (57% vs. 40%, Chi square = 10.07, p = .002).
There were no substantial differences between the four universities regarding the proportion of responders at T3 and responders at T4. Further descriptions of the sample and characteristics of the non-responders are accounted for else where [15].
The study was conducted in collaboration with the Norwegian Medical Association and with the approval of the National Data Inspectorate.
Procedures
Data from all four universities were collected by mail and were anonymous, except for a code number (known only by the National Bureau of Statistics) linking data from all the four occasions to the same person.
Outcome variable
Job satisfaction was assessed at T4 using a 10-item version of the Job Satisfaction Scale [17], which employed a seven-point Likert scale ranging from 1 = 'very satisfied' to 7 = 'very dissatisfied'. The validity and reliability of the scale has been found satisfactory [17]. Some examples of the items are: [how satisfied are you with...] 'the amount of responsibility you are given', 'your fellow workers', 'your hours of work' and 'your opportunity to use your ability'. For the sake of convenience, we inverted the scale so that a high score indicated high satisfaction. A Principal Component Analysis of the 10 items yielded a typical one-factor solution; therefore, the index score from all items was used as the outcome variable. The Kaiser-Meyer-Olkin value was satisfactory (.8) and the Bartlett's Test of Sphericity reached statistical significance. The eigenvalue of this component was: 3.945. Cronbach's α for the whole inventory was .80. The overall mean of Job Satisfaction in the sample was 5.18 (SD = .78). The median was 5.2.
Predictors – bivariate analysis
All predictors are shown in Table 2, with mean, standard deviation (SD), reliability coefficient, and assessment time displayed. Co-linearity-analyses has been conducted for all the included predictors, values did not exceed .40.
Table 2 Mean, SD and assessment time.
Variable Time Mean (SD) and percent
Demographic variables Gender T1 Women 59 %
Men 41%
Age T1 Mean = 21.6 (SD = 2.58)
Father as physician T1 No – 75.7% (n = 159)
Yes – 14.8% (n = 31)
Personality variables Basic Character Inventory T1 Vulnerability: M = 3.8 (SD = 2.22)
Intensity: M = 5.2 (SD = 2.22)
Control: M = 3.2 (SD = 2.12)
Reality weakness: M = 1.8 (SD = 1.67)
Inventory of Interpersonal Problems T2 Submissiveness: M = 0.7 (SD = 0.38)
Aggression: M = 0.6 (SD = 0.37)
Sociability/Withdrawn: M = 0.8 (SD = 0.43)
Symptom checklist T1 Mean = 0.55 (SD = 0.62)
Self esteem T1 Mean = 2.9 (SD = 0.62)
Medical school variables Perceived Clinical Skills T3 Mean = 5.0 (SD = 0.63)
Well-being with peers T2 Mean = 5.3 (SD = 1.00)
Perceived medical school stress T3 Mean = 2.43 (SD = 0.51)
Identification with the doctor's role T3 Mean = 4.9 (SD = 1.06)
Outcome Variable Job satisfaction T4 Mean = 5.2 (SD = .78)
T1, 1993 (beginning of medical school); T2, 1996; T3, 1999 (end of medical school); T4, 2003 (four years after graduation)
Demographic variables
Sex (female = 1/male = 2), age, and the parents' occupations, specifically whether or not the parents were physicians, were recorded.
Personality variables
Personality traits were measured using the 'Basic Character Inventory' [18,19], a 36-item questionnaire with sub-scales of vulnerability (neuroticism), intensity (extroversion), control, and reality weakness, requiring yes/no answers. The data were collected at T1.
One instrument assessed nervous symptoms: the Symptom Checklist-5 [20] with a five-point scale ranging from 0 = 'no problem' to 4 = 'severe problems'. The data were collected at all assessments, but data from T1 were used in this study.
One instrument measured interpersonal functioning: the Inventory of Interpersonal Problems (IIP) (64 items) [21], a five-point scale ranging from 0 = 'no problem' to 4 = 'severe problems'. The data were collected at T2. The 64 items of this inventory were subjected to Principal Components Analysis (PCA). Prior to performing PCA, the suitability of data for factor analysis was assessed. The Kaiser-Meyer-Olkin value was satisfactory (.8) and the Bartlett's Test of Sphericity reached statistical significance, supporting the factorability of the items.
The scree-plot indicated that a three-component solution best fitted the data. The eigenvalues of these three components were: 12.356, 5.180 and 3.960. Together these three components explained a total of 33.6% of the variance. To facilitate the interpretation of these three components, a Varimax rotation was performed. Items with sufficient factor loadings on the first component only constituted an index called 'Submissiveness', consisting of items such as 'It is hard for me to be self-assertive', 'It is hard for me to set limits' and 'It is hard for me to show anger'. Items with sufficient factor loadings on the second component only constituted an index called 'Sociability/Withdrawn', consisting of items such as 'I keep people too much at a distance', 'It is hard for me to trust other people' and 'It is hard for me to really care about another person's problems'. Items with sufficient factor loadings on the third component only constituted an index called 'Aggression' and consisted of items such as 'I lose my temper too much', 'I argue too much' and 'I manipulate others too much'.
Self-esteem is a measurement derived from the vulnerability dimension of the original BCI-136 item version [18,22]. The variable used in the analyses was an index computed from eight items with a four-point scale at T1 ranging from 1 = 'do not agree' to 4 = 'agree'. A typical item was: "I feel most often that others perform better than I do myself".
Medical-school factors (collected at T3)
The curriculum differed in some respects between the four Norwegian medical schools. When the students started their medical education, two sites (Oslo and Bergen) organized their curriculum according to the traditional division between pre-clinical and clinical parts, with a comprehensive exam in between. At the two other sites (Trondheim and Tromsø), the teaching of pre-clinical and clinical subjects was integrated, with no main exam completed prior to data collection at T2. For the multivariate analyses, the four study sites were computed as dummy variables, with one of the integrated schools as the reference value.
Two questions further measured the students' well-being with peers, 'To what degree do you feel secure among your fellow students' and 'To what degree do you feel satisfied with your fellow students'. Both items were measured on a seven-point Likert scale, ranging from 1 = 'to a very little degree' to 7 = 'to a very large degree'.
Perceived medical-school stress (PMSS) was measured at T3 using an instrument developed by Vitaliano et al. [23] and modified by Bramness et al. [24], which has previously shown good reliability and validity. The instrument consists of thirteen items with a five-point scale ranging from 1 = 'low stress' to 5 = 'high stress'.
Identification with the doctor's role at the end of the curriculum was measured using four items, with a seven-point Likert scale ranging from 1 = 'never/little' to 7 = 'always/very much'; an example of the questions is 'I feel like a doctor in the emergency room'. The construction of this variable has been described elsewhere [15].
Self-assessed clinical skills were measured using 'Perceived Clinical Skills' (PCS) (in an earlier study called Perceived Recording Skills) at T3 with six items, of which three items covered confidence in own competence, which has been used and validated earlier [15]. A typical item covering confidence in clinical skills is: 'I feel confident about examinations to be done', using a response scale from 1 = 'never' to 5 = 'always'. For an overview of the measures and instruments, see Table 2.
Multivariate analysis
Block-wise multiple regression analyses were performed, using job satisfaction scores as the dependent variable. The predictors were entered in three blocks: demographics ('sex', 'age' and 'father as physician') were entered in the first block, the only bivariate significant personality variable ('interpersonal problems – withdrawn') was entered in a separate block, and medical school variables ('well-being with peers', 'perceived medical school stress', 'perceived clinical skills' and 'identification with the doctor's role') were entered in the third and final block.
We also conducted identical multiple regression analyses separately for men and women.
Statistics
Means, median, correlations and block-wise multivariate analyses were conducted to investigate the relationship between the independent variables and job satisfaction. A p value < .05 was used as the level of significance. SPSS version 12.0 was used for statistical analyses.
Results
Bivariate correlations between predictors and job satisfaction, and alpha-levels are shown in Table 3. One of the demographic and one of the personality variables were significantly related to job satisfaction four years after graduation: father being a physician (r = .161, p = .028) and the interpersonal index 'withdrawn' (from IIP) (r = -.255, p < .001). Among the medical school variables, students' well-being with peers (r = .221, p = .001), identification with the doctor's role (r = .240, p = .001), perceived medical school stress (r = -.236, p = .001), and perceived clinical skills (r = .240, p < .001) were significantly correlated with job satisfaction.
Table 3 Bivariate correlations between predictors and job satisfaction.
Predictors Job Satisfaction
Background variables
Gender -.026
Age .019
Study location ns.
Parent as physician (father) . 161 * (p = .028)
Personality variables
BCI – Vulnerability -.101 Cronbach's Alpha:.691
BCI – Intensity .050 Cronbach's Alpha: .681
BCI – Control -.036 Cronbach's Alpha: .663
BCI – Reality Weakness .031 Cronbach's Alpha: 59.4
Symptom distress (SCL-5) -.022 Cronbach's Alpha: .802
Interpersonal – dominating -.072 Cronbach's Alpha: .817
Interpersonal – submissive -.110 Cronbach's Alpha: .848
Interpersonal – withdrawn -.255 *** (p < .001) Cronbach's Alpha: .764
Self-esteem (T1) .093 Cronbach's Alpha:..852
Medical school variables
Well-being with peers .221** (p = .001) Jus two items.
Identification with doctor's role .240 ** (p = .001) Cronbach's Alpha: .833
Perceived medical school stress -.236 ** (p = .001) Cronbach's Alpha: .782
Perceived clinical skills .287 *** (p < .001) Cronbach's Alpha: .563
* p < .05 ** p < .01 *** p < .001
Multivariate analyses
The predictors that had a significant bivariate correlation with job satisfaction were entered blockwise in a linear multiple regression analysis, with job satisfaction as the dependent variable (Table 4). In the final model, two predictors independently explained variance in job satisfaction: one was whether father was a physician, the other was level of perceived clinical skills, which together explained a total of 13.9% (R2 .139) of the variance. The change in adjusted R2 was significant from step one to two (F = 12.74, p < .001) and from step two to three (F = 4.63, p = .001).
Table 4 Multivariate analysis of bivariate correlating predictors with job satisfaction
Block 1 Block 2 Block 3
Gender .018 .043 -.004
Age .020 .019 -.001
Father dr. .165* .181* .147*
IIP-W. -.254*** -.113
Well-b. peers .099
PMSS -.112
PCS .171*
Ident .089
Adj. R2 .011 .070 .139
* p < .05 ** p < .01 *** p < .001
We finally conducted the same blockwise analyses in sub-groups of female and male students separately. In these analyses, a different pattern emerged. Among male physicians, only well-being with peers (stand. beta .28, p = .025) contributed significantly, explaining a total of 13.8% (R2 .138) of the variance. 'Father being physician' did not contribute significantly in the male sub-sample (stand. beta .041, p = .728). Among female physicians, 'perceived clinical skills' was the only significant contributor (stand. beta .25, p = .013), explaining a total of 14.4% (R2 .144) of the variance. 'Father being a physician' only reached borderline significance in the female sample (stand. beta .165, p = .076) (not shown in table).
Discussion
The main finding of this study was that young physicians who were the most satisfied in their work were those that had a father who was a physician and those who had a high level of perceived clinical skills at the end of medical school. It may be argued that confidence in own (perceived) clinical skills is reflecting a general self-esteem as a personality trait rather than being linked to real clinical competence. In this study, however, we neither found a significant correlation between the self-esteem index nor the personality variables (BCI) and job satisfaction, indicating that confidence in clinical skills in this respect do measure more than personality characteristics. The impact of self-assessed clinical skills, most probably influencing the management of challenges in medical work, is understandable and consonant with results from other studies [15,19]. An optimal and balanced confidence in clinical skills are not only of utmost importance for the benefit of the patients, but also, as this study shows, significant for job satisfaction after graduation. This finding emphasizes the importance of medical school training of clinical skills that in turn will help promoting an optimal and balanced confidence. In addition, we found that interpersonal functioning is important for job satisfaction, but this factor is most probably goes through perceived clinical skills as this variable is the heaviest influencing one in the block of medical school variables. This might indicate that socially withdrawn students achieve less confidence by clinical training which may reduce their development of clinical competence.
'Father as physician' also contributed significantly to the level of job satisfaction in the final model, and is consonant with a similar influence upon ambition in medical students [12]. This family constellation may reduce the expectations of how satisfying the working situation for a physician should be. When each sex were analysed separately, the effect of 'father as physician' was most important among the female physicians. Being the daughter of a physician may imply a better role model when entering a still male dominant profession, and may also evoke more positive attitudes from colleagues. Although this constellation seems to be a valuable indication of job satisfaction, for many reasons it is still not useful as a criterion for admission to medical school.
When multiple regression analyses were conducted for men and women separately, other interesting differences also emerged. In relation to job satisfaction, perceived clinical skills differentiated women only, while well-being with peers differentiated only men. These findings were, to some degree, unexpected. It can be explained by the assumption that it is more important for female students' satisfaction with work to be competent in clinical skills and feel safe in their relationship with patients, while among men, the satisfaction depends more on their social skills in relating to colleagues. These differences should be further explored.
There are some limitations to this study. Even with the difficulties in assessing an objective non-response rate, there is no doubt that the size of the investigated sample is reduced to at least 60% compared to the student-cohort we intended to follow through the 10 years. As no sex differences in the level of job satisfaction were detected, the higher response rate among women should not bias our results. Although there was a limited response rate, the lack of differences in level of job satisfaction between sexes should not influence the representativity of the sample. Concerning the variable of the father being a physician, the N became small in the separate analyses of men and women, thereby increasing the risk of type II errors. In total, a variance of 14% was explained. This may initially seem small and insignificant. On the other hand, in this study we were mainly occupied with early predictors of job satisfaction, and this probably accounts for the low level of explained variance
Conclusion
We have found that having a father as physician and the level of perceived clinical skills at the end of medical school were related to job satisfaction four years after graduation; this effect remained when controlling for age, sex and personality. Medical schools should therefore emphasize clinical skills training. This is especially important among female students.
Competing Interests
The author(s) declare that they have no competing interests.
Authors' Contributions
KBF acted as the principal investigator. TG and PV contributed substantially to the whole study. EH, RT and OE contributed to the interpretation of data and drafting of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
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| 15932648 | PMC1164416 | CC BY | 2021-01-04 16:30:56 | no | BMC Med Educ. 2005 Jun 2; 5:19 | utf-8 | BMC Med Educ | 2,005 | 10.1186/1472-6920-5-19 | oa_comm |
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BMC NephrolBMC Nephrology1471-2369BioMed Central London 1471-2369-6-51591068710.1186/1471-2369-6-5Research ArticleComparison of two recombinant erythropoietin formulations in patients with anemia due to end-stage renal disease on hemodialysis: A parallel, randomized, double blind study Pérez-Oliva Jorge F [email protected]ález Martha [email protected]ía-García Idrian [email protected]ín Pedro J [email protected] Carmen M [email protected]ández-Montero Tairí [email protected] Marcia [email protected] Yuri [email protected]Ávila-Albuerne Yisel [email protected] Alicia [email protected]ópez Hailen [email protected]és Raúl [email protected]ópez-Saura Pedro A [email protected] Bioequivalence Study of Erythropoietin Group1 Hemodialysis Department, National Institute of Nephrology, Havana, Cuba2 Nephrology Department, "Gustavo Aldereguía Lima" Hospital, Cienfuegos, Cuba3 Clinical Trials Division, Center for Biological Research, Havana, Cuba4 Clinical Laboratory Department, National Institute of Nephrology, Havana, Cuba5 Clinical Trials Department, National Center of Clinical Trials, Havana, Cuba2005 23 5 2005 6 5 5 11 11 2004 23 5 2005 Copyright © 2005 Pérez-Oliva et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Recombinant human erythropoietin (EPO) is used for the treatment of last stage renal anemia. A new EPO preparation was obtained in Cuba in order to make this treatment fully nationally available. The aim of this study was to compare the pharmacokinetic, pharmacodynamic and safety properties of two recombinant EPO formulations in patients with anemia due to end-stage renal disease on hemodialysis.
Methods
A parallel, randomized, double blind study was performed. A single 100 IU/Kg EPO dose was administered subcutaneously. Heberitro (Heber Biotec, Havana, formulation A), a newly developed product and Eprex (CILAG AG, Switzerland, formulation B), as reference treatment were compared. Thirty-four patients with anemia due to end-stage renal disease on hemodialysis were included. Patients had not received EPO previously. Serum EPO level was measured by enzyme immunoassay (EIA) during 120 hours after administration. Clinical and laboratory variables were determined as pharmacodynamic and safety criteria until 216 hours.
Results
Both groups of patients were similar regarding all demographic and baseline characteristics. EPO kinetics profiles were similar for both formulations; the pharmacokinetic parameters were very close (i.e., AUC: 4667 vs. 4918 mIU.h/mL; Cmax: 119.1 vs. 119.7 mIU/mL; Tmax: 13.9 vs. 18.1 h; half-life, 20.0 vs. 22.5 h for formulations A and B, respectively). The 90% confidence intervals for the ratio between both products regarding these metrics were close to the 0.8 – 1.25 range, considered necessary for bioequivalence. Differences did not reach 20% in any case and were not determined by a formulation effect, but probably by a patients' variability effect. Concerning pharmacodynamic features, a high similitude in reticulocyte counts increments until 216 hours and the percentage decrease in serum iron until 120 hours was observed. There were no differences between formulations regarding the adverse events and their intensity. The more frequent events were pain at injection site (35.3%) and hypertension (29%). Additionally, further treatment of the patients with the study product yielded satisfactory increases in hemoglobin and hematocrit values.
Conclusion
The formulations are comparable. The newly developed product should be acceptable for long-term application.
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Background
Erythropoietin (EPO) is a sialoglycoprotein hormone secreted primarily by the mature kidneys in response to tissue hypoxia and/or red cell mass decrease. It stimulates erythrocyte production from the blood marrow [1]. Recombinant human EPO has been used since the 80's in the treatment of anemia associated to end-stage renal disease. Reports in hemodialysis patients indicate an effective erythropoiesis increment, ceasing or decrease of transfusion frequency, and quality of life improvement [2-6]. However, this treatment is still expensive, not affordable to developing countries if an extensive use, for all patients who need it, is sought, considering the same prevalence as in the United States, where there are currently 270 000 patients in dialysis [7,8].
Whether multiorigin recombinant proteins can be clinically interchangeable despite coming from different strains and manufacturing processes is a controversial matter nowadays [9,10]. If this principle is accepted, then a study that shows pharmacological similarity between two products would be enough to indicate its therapeutic interchangeability, given that they are chemically and pharmaceutically equivalent as well.
Recently, a recombinant EPO formulation was obtained in mammalian cells at the Center for Genetic Engineering and Biotechnology (CIGB in Spanish), Havana. Its pharmacokinetic, pharmacodynamic and safety profiles were studied, compared to a similar, previously existing preparation. The reticulocyte count increments and serum iron consumption were used as biological action indicators. Hemoglobin and hematocrit increases after 3-month treatment was used as an additional efficacy criterion as well.
Methods
Subjects
Patients, 18–75 years, both sexes, with anemia (hematocrit ≤ 28% or hemoglobin ≤ 9 g/dL) due to end-stage renal disease on hemodialysis (3 sessions per week), who gave their written, informed consent to participate were included. The exclusion criteria were: previous EPO treatment; pregnancy or nursing; sepsis or active infection; non-treated iron deficiency (serum iron <60 mg/dL, ferritin <100 ng/mL, transferrin saturation index <20%); cancer; hormonal treatment (except thyroid hormone, contraceptive and insulin); liver disease (twice and half the transaminases normal values); diastolic arterial tension ≥ 120 and/or systolic ≥ 180 mmHg; severe psychiatric dysfunction or another limitation that prevented the patient to give his consent; epilepsy; thrombocytosis (≥ 500 000 /mm3); signs of active bleeding; bone marrow aplasia; active collagen disease; severe hyperparathyroidism (serum parathormone > 400 ng/L), and hyperaluminemia (serum aluminum > 50 ng/mL). They were withdrawn from the trial if they died, abandoned voluntarily, had severe adverse reactions, were subjected to renal transplant, used not allowed medications or other EPO formulations, or if any exclusion criteria arose. The trial was in compliance with the Helsinki declaration. The protocol was approved by the Ethics Committee of the participant institutions and by the Cuban Regulatory Authority.
EPO formulations
Formulation A (Heberitro, Heber Biotec, Havana) was in vials containing 2000 IU of human recombinant (rHu) EPO (produced in Chinese hamster ovarian (CHO) cells at CIGB, Havana), 2.5 mg serum human albumin, 0.2 mg polysorbate 20, 2.9 mg NaCl, 4.6 mg NaH2PO4.2H2O, 9.9 mg Na2HPO4, and water for injection to complete 1 mL. Formulation B consisted in a commercially available preparation (Eprex, CILAG AG, Switzerland) presented in pre-filled syringes containing 2000 IU rHuEPO, 0.15 mg polysorbate 80, 2.192 mg NaCl, 0.580 mg NaH2PO4.2H2O, 1.115 mg Na2HPO4.2H2O, 2.50 mg glycine, and water for injection to complete 0.5 mL.
Study design
Subjects were distributed according to a computer-generated random number list, stratified by study center, to receive subcutaneously a single dose of 100 IU/Kg of one of the EPO preparations (A or B) in a parallel design. The study was double blinded. As the presentation of the products differed, blinding was kept by loading formulation A vials in syringes coded with the patients' inclusion numbers, and the nurse that administered the products did not participate in the rest of the trial.
Patients were hospitalized throughout the study under strict medical supervision. EPO administrations and blood sampling were done post-hemodyalisis, when renal chronic failure patients are closer to their ideal dry weight. Antipyretic medication was given orally if flu-like symptoms or pain at the injection site arose. If blood hypertension occurred during follow-up patients were treated according to the investigator's criteria and the precedent individual treatments.
After the 9 days in-hospital follow-up for the pharmacological comparison, both groups of patients continued treatment with formulation A, 30 UI per Kg 3 times per week, subcutaneously, in order to obtain additional efficacy and safety data for this product. Injections were administered after the hemodialysis sessions at the patients' current treatment centers.
Clinical and laboratory evaluations
Blood samples for serum EPO concentration determinations were collected by venipuncture, before and 2, 6, 10, 14, 17, 20, 24, 48, 72, 96, and 120 hours after injection. Pharmacodynamics was assessed by reticulocyte counts before and at 48, 72, 120, 144 and 216 hours, and also by serum iron consumption, before and at 120 and 216 hours. Other hematological determinations (hemoglobin, hematocrit, red blood cell, platelet, and total and differential leukocyte counts) were taken as safety variables, every 24 hours. Alanine aminotransferase (ALT) was measured before and at the end of the in-patient period (216 hours). Patients were regularly checked for vital signs and symptoms, including during the hemodialysis sessions.
EPO was quantified in serum with a highly sensitive enzyme immunoassay (EIA) kit (Quantikine® IVD®, R&D System, Inc, Minneapolis). Serum iron concentrations were determined by a colorimetric test (NITRO-PAPS, CENTIS, Havana). Blood chemistry and hematological counts were done according to usual clinical laboratory procedures. All laboratory analyses were done blindly.
Anti-EPO antibodies were screened at the end of the further treatment and follow-up period by a semiquantitative, "sandwich type", EIA system (EPO; sample; protein A – horseradish peroxidase conjugate) developed and validated at the Center for Biological Research, Clinical Laboratory (manuscript in preparation). For this purpose the blood sample was taken 72 hours after the last EPO administration. If some sample resulted positive it was previewed that the patient's baseline sample would be tested in order to discard spontaneous pre-existing autoantibodies.
Data analysis
The drug disposition data analysis was performed per individual by a non-compartmental method with a combined linear/log – linear trapezoidal rule approach. The linear trapezoidal rule was used up to peak level and the logarithmic trapezoidal rule thereafter. The first-order rate constant associated with the curve terminal (log linear) portion (λ) and elimination half-life (t1/2) were estimated by linear regression of the included terminal data points. Time-to-peak values (Tmax) were determined directly from the experimental data as the time of maximum observed level (Cmax) considering the entire curve. Area under the serum concentration-time curve from 0 to 120 hours (AUC120) was calculated using the linear/log linear trapezoidal rule. Mean residence time (MRT) was also calculated using the moments of the drug disposition curve. Parameters that were extrapolated to infinity, such as AUC (area under disposition curve) and AUMC (area under first moment of the disposition curve), were computed based on the last predicted value from the linear regression performed to estimate λ and t1/2. In addition, other pharmacokinetic parameters were also calculated, such as the systemic clearance (CL), volume of distribution (Vd), and the peak to area ratio value (CAV = Cmax / AUC). The apparent absolute bioavailability (F) was estimated using data from a previously reported study after intravenous dosing of recombinant EPO as the subcutaneous to intravenous AUC ratio [11]. The WinNonlin professional software (Version 2.1, Pharsight Inc., 1997, NC, USA) was used for all these purposes.
Statistical analyses were done using SPSS for Windows version 11.5. Firstly, to test the homogeneity between the treatment groups, the Mann-Whitney's U or Student's t test (depending on the normality assumption) was applied for quantitative control variables and the chi-square or Fisher's exact test for the qualitative ones. Pharmacokinetic parameters were tested for normal distribution by the Shapiro-Wilk's test and for variance homogeneity by the Levene's test. To compare formulations the analysis of variance (ANOVA) and the estimation of confidence intervals (90%) of the ratio were used. Vital signs and laboratory variables were treated using paired analysis (Student's t test or Wilcoxon's test, depending on the normality assumption), taking into account Bonferrony's adjustment for multiple comparisons. Regarding pharmacodynamics, formulations were compared at each time point using the Mann-Whitney's U test and the overall characteristics (mean and maximum effects, and time to reach the latter (T(Rmax)) were evaluated by the Mann-Whitney's U test. Adverse reactions frequencies were compared between formulations using the Fisher's exact test.
Results
Thirty-four patients were recruited at the seven hospitals where they regularly received hemodialysis treatment. Twenty-two of them were included at the National Institute of Nephrology, Havana, where the trial took place for them. The other twelve from "Gustavo Aldereguía Lima" Hospital, Cienfuegos were studied at this same site.
The demographic and baseline characteristics of the patients as well as their toxic habits and causes of renal failure are shown in Table 1. Age and weight were highly variable within each group. Overall, ages ranged from 19 to 75 years and weights from 40 to 87 Kg. Groups were balanced regarding sex and race. Thirty patients had specified the cause of renal failure, prevailing diabetes mellitus and hypertension. The hypothesis of homogeneity between the groups was accepted.
One patient was excluded from pharmacokinetic and pharmacodynamic analysis due to a severe adverse event (ischemic chest pain), 24 hours after the administration of Formulation B. This patient was only included in safety evaluations. The rest of the patients complied with the treatment and follow-up as previewed.
Pharmacokinetic analysis
Except for one patient from each group all had small basal serum EPO levels (0.7 – 22 mIU/mL in group A and 0.9 – 34 mIU/mL in group B). After rHu EPO administration, some individual serum concentrations reached 200 mIU/mL. At 120 hours after the injection, serum EPO concentrations had returned to the initial values, so the AUC120 obtained covered, in all cases, more than 95% of the AUC extrapolated to infinite. The average concentration profiles obtained for both formulations were very similar (Figure 1).
Table 2 shows the results of the EPO pharmacokinetic comparisons. The differences between the means of any of the pharmacokinetic parameters (except Tmax) did not exceed the clinically significant level of 20%. The 90% confidence intervals were generally close to the 0.80 – 1.25 range. A formulation effect was not detected for any of the parameters.
Pharmacodynamic analysis
Average reticulocyte count increments were very similar for both formulations, with the same profiles until 216 hours (Figure 2A). The larger increments were approximately 3 × 109 cells/L. The paired analysis yielded statistically significant reticulocyte count increments from 120 to 216 hours with respect to time 0. Both formulations behaved similarly (Table 3). The overall characteristics of the curves (maximum, means, and time to reach the maximum effect) did not show differences likewise (Table 4).
Figure 2B shows the percentage mean reduction in serum iron. A significant reduction was observed at 120 hours (paired analysis; Table 3). It was also comparable for both formulations (43 % for A and 36 % for B). A smaller percentage reduction appeared at 216 hours for formulation B. Quantitative analysis of the data also evidenced similar performance for both products (Table 4).
Safety analysis
Twenty-three patients (67.6%) presented at least one adverse event during the study, 11 that received Formulation A (64.7%) and 12 Formulation B (70.6%). There were no differences between formulations concerning the adverse events, except for edemas, which were significantly more frequent with Formulation B (Table 5). The more frequent adverse events were pain at the site of injection (35.3%) and hypertension (29%). The events were mild to moderate and well controlled in general. Just one patient had a serious episode of angina 24 hours after Formulation B administration. This event was considered as "not related" with the product because the single dose applied does not justify an erythrocyte increment that could lead to this syndrome. Additionally, this patient had antecedents of ischemic cardiopathy. Differences in vital signs and other laboratory evaluations were not significant between formulations (data not shown). Hemoglobin and hematocrit were not affected with the single dose administered.
Further follow-up
Further treatment with formulation A induced significant increases in hemoglobin and hematocrit (Table 6). All the patients, except 4 from both groups, were considered as responders since they increased at least the hematocrit in 5% and/or hemoglobin in 2 g/dL, after 3 months of treatment. During this period, the EPO-related adverse events were fever, chills and headache, each one recorded in one patient. No serum anti-EPO antibodies were detected in any patient.
Discussion
This trial indicates that the compared formulations have similar pharmacokinetic and pharmacodynamic characteristics. The dose selected was adequate, since EPO titers in serum were easily detected and the treatment was quite well tolerated. Differences between formulations were not statistically significant for any of the calculated parameters. Regarding serum EPO profiles, differences were not statistically significant at any time point; the curves overlap. The fact that more than 80% of the AUC could be covered by the AUC120 obtained indicates that internal validity of the data is high. For pharmacokinetic parameters, except for CL, the 90%confidence intervals of the ratio between formulations were outside the 0.80 – 1.25 limits accepted for bioequivalence, but close to it. Taking into consideration that it was a parallel trial, using biotechnological products, in patients with anemia due to end-stage renal disease on hemodialysis and not in healthy subjects, a higher inter-individual variability could be expected. For example, patients' age range was quite wide. Then, a more flexible approach for bioequivalence criterion, at least 0.7 – 1.3, could be justified.
This variability occurred in the calculated pharmacokinetic parameters as well. Main parameters such as AUC, AUC120 or Cmax showed very small punctual differences (5% or less), but standard deviations, although similar between treatment groups, were large (coefficients of variation around 50%). Consequently, it was not possible that 90% confidence intervals fulfill with the 0.80 – 1.25 range.
This kind of study is usually done in healthy volunteers [12-15], since under a more controlled situation a smaller variability of the results is obtained. However, in this case investigators preferred to do it in the target patients since their renal failure condition would have considerable influence on EPO pharmacokinetics and pharmacodynamics. Besides, there was a risk due to EPO-dependent blood viscosity increase and hypertension [16], and Ethics Committees could have been reluctant to approve the protocol. In fact, there is very little safety information in the above cited reports on healthy volunteers.
On the other hand, for bioequivalence trials crossover treatment groups with an inter-treatment washout period are preferred. This leads to estimate and to reduce the individual variability as the main variation source; therefore, statistical analyses have more power. However, anemic patients with renal chronic failure need continuous EPO for their red cell synthesis. To submit them to a washout period implied to wait again for the irruption of anemia and this procedure was not ethically acceptable. After this trial, the patients continued EPO treatment under phase II trial conditions. They were monitored during this period and the results show that the study product was effective in increasing their hemoglobin and hematocrit values significantly.
In some situations, the intra and inter-subject variability can be equally or more important than the variation from an innovator product to a similar. In fact, the importance of the individual bioequivalence concept is recently being claimed, which would allow the scaling of the established intervals based on the individual comparison and the subject – formulation interaction [17,18].
For both formulations, Tmax was within the 12 – 18 hours reported for EPO, although a great variability is reported for this variable in renal patients [11,19-23]. Tmax is a discrete variable that depends on the experimental design so it cannot be interpreted as a continuous value and thus the small punctual difference observed looses practical significance. The European Guidelines recommend its analysis by non-parametric procedures [24]. Values for F, t1/2 and MRT were according to other studies, 18 – 49%, 15–25 hours and 25 – 45 hours, respectively [11,12,19,25-28]. Cmax, although continuous, depends much on the times at which samples are taken. Several reports propose to expand its bioequivalence range up to 30% [29-31], and in some cases this wider 90% CI can be accepted [24]. Cmax is considered either as a magnitude or as a rate of absorption parameter but does not accurately describe either process. Other parameters such MRT and CAV best describe absorption rates [32,33].
The results of pharmacodynamic variables (reticulocyte counts and serum iron) predict a possible therapeutic equivalence between the formulations, given their similitude. It is not possible to achieve significant increments in hematocrit or hemoglobin (main clinical endpoints) with a single EPO dose, but the reticulocyte count increase correlates with the clinical effect. Stable increments in hemoglobin and hematocrit are only reached after prolonged treatment, as was found in these patients. A multiple dose design is costly and complicated for this kind of trial. It is only justified for drugs with specific pharmacological characteristics (e.g. non-linear pharmacokinetics). Additionally, any eventual blood transfusion, which is often in these patients, would interfere with the results.
The pharmacodynamic curves were not well characterized by using pharmacokinetic-like parameters due to ethical reasons, since some patients needed to be transfused. Additionally, EPO receptor-binding dependent biological effects attain their maximum several days after administration, when EPO serum levels have returned to baseline [14,34]. Therefore the dissipation phases of the effects were not well covered.
The integration of pharmacokinetic and pharmacodynamic characterizations into drug development provides a scientific framework for its rational and efficient application [35,36], as has already been done for EPO in athletes [37]. Repeated administration of EPO to healthy subjects was more effective in stimulating a reticulocyte response than single-dose administration of the same total amount [14]. However, similar pharmacodynamic effects with dissimilar doses or schedules were also reported in healthy individuals [38].
Concerning safety, treatment was well tolerated, except for an episode of angina. The number of patients that presented adverse events was almost the same for both formulations. Many adverse events were observed during hemodialysis sessions or few hours after. Thus, it is very difficult to predict if those events were EPO- or dialysis-related. The most frequent event observed with recombinant EPO treatment is hypertension, but headache, flu-like syndrome, rash, vascular thrombosis and others can appear [2-4,16,39,40]. As a rule they appear after longer treatments, higher doses or due to a rapid hematocrit increase. During the follow-up period very few drug-related adverse events appeared, mostly mild.
The introduction of "biogenerics" to the market is a controversial matter nowadays. Two recombinant EPO molecules could be similar from the physicochemical point of view, but it is always possible that differences in producing strains and/or manufacturing processes turn out into small differences in composition (isoforms, host contaminants, ingredients, etc.) that could have clinical repercussion in terms of safety or efficacy. However the "process = product" dogma has been broken in the last years and the attitude of regulatory agencies is more towards accepting, on a case by case basis, the possibility of interchangeability of biological – derived products, mostly when they are highly purified and characterized molecules. In that sense, some examples exist already in the market such as different insulins, somatotrophins, and CHO cells-derived interferon beta from different manufacturing sites [41]. In fact, two EPREX formulations with different stabilizers were pharmacokinetically equivalent [42]. The different EPO preparations have been in the clinics for several years and substantial information concerning their safety and efficacy in their various indications has been gathered. It is thus unlikely that any unexpected general safety or efficacy issue can arise from the use of a well characterized EPO preparation.
The main concern has been the development of anti-EPO neutralizing antibodies and consequently, aplastic anemia [43]. This phenomenon has been related to packaging and storage conditions [44], but should be monitored carefully since it still appears, even at very low frequency. Canadian authors found very low frequency and concluded that a general screening is not justified, but only on resistant patients [45]. No serum anti-EPO antibodies were detected after 3 months of treatment in our study. However this is still a short period to derive any conclusion on the immunogenicity of the molecule.
The possibility that a home-made EPO reaches the market in a developing country is an attractive alternative in order to expand its use, given the high prices that the proprietary drugs have. The cost of EPO treatment is approximately 500 USD per month [46] in the United States using commercially available products. For example, in Cuba (11 million inhabitants) there are approximately 2000 renal patients in hemodialysis, but this figure should increase after an investment policy where new hemodialysis facilities are set up throughout the country. If EPO had to be imported to cover this population, the country should spend not less than 12 million USD per year for this purpose (not counting additional freight and commercial charges). To use the national product would permit a significant impact on health care, even if the "biogenerics" concept is not accepted. In the case of newly developed EPO preparations, the comparable pharmacokinetics and pharmacodynamics data can be the basis for its introduction in the national market. Then further efficacy information can be gathered as well as immunogenicity monitoring.
Conclusion
Given that both formulations have similar pharmacokinetic, pharmacodynamic and short-term safety profiles, the newly developed product can be used in the clinics, with an appropriate efficacy, safety and immunogenicity monitoring.
Competing interests
Authors IGG, PJPM, CMVS, HBL, and PALS are employees of the Center for Biological Research, which is part of the Center for Genetic Engineering and Biotechnology, Havana network, where human recombinant EPO is produced. The rest of the authors have no competing interests at all.
Authors' contributions
JFPO and MCG and RHV took care of patient recruitment, management, and follow-up. IGG participated in the study design and coordination, EPO EIA determinations and wrote the manuscript draft. PJPM conceived the study and participated in the study design and coordination. CMVS participated in the study design and statistical analysis. THM and MLA carried out blood sampling and laboratory determinations. HLB developed the anti-EPO antibodies assay, its validation, and tested the patient's samples. YCK, YAA and AVB assisted as monitors. PALS took part in the design, results analysis and manuscript writing. All authors read and approved the final manuscript.
Appendix
The other members of the Bioequivalence Study of Erythropoietin Group are as follows: Emilio Fors-López ("Carlos J. Finlay" Hospital, Havana), Rafael Cruz-Peinado ("Miguel Enríquez" Hospital, Havana), Diego Falcón ("Calixto García" Hospital, Havana), Eusebio González-Alvarez, Ortelio González-Flebes, Oscar E. Hernández-Díaz ("Aleyda Fernández Chardiet" Hospital, Güines), Isabel Herrera, Zenaida Hernández, Leonel Soto-León ("Abel Santamaría" Hospital, Pinar del Río), Hortensia Gautier-du Defaix, Delfina Pedroso-Comino, Tsunami Alvarez, Maiker Bello, Yanet Santos, (National Institute of Nephrology, Havana), Orelys Olivert-Hernández, Pedro Muñiz-Olite, Aldo González-Pérez, René Rosales-González, Carmen L. Rodríguez-Santiago, Francisca González ("Gustavo Aldereguía Lima" Hospital, Cienfuegos), Gisou Díaz-Rojo, Elizeth García-Iglesias, Giselle Pentón-Rol, Damián Mainet-González (Center for Biological Research, Havana), Pedro R. Casanova-Arias, Maité Reyes-Cañedo (Center for Genetic Engineering and Biotechnology, Havana), Roger Delgado-Figueroa, Karina Alfonso-Alfonso (National Center of Clinical Trials, Havana), Jorge Ducongé-Soler (University of Havana, Institute of Pharmacy and Food, Havana, Cuba).
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors wish to thank the nurses Mirtha Mariño, María E. Silva, Sandra Cruz, Onidia Téllez, Dianelys González, Yanet Blanco, Mariela Ruiz, Juan Valdés, Mariela Puerto, Dayaneisy Rodríguez, Lídice Pérez, Mariela Gregorio, Lázara Puente, Reinaldo Moya, Omar Lozada, Yanet Pérez, Iluminada Alfonso, Esther Cordero, Adrián Rodríguez, Aracelis Campos, Miriam Hidalgo, Teresa Arencibia, Magdelis Acosta, Alexis Fernández, Yaneya Lorente, Nivaldo Becerra, Nereida Cabrera for their participation in the clinical work. They also thank Drs. Francisco Machado, Elías N. Rodríguez, Francisco Hernández, Idania Baladrón, Ena Infante, and Iraldo Bello and the technicians Cimara Bermúdez, Susette Machado, Leovaldo Álvarez and Ketty Cruz for their assistance, Dr. Eduardo Fernandez for his advice, and especially the 34 patients who were included in this study. The authors received both formulations free from Heber Biotec, Havana, Cuba.
Figures and Tables
Figure 1 Average EPO concentration in serum. Data correspond to patients who received 100 IU/Kg of Heberitro (solid line) or Eprex (dashed line) at time 0. Standard deviations are not shown for the sake of simplicity of the illustration. Curves were not significantly different at any point in a multiple determinations ANOVA with Bonferrony's correction.
Figure 2 Pharmacodynamic variables. Data correspond to patients who received 100 IU/Kg of Heberitro (solid line) or Eprex (dashed line). (A): average reticulocyte counts increments; (B): percentage mean reduction in serum iron. Standard deviations are not shown for the sake of simplicity of the illustration.
Table 1 Demographic and baseline characteristics of the patients
Variables Formulation A Formulation B Total p
N 17 17 34
Age (years) 52 ± 15 (19–72) 52 ± 16 (23–75) 52 ± 15 (19–75) 0.90*
Weight (Kg) 60 ± 12 (40–87) 59 ± 13 (42–83) 59 ± 12 (40–87) 0.82*
Sex Male 11 (64.7%) 8 (47.1%) 19 (55.9%) 0.30Δ
Female 6 (35.3%) 9 (52.9%) 15 (44.1%)
Race White 8 (47.1%) 10 (58.8%) 18 (52.9%) 0.73!
Non-white 9 (52.9%) 7 (41.2%) 16 (47.1%)
Smoking 4 (23.5%) 1 (5.9%) 5 (14.7%) 0.33!
Alcohol consumption 4 (23.5%) 1 (5.9%) 5 (14.7%) 0.33!
Coffee drinking 7 (41.2%) 7 (41.2%) 14 (41.2%) 1.00Δ
Causes of renal failure
Diabetes Mellitus 7 (46.7%) 6 (40.0%) 13 (43.3%) 0.71Δ
Hypertension 5 (33.3%) 4 (26.7%) 9 (30.0%) 1.00!
Other glomerulopathies 1 (6.7%) 1 (6.7%) 2 (6.7%) 1.00!
Cystic disease 1 (6.7%) 3 (20.0%) 4 (5.6%) 0.60!
Data are reported as mean ± standard deviation (range) or number of patients (%).
*: Student's t test; Δ: chi-square test; !: Fisher's exact test
Table 2 Pharmacokinetic parameters calculated from the EPO concentration in serum
Parameter Formulation A N = 17 Formulation B N = 16 Difference (%) p 90 % CI
AUC120(mUI·h/mL) 4500 ± 2281 4654 ± 2221 5.1 0.49 (0.71 – 1.27)
AUC (mUI·h/mL) 4667 ± 2319 4918 ± 2113 3.3 0.66 (0.71 – 1.32)
Cmax (mUI/ml) 119.1 ± 56.4 119.7 ± 60.5 0.5 0.96 (0.73 – 1.35)
Tmax (h) 13.9 ± 9.9 18.1 ± 14.9 23.2 0.08 -
λ(h-1) 0.040 ± 0.018 0.037 ± 0.015 10.8 0.49 (0.89 – 1.32)
t1/2 (h) 20.0 ± 7.9 22.5 ± 12.1 11.1 0.29 (0.69 – 1.14)
CAV 0.027 ± 0.008 0.024 ± 0.007 8.0 0.38 (0.94 – 1.31)
F 0.29 ± 0.14 0.31 ± 0.14 6.4 0.53 (0.75 – 1.17)
MRT (h) 33.9 ± 10.7 42.3 ± 20.8 19.8 0.15 -
CL (mL/h·Kg) 6.3 ± 0.3 6.3 ± 0.4 0.5 0.55 (0.98 – 1.03)
Vd (mL/Kg) 182 ± 74 202 ± 107 10.0 0.67 (0.70 – 1.15)
Data are reported as mean ± standard deviation
CI: confidence intervals of the mean ratio; for Tmax and MRT, 90 %CI could not be estimated for a parallel design.
p: ANOVA between groups
Table 3 Reticulocyte Counts Increments and Percentage Reduction of Serum Iron during the study
Formulation A N= 17 Formulation B N= 16 p A vs. B
Reticulocyte counts Increments (×109/L) (p for paired test) significance level p = 0.010
T48 -T0 2.5 ± 4.2 (0.109) 2.4 ± 3.4 (0.028) 0.694
T72 -T0 2.4 ± 3.4 (0.078) 2.5 ± 3.7 (0.011) 0.520
T120 -T0 2.8 ± 2.9 (0.001) 2.9 ± 2.8 (0.002) 0.986
T144 -T0 3.2 ± 2.8 (<0.001) 2.8 ± 2.7 (0.002) 0.552
T216-T0 2.8 ± 2.2 (< 0.001) 2.5 ± 2.4 (0.001) 0.407
Serum iron % Reduction (p for paired test) significance level: p = 0.025
T120 -T0 42.7 ± 20.5 (0.001) 35.7 ± 17.1 (0.001) 0.205
T216-T0 37.0 ± 21.4 (0.050) 17.3 ± 19.4 (0.541) 0.805
Data are reported as mean ± standard deviation.
Paired tests vs. baseline were done by the Wilcoxon's test and comparison between formulations at each time by the Mann-Whitney's U test.
Table 4 Overall quantitative analysis of reticulocyte counts (×109/L) and serum iron percentage reduction
Analysis Formulation A N= 17 Formulation B N= 16 p (Mann-Whitney's U test)
Reticulocyte counts (×109/L)
Mean 2.7 ± 2.9 2.6 ± 2.7 0.678
Maximum 4.3 ± 3.9 3.9 ± 3.9 0.397
T(Rmax) (hours) 146.8 ± 59.9 130.5 ± 51.8 0.201
Serum iron percentage reduction
Mean 39.8 ± 18.4 26.5 ± 13.1 0.265
Maximum 47.3 ± 19.2 39.2 ± 15.2 0.355
T(Rmax) (hours) 160.9 ± 61.6 148.5 ± 53.5 0.365
Data are reported as mean ± standard deviation.
These data correspond to the 34 patients who received one of the EPO formulations.
T(Rmax): Time to reach maximum response.
Table 5 Frequency of adverse events during the study
Variable Formulation A N = 17 Formulation B N = 17 p (Fisher's test)
Adverse events Yes 11 (64.7%) 12 (70.6%) 1.00
No 6 (35.3%) 5 (29.4%)
Specific adverse event Chest pain 0 1 (5.9%) 0.50
TGP increase 1 (5.9%) 0 0.50
Tachycardia 1 (5.9%) 4 (23.5%) 0.17
Headache 4 (23.5%) 3 (17.6%) 0.50
Abdominal pain 0 1 (5.9%) 0.50
Asthenia 1 (5.9%) 3 (17.6%) 0.30
Diarrhea 0 2 (3.6%) 0.24
Dyspnea 0 2 (3.6%) 0.24
Somnolence 0 1 (1.8%) 0.50
Neck pain 1 (5.9%) 0 0.50
Hypertension 6 (35.3%) 4 (23.5%) 0.35
Pain at site of injection 6 (35.3%) 6 (35.3%) 1.00
Bone pain 0 1 (5.9%) 0.50
Edema 0 6 (35.3%) 0.009*
Fever 2 (11.8%) 5 (29.4%) 0.20
Hypotension 5 (29.4%) 2 (11.8%) 0.20
General malaise 1 (5.9%) 1 (5.9%) 0.50
Pneumonia 0 1 (5.9%) 0.50
Pruritus 1 (5.9%) 3 (17.6%) 0.30
Chills 2 (11.8%) 2 (11.8%) 0.70
Vomiting 3 (17.6%) 6 (35.3%) 0.22
Data are presented as number of individuals with each adverse reaction (%).
Table 6 Efficacy evaluation during further follow-up. Both groups of patients received formulation A, 30 UI per Kg 3 times per week, subcutaneously.
Variable Day Group A Group B
Hemoglobin (g/dL) 0 8.8 ± 1.7 9.0 ± 1.4
90 10.5 ± 1.4 11.4 ± 1.6
p (90 vs. 0) 0.027 0.004
Hematocrit (%) 0 25.6 ± 4.4 26.9 ± 5.9
90 35.5 ± 3.2 36.4 ± 5.1
p (90 vs. 0) < 0.001 0.001
Data are reported as mean ± standard deviation.
Paired tests vs. day 0 were done by paired t test.
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BMC Nucl MedBMC Nuclear Medicine1471-2385BioMed Central London 1471-2385-5-31595339510.1186/1471-2385-5-3Technical AdvanceUse of segmented CT transmission map to avoid metal artifacts in PET images by a PET-CT device Mirzaei Siroos [email protected] Michel [email protected] Christopher [email protected] Peter [email protected] Michel [email protected] Peter [email protected] Centre National PET, Clinique Ste. Thérèse, Luxembourg2 Wilhelminenspital, Institute of Nuclear Medicine, Vienna, Austria3 Philips Medical Systems, BeNeLux2005 14 6 2005 5 3 3 25 10 2004 14 6 2005 Copyright © 2005 Mirzaei et al; licensee BioMed Central Ltd.2005Mirzaei et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Attenuation correction is generally used to PET images to achieve count rate values independent from tissue densities. The goal of this study was to provide a qualitative comparison of attenuation corrected PET images produced by a PET-CT device (CT, 120 kV, 40 mAs, FOV 600 mm) with and without segmentation of transmission data (ACseg+ and ACseg-respectively). Methods: The reconstructed images were compared to attenuation corrected images obtained with a high-energy transmission source (Cs-137 – 662 keV).
Thirty oncologic patients were studied using CT and 137Cs for attenuation correction. All image data were acquired using the Gemini PET-CT scanner (Philips Medical Systems). It is an open PET-CT system that consists of the MX8000 multislice CT and the Allegro PET scanner arranged in a separable configuration. Images with ACseg+ and ACseg- were analyzed simultaneously in coronal, sagittal and transaxial planes. Two nuclear medicine physicians reviewed the image sets. Results: The image quality in the area of metal implants was better with ACseg+ than ACseg-, without metal induced artifacts generally observed in CT corrected images. Further the images with ACseg+ were qualitatively comparable to those obtained with 137Cs attenuation correction. Conclusions: In case of metal implants, PET studies corrected by CT should preferably use the ACseg+ method to avoid the image artifacts.
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Background
Attenuation correction (AC) is generally applied to achieve count rate values independent from tissue densities. Transmission scanning was already suggested in 1952 by Myneord [1]. The transmission scanning was used to match anatomical contours to the radionuclide distribution measured by a rectilinear scanner [2]. Different isotopes have been used to acquire transmission data as earlier I-125, I-131, Tm-170 and later Tc-99 m, I-131, Ba-133, Cs-137 and Ge-68. With the development of SPECT and PET systems the transmission scanning was used for the purpose of AC and various methods for the use of transmission data were developed [3,4]. The PET transmission scan is ideally acquired before the radiopharmaceutical is administered, but would lead to long clinical protocols which are not suitable for routine use. Today, for PET-investigations post-injection transmission scans are performed using electronically windowed rotating rod sources as Ge-68, or rotating point sources as Cs-137 or CT in PET-CT systems. The combined PET-CT systems have the advantage to provide anatomical information while CT transmission data can be used for AC of the emission scan. This results in getting structural and metabolic information in the same session and a joint report of a nuclear medicine physician and a radiologist is then sent to the referring physician. However the PET examinations corrected for attenuation by CT images may be hampered by artifacts which are not usually seen in only PET images, as respiration [5], truncation [6], CT contrast agents and metal implants [7-9]. It is known that the high atomic numbers of contrast agents or metal implants result in increased fraction of photelectric interactions, yielding increased Housfield units (HU). With the current used reconstruction algorithms, contrast can be therefore missclassified as high density bone and thus associated with an incorrect scaling factor [10]. The goal of this study was to provide a qualitative comparison of attenuation corrected PET images of patients with metal implants produced by a PET-CT device with and without segmentation of CT.
Methods
Thirty oncologic patients with metal implants (hip prosthesis, dental implants, pacemaker) were studied using CT and Cs-137 transmission scans for attenuation correction. None of the included patients had clinical signs of local inflammation of the implants. All image data were acquired using the Gemini Dual PET-CT scanner (Philips Medical systems). The GEMINI Dual is an open PET-CT system that combines a helical dual slice CT and a 3D PET scanner equipped with its own transmission source [11]. Gantries are arranged in a separable configuration that allows the use of the 2 scanners separately when desired. The MX8000 EXP CT scanner is a dual slice system whose detector consist of 1344 Cadmium Tungstate elements. Gantry allows a patient port of 70 cm. Minimum scan time per full rotation is 0.5 s. and slice thickness can range between 0.5 mm and 10 mm. The Allegro 3D PET scanner works exclusively in 3D detection mode (no septa). It is comprised of 29 arrays of 616 GSO (gadolinium oxyorthosilicate) crystals each. Crystal dimensions are 4 × 6 × 20 mm3. The axial field of view is 180 mm and patient port is 63 cm. The rotating point source of Cs-137 located in the gantry allows acquisition of transmission scans in 3D detection mode. Cs-137 emits single photons with energy of 662 KeV.
Patients were scanned 1 hour after injection of 4 MBq/Kg FDG. A low dose CT helical scan was performed first (scan field of 600 mm, increment of 5 mm, slice thickness 6.5 mm, pitch of 1.5, 0.75 second per rotation, matrix 512 × 512, 120 KV, 40 mAs), followed by the Cs-137 transmission scan. The high activity of the Cs-137 source (750 MBq – half-life 30 years) and the detection of single events allows fast transmissions scan. Total duration of Cs-137 transmission scan is about 4 minutes (100 cm scan length). PET emission scan is automatically started at the end point of transmission. Emission scan consists of 8 to 11 bed positions of 3 minutes each which allows to cover 77 cm to102 cm. Randoms are online subtracted during acquisition (time-delayed events). Total acquisition time per patient varied from 30 to 40 minutes.
4 sets of data were reconstructed for each patient: no AC, AC with Cs-137 point source, AC with non-segmented CT image (Acseg-) and AC with segmented CT (Acseg+). Acquisition data were processed with standard package delivered with the system (Petview software – Philips Medical Systems). Reconstruction without AC was performed with RAMLA 2D iterative algorithm [12,13]. All reconstructions with AC were performed with RAMLA 3D iterative algorithm [12,14]. Voxel size after reconstruction is 4 × 4 × 4 mm3. Scatter correction is performed by fitting the tails of activity outside patient with a parabolic curve. AC with Cs-137 source is performed by segmentation of original transmission data set. Segmentation is used to divide the CT image into regions representing different tissue types (lung, soft tissue, bone). All pixels in regions corresponding to different tissues are assigned the value associated with these particular tissue types. Regions with higher attenuation coefficients than bone tissues (metal implants) are assigned to soft tissue attenuation coefficient. Natural variations in densities are reflected by merging [15,16] segmented image with the measured attenuation map that is first corrected such that the 'average' values of lung tissue's and soft tissue's attenuation coefficients are equal to nominal values set in reconstruction protocol file. Merging is controlled by lung and soft tissues attenuation coefficients (user defined uniformity parameters). With values set to 1.0, only the segmented image is used and no merging is performed. With values set to 0.0, only an over-smoothed mean-adjusted image of measured transmission is used. Intermediate values result in a mix between the two. Default values were used (0.50 for lungs and 1.0 for soft tissues). Computation of AC with low dose CT data requires first the down-sampling of CT scan to PET matrix size and pixel size. Then CT truncation compensation is applied to compensate artifacts resulting in using a FOV of 600 mm [17]. Resolution matching is then performed by smoothing CT images. Gaussians filters are used with FWHMs that match PET resolution in axial and transaxial directions. CT images with pixels in Hounsfield Units (HU) are then converted to linear attenuation coefficients at 511 KeV [18,19]. Converted CT images are segmented to perform AC by dividing maps into regions representing tissue types, as described before. Images with ACseg+ and ACseg- were displayed simultaneously in three different planes (coronal, sagittal, transaxial). Two nuclear medicine physicians reviewed the data sets with a specific multi-modality registration software (Philips Syntegra). PET and CT images can be visualized independently as well as registered on the same screen in coronal, sagittal and transverse planes. Layout can be customized and different presets are available to scale the CT images (lungs, soft tissues, bone, abdomen ...). Segmented transmissions scans obtained with Cs-137 and CT were also compared.
Results
The image quality in the area of metal implants was in all cases better with ACseg+ than ACseg-, without artifacts seen in images with ACseg- (Figure 1, 2, 3, 4, 5). Further the emission images with ACseg+ were qualitatively comparable to reconstructed data obtained with Cs-137 AC.
Figure 1 FDG-PET image of a patient with a hypermetabolic retrosternal mass. Artifact (arrow) due to a dental implant in CT corrected images without segmentation.
Figure 2 The same patient as in Fig. 1: The Artifact due to dental mass (Fig. 1) is not visible in the CT-corrected images with segmentation of transmission data.
Figure 3 FDG-PET image of a patient with lymphoma involvement in the left cervical region. Note the periprosthetic artifact (arrow) in the region of the right hip in CT corrected images without segmentation.
Figure 4 The same patient as in Fig. 3: There is no pathological uptake in the periprosthetic area of the right hip in the CT-corrected images with segmentation of transmission data.
Figure 5 The same patient as in Fig. 3: There is no pathological uptake in the periprosthetic area of the right hip in the images corrected with AC by Cs-137.
As shown in images 1–5 there are hypermetabolic areas seen in the ACseg- images in the area of metal implants which are not visible on ACseg+ reconstructed images. Further we assessed the images without AC. Also in these images the artifacts due to metal implants were not visible, but the image quality was inferior to that of images with both methods of AC.
Discussion
For better anatomic localization of areas with increased tracer uptake in PET images, anato-metabolic imaging has been proposed during the same session, which has lead to manufacturing combined PET/CT devices [20]. This modality allows to obtain independently high quality PET and CT images as well as to merge both data sets to evaluate the morphological and functional information. For abdominopelvic evaluation, CT has been suggested as procedure of choice with PET imaging [21]. The CT transmission images are at the same time used for purpose of AC which is an indispensable part of image reconstruction and makes it also possible to get quantitative information in the areas of interest. There are three methods to generate attenuation maps from a CT image including segmentation, scaling and dual energy CT scans [22]. Oncologic patients often have artificial metal implants, such as metal braces in the spinal region, chemotherapy ports, dental fillings or implants, pacemaker, that can sometimes be mistaken for other pathologies [23,24]. PET transmission scans using point source of Cs-137 show little or no artifacts. With the significantly higher photon absorption at CT energy, however, these artifacts are present, which are not yet corrected in standard PET-CT protocols. Therefore the reconstruction of emission images without AC has been suggested [22,25]. As we could demonstrate in this study, the artifacts due to metal implants could be avoided using segmentation algorithm for AC with CT data. However, both methods for AC resulted in much better image quality compared to those without AC. In our center we did not make use of contrast medium for the CT part of the examination. But since the artifacts due to contrast medium in PET/CT images [26,27] are caused in the same way as CT hyperintense metal implants, we assume that these would also be avoided using AC with segmentation as described in our study.
Conclusion
Purpose of this study was to provide a qualitative comparison of attenuation corrected PET images of patients with metal implants produced by a PET-CT device with and without segmentation of CT. It is known that based on reconstruction methods there are differences in quantitative measurements because of the way transmission data are processed [28,29]. Further investigations should be performed to quantitatively estimate the effect of segmentation algorithms used for CT AC.
In case of metal implants, we suggest that PET studies corrected by CT should preferably use the ACseg+ method to avoid the image artifacts.
Abbreviations
AC = Attenuation correction
Acseg- = AC with non-segmented CT image
Acseg+ = AC with segmented CT image
CT = Computed Tomography
HU = Hounsfield Units
PET = Positron Emission Tomography
Competing interests
The article-processing charge will be refunded by Philips. Dr Guerchaft is an employee of Philips Medical Systems. All other authors declare that they have no competing interests.
Authors' contributions
SM performed different reconstruction methods and drafted the manuscript. MG participated in drafting the manuscript and provided technical support. CB carried out the studies and participated in the design of the study. PK provided technical support. MD and PB participated in the design of the study. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
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| 15953395 | PMC1164418 | CC BY | 2021-01-04 16:30:51 | no | BMC Nucl Med. 2005 Jun 14; 5:3 | utf-8 | BMC Nucl Med | 2,005 | 10.1186/1471-2385-5-3 | oa_comm |
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BMC PediatrBMC Pediatrics1471-2431BioMed Central London 1471-2431-5-121590721610.1186/1471-2431-5-12Research ArticleBupivacaine versus lidocaine analgesia for neonatal circumcision Stolik-Dollberg Orit C [email protected] Shaul [email protected] Department of Anesthesiology, Sheba Medical Center, Tel Hashomer, Israel2 Department of Neonatology, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel3 Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel2005 22 5 2005 5 12 12 12 12 2004 22 5 2005 Copyright © 2005 Stolik-Dollberg and Dollberg; licensee BioMed Central Ltd.2005Stolik-Dollberg and Dollberg; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Analgesia for neonatal circumcision was recently advocated for every male infant, and its use is considered essential by the American Academy of Pediatrics. We compared the post-operative analgesic quality of bupivacaine to that of lidocaine for achieving dorsal penile nerve block (DPNB) when performing neonatal circumcision.
Methods
Data were obtained from 38 neonates following neonatal circumcision. The infants had received DPNB analgesia with either lidocaine or bupivacaine. The outcome variable was the administration by the parents of acetaminophen during the ensuing 24 hours.
Results
Seventeen infants received lidocaine and 19 received bupivacaine DPNB. Ten infants in the lidocaine group (59%) were given acetaminophen following circumcision compared to only 3 (16%) in the bupivacaine group (P < 0.01). Regression analysis showed that the only significant variable associated with the need for acetaminophen was the use of lidocaine (R2 = 20.6; P = 0.006).
Conclusion
DPNB with bupivacaine for neonatal circumcision apparently confers better analgesia than lidocaine as judged by the requirement of acetaminophen over the ensuing 24-hour period.
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Background
The issue of whether or not to circumcise a male infant one week after birth (when medically permissible) is essentially nonexistent in Israel. In an atmosphere of celebration and feasting, the procedure ("brit mila") has been carried out for over 4,000 years by non-physicians ("mohelim"). A mohel uses no anesthesia when performing the circumcision. The baby is given a few drops of sacramental wine and the mohel applies tight bandaging to the wound. The past few years, however, have witnessed a small but growing trend of young couples who seek to have their newborns circumcised by doctors using some kind of anesthesia in order to obviate the baby's pain and discomfort as well as their own anxiety. This cultural change has elicited interest among Israeli doctors in seeking optimal management of their newborn patients.
Anesthesia is not routinely administered for neonatal circumcision for a variety of reasons, among them the relatively short duration of the intervention, the perceived lack of importance of the pain, and concerns of toxicity from the medications [1]. It is now recognized that neonates are capable of both perceiving and exhibiting reproducible responses to pain, and that pain in neonates may have long-term effects (e.g., "pain memories") [2,3]. The routine use of analgesia during neonatal circumcision is now considered essential by the American Academy of Pediatrics [4].
A topical application of eutectic mixture of the local anesthetics, lidocaine and prilocaine (EMLA, Astra Pharma Inc. Sweden) had achieved considerable popularity for its ability to diminish pain associated with circumcision. In a review of the Cochrane database [5], however, its use was not shown to have any special advantage over other analgesic techniques with proven efficacy, such as regional nerve block with local anesthetic medications (e.g., lidocaine injection). We had been using lidocaine for dorsal penile nerve block (DPNB) in our service, and recently introduced bupivacaine because it has the important advantage of a longer duration of action compared to other local anesthetics [6]. In order to evaluate the clinical significance of this modification, we used the requirement of post-operative analgesia as a tool to assess long-term analgesic efficacy of analgesic medications in general [7], as well as specifically the requirement of acetaminophen after neonatal circumcision [8]. In the current study, we evaluated the duration of the long-term analgesic effect of bupivacaine in infants undergoing neonatal circumcision. We hypothesized that infants treated with bupivacaine will require less acetaminophen than infants treated with lidocaine.
Methods
All male infants under the age of 2.5 months with normal penile anatomy were eligible for the study. By design, we excluded the infants with abnormal penile anatomy (e.g., hypospadias), those whose parents did not apply topical EMLA cream as instructed or when parents did not keep the scheduled follow-up appointment. The circumcision was performed by a board certified neonatologist and a board certified anesthesiologist in an office setting equipped with all necessary resuscitation equipment. Parents were instructed to apply approximately 2.5 grams of EMLA cream on the shaft of the penis and 1 cm around its base one hour prior to the appointed time for circumcision. In order to avoid absorption of the EMLA cream into the disposable diaper, parents were instructed to apply a 20 × 20 cm piece of household plastic wrap on the inside of the disposable diaper over the penis. Upon arrival to the office, the infants received 25–30 mg/kg acetaminophen, either orally or per-rectum, as preemptive analgesia (Acamoli syrup 120 mg/ml or Acamoli suppository 150 mg, Teva Medical, Petah Tikva, Israel). Oral doses of 40/kg body weight of acetaminophen have been used for post-operative pain treatment in infants [9]. Since acetaminophen has a large volume of distribution, a relatively large initial dose is required irrespective of whether treatment is administered orally or rectally. Tréluyer et al. found that the optimum oral dose was 30 mg/kg [10]. From 0.3–0.5 ml 30% sucrose-solution was given orally just prior to the analgesic injection [11]. All study infants received an injection of lidocaine 1% 6–8 mg as a subcutaneous ring block to the base of the penis by means of a hypodermic 27 G beveled needle (Becton Dickinson, Drogheda, Ireland) [12]. The infants were then injected with either lidocaine 1% (Ezracaine 1%, Rafa Laboratories, Jerusalem, Israel) 4–5 mg/kg (the lidocaine group which was comprised of infants who underwent the circumcision before May 18, 2003) or bupivacaine 1.5–2 mg/kg (Marcaine, Astra, Sweden), (the bupivacaine group consisting of infants who were circumcised after May 18, 2003) in a DPNB using a 25 G needle (Becton Dickinson) [13]. In order to avoid inadvertent intravenous injection, suction was applied to the syringe handle prior to injection, ensuring that the tip of the needle was not inside a blood vessel.
Circumcision was performed 4–5 minutes after analgesia administration. An additional dose of 0.3–0.5 ml 30% sucrose solution was dripped orally. The infants lay on a padded surface and circumcision was performed using the Mogen circumcision clamp (all procedures were carried out by S.D.) [14]. The infants were observed for adverse effects of analgesia or circumcision for at least 15 minutes and underwent a physical examination.
The parents were given an instruction sheet and verbally instructed by the physician to administer a dose of 30 mg/kg liquid acetaminophen 4 hours after the procedure if they subjectively felt that the infant displayed any signs they perceive as pain. One repeat dose was also recommended 4 hours after that if symptoms of pain or discomfort reappeared. The parents were instructed to contact the physician if pain persisted thereafter. A follow-up appointment was scheduled within 2–5 days after the circumcision, and the number of acetaminophen doses administered to the infants during the 24-hour period after the circumcision was routinely recorded. Consent was obtained from both parents.
Statistical methods included the t-test, Chi-square test, and regression analysis. Data are presented as mean ± SD. A P value of <0.05 was considered significant.
Results
Thirty-eight consecutive infants who underwent neonatal circumcision between March 1 and June 30, 2003 were included in this study. Four other infants were excluded, 3 because the parents did not apply EMLA cream and one because his parents failed to keep the scheduled post-circumcision appointment. Of these 38 infants, 17 had DPNB with 1% lidocaine and 19 with 0.5% bupivacaine. There was no significant difference in birthweight (3065 ± 635 g vs. 3081 ± 570 g for the lidocaine and the bupivacaine groups, respectively), postnatal age (13.3 ± 7.2 days vs. 12.9 ± 8.3 days), or weight at circumcision (3256 ± 570 g vs. 3285 ± 517 g). None of the studied infants showed any clinical signs of cardiac or neurological toxicity after either analgesia.
Nine of the seventeen infants in the lidocaine group were given an additional dose of acetaminophen and another one received 2 doses (59%). In the bupivacaine group, 1 of the 19 infants received 1 dose and 2 were given two doses of acetaminophen (16%, χ2 = 7.2; P < 0.01). None of the study infants was given more than 2 doses and none of the parents contacted the physician because they detected signs that their infant was experiencing pain or discomfort. Regression analysis, taking into account the need for acetaminophen as the dependent variable and the birthweight, current age, current weight and bupivacaine or lidocaine as the administered drug, showed that only the type of drug was significant in the regression model (R2 = 20.6; P = 0.006).
Discussion
Infants who were treated with bupivacaine injected as a DPNB had significantly better post-operative analgesia compared with lidocaine, as judged by their lower requirement of acetaminophen 24 hours after circumcision. Despite the recommendation to use 0.5% bupivacaine for DPNB in the previous edition of a major textbook of regional analgesia [15], a Medline search failed to identify any reports on the use of bupivacaine in DPNB for neonatal circumcision.
The use of bupivacaine for pediatric analgesia for circumcision was reported in two recently published studies performed on children older than 1 year. A study by Choi et al [6] compared the use of topical EMLA cream with bupivacaine in a randomized placebo-controlled manner and concluded that, despite no difference in the score obtained using a pain scale between the two groups, bupivacaine DPNB resulted in significantly longer analgesia. A second recently published study by Gauntlett [8] compared bupivacaine in DPNB with caudal bupivacaine with ketamine in 60 boys and reported no immediate adverse effects.
The use of bupivacaine may be potentially more hazardous than lidocaine in cases of accidental intravenous injection. Cardiac toxic effects of high doses or unintentional intravascular injection may lead to high plasma levels and related depression of the myocardium, decreased cardiac output, heart block, hypotension, bradycardia, ventricular arrhythmias and cardiac arrest [16]. Adverse central nervous system effects include restlessness, anxiety, dizziness, tinnitus, blurred vision or tremors and sometimes convulsions [16]. The safety of DPNB was recently studied in 3,909 pediatric patients undergoing circumcision. The authors did not report the type of medication and dosage that had been used and also failed to note if there had been any patients with accidental intravenous injection of the local anesthetic medication [17]. An additional study of neonatal circumcision with DPNB in 491 patients found that the only complication was bruising at the injection site in 11% of patients [18]. The type of drug used in this study was not specified in this study.
The mechanism of the reduced requirement of acetaminophen after bupivacaine injection is not clear. High protein binding, better lipid solubility and high pKa all contribute to the longer duration of analgesia, reported mean elimination half-life of 8.1 hours versus 3.2 hours of lidocaine [19]. It is possible that, in addition, the longer period of blocked nociception is sufficient to blunt the hypersensitization associated with persistent postoperative pain input. This secondary hyperalgesia is mediated in the spinal cord and contributes to postoperative pain [20].
Some potential limitations of our study should be mentioned. The data for this study were collected retrospectively and patient allocation was in a sequential fashion. This approach may introduce a potential bias: for example, while it is theoretically possible that the performance of the analgesia improved over time, thereby resulting in a better outcome in the bupivacaine group, the study was performed during a relatively short time frame and this drawback is not very likely. Secondly, the assessment of pain in the infants was done subjectively by the parents. Crying may have been misinterpreted by the parents as pain, but could have stemmed from other reasons, and could have been managed by some of the parents by modalities other than acetaminophen (e.g., feeding, diaper changing, rocking the infant). Since the parents in both groups were instructed in an identical manner (verbal and written instructions) to give acetaminophen if they felt that their son was in pain, this potential bias was probably not significant.
Conclusion
Given the rarity of accidental intravenous injection and its longer analgesic effect, we conclude that bupivacaine is superior to lidocaine for DPNB when performing neonatal circumcision.
Abbreviations
DPNB – Dorsal penile nerve block
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Both authors (Orit Stolik-Dollberg and Shaul Dollberg) 1) have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) have been involved in drafting the article or revising it critically for important intellectual content; and 3) have given final approval of the version to be published.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Esther Eshkol is thanked for editorial assistance.
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-511594388210.1186/1471-2458-5-51Research ArticleSelf-rated health in Pakistan: results of a national health survey Ahmad Khabir [email protected] Tazeen H [email protected] Nish [email protected] Clinical Epidemiology Unit, Department of Community Health Sciences, Aga Khan University, Karachi, Pakistan2 Section of Ophthalmology, Department of Surgery, Aga Khan University, Karachi, Pakistan3 Section of Nephrology, Department of Medicine, Aga Khan University, Karachi, Pakistan, and Division of Nephrology, Department of Medicine, Tufts-New England Medical Center, Tufts University Medical School, Boston, MA, USA4 Department of Epidemiology and Public Health, Imperial College of Medicine at St. Mary's, UK2005 19 5 2005 5 51 51 30 11 2004 19 5 2005 Copyright © 2005 Ahmad et al; licensee BioMed Central Ltd.2005Ahmad et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Self-rated health (SRH) is a robust predictor of mortality. In UK, migrants of South Asian descent, compared to native Caucasian populations, have substantially poorer SRH. Despite its validation among migrant South Asian populations and its popularity in developed countries as a useful public health tool, the SRH scale has not been used at a population level in countries in South Asia. We determined the prevalence of and risk factors for poor/fair SRH among individuals aged ≥15 years in Pakistan (n = 9442).
Methods
The National Health Survey of Pakistan was a cross-sectional population-based survey, conducted between 1990 and 1994, of 18 135 individuals aged 6 months and above; 9442 of them were aged ≥15 years. Our main outcome was SRH which was assessed using the question: "Would you say your health in general is excellent, very good, good, fair, or poor?" SRH was dichotomized into poor/fair, and good (excellent, very good, or good).
Results
Overall 65.1% respondents – 51.3 % men vs. 77.2 % women – rated their health as poor/fair. We found a significant interaction between sex and age (p < 0.0001). The interaction was due to the gender differences only in the ages 15–19 years, whereas poor/fair SRH at all older ages was more prevalent among women and increased at the same rate as it did among men. We also found province of dwelling, low or middle SES, literacy, rural dwelling and current tobacco use to be independently associated with poor/fair SRH.
Conclusion
This is the first study reporting on poor/fair SRH at a population-level in a South Asian country. The prevalence of poor/fair health in Pakistan, especially amongst women, is one of the worst ever reported, warranting immediate attention. Further research is needed to explain why women in Pakistan have, at all ages, poorer SRH than men.
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Background
South Asia, home to over 1.5 billion people (a quarter of the world's population) has one of the worst health indicators in the world [1]. Around 34% of the world's childhood deaths occur in this region, which also has unacceptably high rates of maternal deaths [1,2]. In addition, South Asia is also facing a growing burden of cardiovascular disease (CVD)[3]. South Asians, even if they live in developed countries, have substantially higher morbidity and mortality rates than the local majority populations from CVD. Further, compared to native Caucasian populations, migrants of South Asian descent have substantially poorer self-rated health (SRH)[4] which is a robust predictor of key health outcomes, including mortality and morbidity [5]. Out of all South Asian ethnic subgroups in the UK, Pakistanis and Bangladeshis have the poorest SRH[4]. This is particularly striking, as South Asians largely migrate to the UK in search of employment, and should thus be subject to the healthy migrant effect.
Despite its popularity in developed countries as a useful public health tool and its validation among migrant South Asian populations[4], the SRH scale has not been used at a population level in countries in South Asia. SRH was assessed for the first time at a population level in the National Health Survey of Pakistan (NHSP)-1990–94. We analysed NHSP data to estimate the prevalence of and identify risk factors for poor/fair SRH among individuals aged ≥15 years in Pakistan (n = 9442).
Methods
NHSP's design is described in detail elsewhere[6,7]. Briefly, the NHSP was commissioned by the Pakistan Medical Research Council (PMRC), and conducted between 1990–1994, under the technical assistance of the United States National Center for Health Statistics. The design of the survey was a modification of United States' Third National Health and Nutrition Examination Survey (NHANES III). Using a two-stage stratified design, the NHSP was a nationwide household survey that collected information on health and nutritional indicators from a representative sample (18,135 individuals aged 6 months or older). Data collection involved the use of a questionnaire which had been validated in local languages. NHSP included an interview conducted at the participant's home by trained health workers, followed by a standardized physical examination in a well-equipped clinic by physicians.
Our primary outcome measure was SRH which was assessed using the question: "Would you say your health in general is excellent, very good, good, fair, or poor?" SRH was dichotomized into poor/fair and good (excellent, very good, or good). Household socioeconomic status (SES; high, middle or low) was defined on the basis of the number of material items owned (TV, bicycle, motorcycle, refrigerator, car, etc). Tobacco use (cigarettes, beddies, huqqa, chillum, and tobacco chewing) was dichotomized into current use or not. Respondents were asked, "Have you smoked at least 100 cigarettes or beddies during your entire life?" Those who replied "yes" were asked, "Do you smoke now?" Current smokers were persons who reported current smoking and having smoked at least 100 cigarettes or beddies during their lifetime. Respondents were also asked, "Have you chewed tobacco or used snuff at least 100 times during your entire life?" Those who said "yes" were asked, "Do you chew tobacco or use snuff now?" Those respondents who replied they did were reported as current tobacco chewers/snuff users.
Literacy was defined as the ability to read. There are four provinces in Pakistan: Punjab, Sindh, North West Frontier Province (NWFP) and Balochistan, each having its distinct culture, languages, and climate. Urban was defined as any area which had any of the following local government institutions at the time of 1981 population census: metropolitan corporation, municipal corporation, municipal committee, town committee, or cantonment board. All other areas were defined as rural.
Analyses were performed with the SPSS statistical package (version 10.0). Means and standard deviations were computed for continuous variables, while proportions and 95% CIs for categorical variables. The association between poor/fair SRH and age, sex, literacy status, province of residence, area of residence (urban vs. rural), SES, marital status, hypertension, and current tobacco use was investigated, using univariate logistic regression. Variables that were associated with poor/fair health at p-value < 0.2 at the univariate logistic regression analysis were considered for the final multivariate logistic model (Table 2). Factors that were included in the final model were sex-age interaction, province of living, area of living (urban vs. rural), literacy, SES, and current tobacco use. Tests for interactions between sex and other independent variables were performed. The interaction between sex and age was assessed first using age as a continuous variable. However, based on the plot of association between poor/fair SRH and age by gender (Fig 1), age was used as a categorical variable (15–19 years and ≥20 years) in the final model.
Table 2 Unadjusted odds ratio for poor/fair self-rated health status for individuals aged 15 years and older in the first National Health Survey of Pakistan
Variable Total Individuals with poor/fair SRH (%) Unadjusted Odds ratio for poor/fair SRH (95% CI) P-value
Number of individuals 9373 6101 (65.1)
Age 15–19 1636 866(14.2) 1.0 <0.0001
≥ 20 7737 5235(85.8) 1.86(1.67,2.07)
Sex Female 4995 3856 (63.2) 3.22 (2.94, 3.51) <0.0001
Male 4378 2245 (36.8) 1.0
Marital status Never married 2239 1197 (20.6) 1.0 <0.0001
Married 5980 4043 (69.4) 1.82 (1.65, 2.01)
Widow/widower/ divorced/separated 708 582 (10.0) 4.02 (3.26, 4.96)
Dwelling Rural 5995 4087 (67.0) 1.45 (1.33, 1.58) <0.0001
Urban 3378 2014 (33.0) 1.0
Literacy Literate 3012 1570 (25.7) 1.0 <0.0001
Illiterate 6361 4531 (74.3) 2.27 (2.08, 2.49)
Socioeconomic status (SES) Low SES 3103 2228 (36.5) 1.81 (1.59, 2.04) <0.0001
Middle SES 4592 2891 (47.4) 1.21 (1.08, 1.35)
High SES 1678 982 (16.1) 1.0
Province Punjab 4858 4015 (65.8) 9.20 (8.17, 0.36) <0.0001
NWFP 1468 684 (11.2) 1.69 (1.47, 1.94)
Balochistan 1074 729 (11.9) 4.08 (3.48, 4.78)
Sindh 1973 673 (11.0) 1.0
Currently use tobacco Yes 2307 1433 (23.5) 0.84 (0.76, 0.93) <0.01
No 7066 4668 (76.5) 1.0
Hypertension Normal 7522 4832 (80.1) 1.0 <0.01
Hypertensive 1772 1203 (19.9) 1.18 (1.05, 1.31)
Figure 1 Graphic presentation of the association between poor/fair self-rated health and age by gender.
Results
The overall agreement to be interviewed was 92.6%. A total of 9442 individuals interviewed were aged ≥15 years. The mean age (± SD) of the respondents was 36.3 years (± 17.1) [men: 37.3(± 17.9) vs. women 35.5(± 16.3)]. The mean missing data rate was 0.7 %. The missing data rates were 0 % for age, dwelling, literacy, SES, tobacco use and province; and 4.8 %, 0.9%, and 0.7 % for marital status, hypertension and self-rated health, respectively. Majority of the respondents lived in rural areas (64.1 %), were illiterate (68.0) and belonged to either low (33.1) or middle (49.0) socioeconomic group. As much as 7.6 % respondents (10.4 women vs. 4.6 % men) were widows, widowers, separated or divorced. Data on SRH were missing for 69 individuals (0.7%) so the final sample size was 9373 people. Of these, 65.1% respondents, 51.3 % men vs. 77.2 % women, rated their health as poor/fair (Table 1 and 2). In the Punjab province, 82.6 % people (69.6 % men vs. 94.6 % women) perceived their health to be poor/fair. Women had, at all ages, poorer SRH than men (Figure 1). Multiple logistic regression analysis showed a significant interaction between sex and age (p < 0.0001); but as shown in Fig 1 the interaction was due to the gender differences only in the ages 15–19 years, whereas poor/fair SRH at all older ages was more prevalent among women and increased at the same rate as it did among men. There was no significant interaction for ages 20 and above (p = 0.26). Women aged 15–19 years (adjusted OR 2.79, 95 % CI: 2.21–3.51), women aged ≥20 years (7.60, 6.23–9.27) and men aged ≥20 years(1.37, 1.14–1.64) were more likely to report having poor/fair health compared with men aged 15–19 years. Individuals living in Punjab had the highest odds (13.34, 95 % CI: 11.63–15.29) of poor/fair health. Other independent determinants of poor/fair health were low or middle SES, rural dwelling, literacy, and current tobacco use (Table 3).
Table 1 The distribution of self-related health status and other characteristics among men and women aged 15 years and older in the first National Health Survey of Pakistan
Variable Men (n = 4414) % Women (n = 5028) % All (n = 9442) %
Self-rated health status Excellent 0.6 0.3 0.4
Very good 8.3 2.1 5.0
Good 39.8 20.4 29.5
Fair 37.0 39.4 38.3
Poor 14.3 37.8 26.8
Age <20 18.1 16.9 17.4
20–29 23.8 27.1 25.5
30–39 18.8 20.3 19.6
40–49 13.3 14.4 13.9
≥ 50 26.1 21.4 23.6
Dwelling Rural 63.6 64.5 64.1
Urban 36.4 35.5 35.9
Literacy Illiterate 53.1 81.1 68.0
Literate 46.9 18.9 32.0
Socio Economic Status (SES) Low SES 33.7 32.6 33.1
Middle SES 49.0 49.1 49.0
High SES 17.3 18.3 17.9
Currently use tobacco No 60.3 88.8 75.5
Yes 39.7 11.2 24.5
Province Sindh 20.7 21.2 20.9
Punjab 53.3 50.9 52.0
NWFP 15.5 15.7 15.6
Balochistan 10.6 12.3 11.5
Marital status Never married 31.2 19.6 25.1
Married 64.1 69.5 66.9
Widowed/widower 4.5 10.4 7.6
Divorced 0.2 0.3 0.2
Separated 0.1 0.2 0.2
Hypertension Normal 79.8 82.0 80.9
Hypertensive 20.2 18.0 19.1
Table 3 Adjusted odds ratio for poor/fair self-rated health status for individuals aged 15 years and older in the first National Health Survey of Pakistan
Variable Adjusted Odds ratio for poor/fair SRH (95% CI)
Male 15–19 years 1.0
≥ 20 years 1.37(1.14–1.64)
Female 15–19 years 2.79(2.21–3.51)
≥ 20 years 7.60(6.23–9.27)
Dwelling Rural 1.25(1.11–1.39)
Urban 1.0
Literacy Literate 1.33(1.17–1.50)
Illiterate 1.0
Socioeconomic status (SES) Low SES 1.56(1.34–1.82)
Middle SES 1.17(1.02–1.35)
High SES 1.0
Province Punjab 13.34(11.63–15.29)
NWFP 1.93(1.66–2.25)
Balochistan 4.52(3.79–5.39)
Sindh 1.0
Currently use tobacco Yes 1.33(1.17–1.51)
No 1.0
Discussion
The prevalence of poor/fair health in Pakistan (65.1%) is far higher than that in Japan (9.8), Canada (11.7), United States (14.5) and the UK (25.3) in almost comparably aged populations [8-11]. However, it is closer to that obtained in a study in Russia in 1996 where the prevalence was higher, mainly because of major social changes and political and economic uncertainties after the collapse of the erstwhile Soviet Union [12].
South Asians, regardless of whether they live in their home countries or overseas have high morbidity and mortality rates. The fourth National Survey of Ethnic Minorities (1993–1994) in the UK that assessed the association between SRH and ethnicity found that people of Pakistani and Bangladeshi origin living in the UK had the poorest SRH, followed by Indians.
Our most striking finding was the substantially poorer health status, at all ages, of women compared to men in Pakistan. To our knowledge, this is the first study on SRH reporting a significant interaction between age and sex (p < 0.0001) – although the interaction was due to the gender differences only in the ages 15–19 years and not in older ages. For individuals aged ≥20 years, poor/fair SRH was more prevalent among women and increased at the same rate as it did among men. A limitation of the NSHP was that it did not assess mental health status, which is an important determinant of SRH. Studies in both developed and developing countries have shown than women have a higher prevalence of depression and anxiety than men. These disparities are strongly age-related. So differences in the rates of poor/fair SRH between women and men in our analysis may be due to possible age-related sex differences in the prevalence of common mental disorders in Pakistan. According to the WHO, an important reason for why women have higher rates of depression and anxiety than men is the high rates of domestic and sexual violence to which women are increasingly subjected [13]. Violence against women is endemic in Pakistan [14]as are mental disorders such as depression and anxiety [15].
The relationship between SES and the health status is well-established [16-18] although the potential for reverse causation (people may be poor because they are ill) makes such interpretations difficult. In the NHSP, SES was determined by the number of household items owned. In our analysis, even in the adjusted model people with low and middle SES were 1.56 and 1.17 times more likely to report poor/fair health compared with their counterparts with high SES. This is consistent with the findings of a study conducted in Poland and Hungary where as the number of household items owned increased, the odds of poor health decreased [19]. It is argued that material aspects of ownership of household items are important, but equally so may be the psychosocial aspects. For example, many household items not only have a direct protective effect on health, they also are marker of a higher status in the society. They may affect psychosocial well-being by psychosocial processes [20].
Another important finding is that tobacco use was independently associated with poor/fair SRH, although the cross-sectional design of the study limits the causal interpretation of this association. Several other studies have also reported a significant association between tobacco use and suboptimal SRH [21-23]. We also found province of dwelling and rural dwelling to be independently associated with poor/fair SRH.
Conclusion
This is the first study reporting on the prevalence of and risk factor for poor/fair SRH at a population-level in a South Asian country. Our findings may be generalisable to several other countries in South Asia given similarities in physical environment, culture, socioeconomic conditions and public investment in health. The prevalence of poor/fair SRH in Pakistan is one of the worst ever reported globally, warranting immediate attention. Further research is needed to explain why women in Pakistan have, at all ages, poorer SRH than men.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
KA conceived the report, performed the statistical analysis, and drafted the manuscript. TJ and NC contributed to review, and to the revision of the report. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Supported by grants from Fogarty International Center, NIH (Drs. Jafar/Chaturvedi). The funding source had no role in the analysis, or interpretation of the data or in the decision to submit the paper for publication. We thank members of the Pakistan Medical Research Council, and the United States Department of Health and Human Services for their assistance in acquisition of the NHSP data.
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-581593509610.1186/1471-2458-5-58Research ArticleThe use of simplified verbal autopsy in identifying causes of adult death in a predominantly rural population in Ethiopia Lulu Kidest [email protected] Yemane [email protected] Department of Community Health, Jimma University, Jimma, Ethiopia2 Department of Community Health, Faculty of Medicine, Addis Ababa University, P.o. Box 2077, Addis Ababa, Ethiopia2005 3 6 2005 5 58 58 15 9 2004 3 6 2005 Copyright © 2005 Lulu and Berhane; licensee BioMed Central Ltd.2005Lulu and Berhane; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Information on adult mortality is essentially non-existent in Ethiopia particularly from rural areas where access to health services is limited and most deaths occur at home. This study was conducted with the aim of identifying causes of adult death in a rural population of Ethiopia using a simplified verbal autopsy instrument.
Methods
All deaths in the age-group 15–49 years during the period of 1995–99 were taken from computerized demographic surveillance database maintained by the Butajira Rural Health Program. Data on the causes of death were collected from close relatives of the deceased persons by lay interviewers. Causes of death were diagnosed using "expert algorithm" programmed onto a computer.
Results
The major causes of death were acute febrile illnesses (25.2%), liver diseases (11.3%), diarrheal diseases (11.1%), tuberculosis (9.7%) and HIV/AIDS (7.4%). Overall communicable diseases accounted for 60.8% of the deaths. The high levels of mortality from communicable diseases reflect the poor socioeconomic development of the country, and the general poor coverage of health and education services in rural Ethiopia. The tools used in this study can easily be added-on to the numerous health surveys conducted in the country.
Conclusion
The simplified approach to verbal autopsy diagnosis can produce useful data that can effectively guide priority health interventions in rural areas where routine information system is either very weak or non-existent.
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Background
Mortality statistics have several advantages when compared to other sources of health information such as morbidity data as death is a unique event that is remembered for long time and because of its finality it is clearly defined. Thus, statistics on cause of death are reliable and useful in guiding priority interventions in public health [1].
In developing countries, where most deaths are neither attended by doctors nor medically certified, information on causes of death is usually incomplete and of poor quality [2]. To alleviate the problem in resource poor countries an indirect method called verbal autopsy (VA), which makes use of lay reporters, has been adopted to identify causes of death. It uses information on the circumstances leading to death, symptoms and signs during the terminal illness, obtained from the family of the deceased, to assign cause of death. VA data are generated through retrospective questioning in surveys or in demographic surveillance systems [3-6].
The VA technique is based on the assumption that most causes of death have distinct symptom complexes that can be recognized, remembered and reported by lay respondents. It assumes that it is possible to classify deaths based on the reported information into useful categories of causes of death. The validity of VA is influenced by the type of illness leading to death, characteristics of the deceased person, and other factors related to the classification of causes of death, as well as the design and content of the questionnaire and field procedures [5,7,8].
Factors that affect the validity of a VA include the approach to mortality classification, the design of the questionnaire, the methods of deriving a diagnosis, and the number and combinations of categories of causes of death assigned. The choice of categories of causes of death determines the complexity of diagnostic algorithms. A classification approach using fewer categories will group causes of death with closely related symptom complexes together and hence tends to increase the validity of the VA but provides less detailed information. The other advantage in broadly classifying mortality is that the classification can be used in a wide range of settings with minor modifications [5].
The type of questionnaire also influences the validity of the VA outcome. An open format VA questionnaire would require skilled and possibly medically trained interviewers, and would also have a higher inter-interviewer variability. A checklist with filters on the other hand would not need medically trained interviewers, and would be more efficient for data collection. But, a false negative answer to a filter question will result in omitting one whole disease category and hence decrease the sensitivity of the VA. A checklist without filters would not require a medically trained person and would reduce interviewer bias, but it may not capture all details of the symptoms leading to death and may increase the number of falsely reported symptoms [5].
Different validation studies for the VA method have been done for deaths in children [9-12], and adults [6,13] suggesting that the VA method is an important tool for diagnosing causes of death, especially in developing countries. Studies in Tanzania [14] and in Ethiopia [15] have shown the robustness of VA method in identifying cause of death using lay interviewers.
Trends in cause-specific mortality over time, differentials of mortality between different population groups, or evaluation of the effect of interventions can be studied from data using the VA method. Additionally, they can be used to establish the relative public health importance of different causes of death in the populations in order to identify priorities and appropriate interventions [5,16]
Ethiopia is a country where low standards of living, poor environmental conditions and under developed social services have caused serious impact on the health status of the population [17,18]. Although adults are the pillar of the society as economically productive, biologically reproductive, and responsible for the support of children and elderly dependents their health needs receive little attention in developing countries [19]. Information on adult health conditions is essentially non-existent in most developing countries as the routine system does not cover large segment of the population, especially those living in the rural areas [2,20,21]. In Ethiopia, due to the absence of vital event registration system and inaccessibility of health facilities most deaths occur at home and pass undocumented. This study was done with the objective of identifying the causes of death in a predominantly rural population where almost all deaths are occurring at home. The paper tries to demonstrate a possibility of using a relatively simple and inexpensive verbal autopsy method for obtaining valuable data in improving health services to the rural underserved populations.
Methods
The study district, Meskan and Mareko, is located in the central-south part of Ethiopia at an average altitude of 2,100 meters above sea level with a range of 1,750 m in the low lands to 3,400 m in the mountainous areas. The actual study sites were the nine rural and one urban study kebeles (Kebele is the smallest administrative unit in Ethiopia often having an average of 500 households, the size varies significantly from district to district) that are under continuous demographic surveillance by the Butajira Rural Health Program (BRHP). These sites were randomly selected using the probability proportionate to size technique. The BRHP registers events such as birth, death, marriage, new household, out-migration, in-migration, and internal move. Information on the circumstances leading to death was not routinely collected by the surveillance system. Data on mortality as well as the above mentioned events were collected monthly, by trained village-based enumerators, through household visits. The total population of the area was estimated to be 257,000 in 1999. Detailed description of the study area is given elsewhere [22].
All adult deaths in the age group 15 to 49 year that were registered by the BRHP from January 1995 to December 1999 were identified from the longitudinal surveillance database and considered for this study. The age-group 15–49 was selected for this study in order to identify the causes of premature adult death, since life expectancy at birth in Ethiopia is 53 years for females and 50.9 years for males [17,23].
Data were collected using a simplified verbal autopsy questionnaire administered to relatives of the deceased. The questionnaire for this study was developed based on the WHO questionnaire for identifying cause of death in children and modified to address adult signs and symptoms of diseases [8]. It was initially prepared in English and then translated to Amharic (the national language). The first part of the questionnaire has questions on the socio-demographic characteristics of the subjects such as area of residence, age, sex, marital status, education, economic status, health service usage; and their personal habits such as smoking and alcohol drinking. The second part has questions on circumstances surrounding mortality such as signs and symptoms of illness, duration of illness, health care seeking, medications used, and admission to a health facility. The interview on average took about 20 minutes. The VA questionnaire used in this study is short and simplified. That was done for the following reasons: to develop a less complicated questionnaire that can be used by lay interviewers with minimal training; to get adequate response to all questions from rural and illiterate people; to make the questionnaire suitable for annexing into routine surveillance systems in the future; to facilitate the development of an uncomplicated algorithm for identifying broad categories of cause of death; and to obtain fairly complete information about each death. Data collectors and supervisors were recruited from the study area. The data collectors were all high school graduates with good experience in a similar type of fieldwork. Data collectors went house to house to collect information about the deceased person identified from the study base. In the event respondents were not available on first visit, repeated visits were arranged on at least two occasions.
Data entry and analysis were done using Epi Info version 6.04 statistical package. Causes of death were analyzed using an expert algorithm programmed into a computer. The algorithm does not include cancer as one category mainly because health records did not show cancer as major causes of either hospital admission or hospital death for adults in Ethiopia [17,23]. The second reason is that because of the varied manifestations for different types of cancers, it would be difficult to accommodate deaths due to cancer into the simple questionnaire and algorithm used in this study. The algorithms used are shown in Table 1.
Table 1 Algorithm used for the diagnoses of cause of death, in Meskan and Mareko district, Ethiopia. 2000.
Algorithm Diagnosis
Duration of illness < 30 days + Accidents (intentional or unintentional) Injuries
Female sex + (Pregnant or In labor or in the puerperal period) Maternal causes
Duration of illness > 30 days + Cough + Weight loss + (Bloody sputum or Fever or Ascites) + no diarrhea Tuberculosis
Duration of illness > 30 days + Cough + Diarrhea + Fever + Weight loss AIDS
Duration of illness > 15 days + (Edema of legs or Ascites) + Jaundice Liver diseases
Duration of illness < 15 days + Abdominal swelling + Repeated vomiting + No diarrhea Acute abdomen
Duration of illness > 30 days + cough + Dyspnea + Wheezing + No bloody sputum Chronic obstructive lung diseases
Duration of illness < 30 days + Diarrhea + No cough Diarrheal diseases
Duration of illness < 15 days + Fever + Headache Acute febrile illness
Duration of illness < 15 days + Fever + Headache + Neck stiffness Meningitis
Duration of illness < 30 days + Cough + Fever + (Dyspnea or Chest pain) Pneumonia
Duration of illness < 30 days + Dyspnea + Palpitation + (Edema of legs or Ascites) Cardio Vascular diseases
Results
A total of 819 adult (15–49 years) deaths were identified from the BRHP computer database for the period 1995–1999. In the field appropriate respondents (family members and close relatives) were identified for 515 (63%) of the deceased persons. The reasons for not identifying the rest include out-migration of the entire family of the deceased person, unavailability of relatives on repeated visits, wrong recorded information about the deceased, and double recording. The highest proportion of the missed cases (59.7%) was for those who died in the initial one and half years of the study. From those who died in the last one and half years of the study only 19.4% of the cases were missed. Communicable diseases accounted for 60.8% of the deaths, non-communicable for 24.1%, maternal causes 7%, injuries 1.7%, and undetermined causes consisted of 6.4%. The highest number of deaths was due to acute febrile illnesses (AFI) comprising 25.2% of all causes. AFI, diarrheal diseases, tuberculosis, AIDS, and liver diseases together accounted for 64.7% of the deaths (Table 1). AFI was the cause for 28% and 25.5% of the deaths in the rural highland, and lowland areas respectively, while it accounted to only 15.5% of the urban deaths. Diarrheal diseases accounted for 7% of the deaths in the urban area, 10.6% in the rural highland and 13% in the rural lowland. However, HIV/AIDS in the urban area accounted for 11.3% in contrast to 8.5% and 4.8% in the rural highland and lowland (Table 2).
Table 2 Possible verbal autopsy based causes of death by area of residence in Meskan and Mareko district, Ethiopia. 1995–1999.
Possible cause Urban Rural highland Rural lowland Total
No. (%) No. (%) No. (%) No. (%)
I. Communicable Diseases 37 (52.1) 150 (63.6) 126 (60.6) 313 (60.8)
Acute Febrile illnesses 11 (15.5) 66 (28.0) 53 (25.5) 130 (25.2)
Diarrheal diseases 5 (7.0) 25 (10.6) 27 (13.0) 57 (11.1)
All forms of tuberculosis 6 (8.5) 23 (9.7) 21 (10.1) 50 (9.7)
HIV/ AIDS 8 (11.3) 20 (8.5) 10 (4.8) 38 (7.4)
Pneumonia 4 (5.6) 14 (5.9) 10 (4.8) 28 (5.4)
Meningitis 3 (4.2) 2 (0.8) 5 (2.4) 10 (1.9)
II. Non-communicable Diseases 20 (28.2) 56 (23.7) 48 (23.1) 124 (25.4)
Liver diseases 8 (11.3) 26 (11.1) 24 (11.5) 58 (11.3)
Cardio-vascular diseases 6 (8.5) 13 (5.5) 13 (6.3) 32 (6.2)
Chronic obstructive lung diseases 4 (5.6) 14 (5.9) 9 (4.3) 27 (5.2)
Acute abdomen conditions 2 (2.8) 3 (1.3) 2 (0.9) 7 (1.4)
III. Maternal causes 3 (4.2) 19 (8.1) 14 (6.7) 36 (7.0)
IV. Injuries 2 (2.8) 6 (2.5) 1 (0.5) 9 (1.7)
Undetermined 9 (12.7) 5 (2.1) 19 (9.1) 33 (6.4)
Total 71 (100) 236 (100) 208 (100) 515 (100.0)
In general, deaths from communicable diseases was greater in males (62.8%) than in females (58.7%); while deaths from non-communicable diseases was greater in females (35.4%) than in males (26.8%). Maternal mortality accounted for 7% of all deaths, and 14.2% of the female deaths (Table 3). Of the 36 women identified to have died from maternal causes; 17 (47.2%) had fever, 10 (27.8%) had prolonged labor, and 4 (11.1%) had hemorrhage.
Table 3 Possible verbal autopsy causes of death by sex in Meskan and Mareko district, Ethiopia. 1995–1999.
Possible cause Females Males Total
Number (%) Number (%) Number (%)
I. Communicable Diseases 149 (58.7) 164 (62.8) 313 (60.8)
Acute Febrile illnesses 63 (24.8) 67 (25.7) 130 (25.2)
Diarrheal diseases 28 (11.0) 29 (11.1) 57 (11.1)
All forms of tuberculosis 21 (8.3) 29 (11.1) 50 (9.7)
HIV/ AIDS 19 (7.5) 19 (7.3) 38 (7.4)
Pneumonia 17 (6.7) 11 (4.2) 28 (5.4)
Meningitis 1 (0.4) 9 (3.4) 10 (1.9)
II. Non-communicable Diseases 54 (21.2) 78 (26.8) 124 (25.4)
Liver diseases 26 (10.2) 32 (12.3) 58 (11.3)
Cardio-vascular diseases 14 (5.5) 18 (6.9) 32 (6.2)
Chronic obstructive lung diseases 10 (3.9) 17 (6.5) 27 (5.2)
Acute abdomen conditions 4 (1.6) 3 (1.1) 7 (1.4)
III. Maternal causes 36 (14.2) --- 36 (7.0)
IV. Injuries 3 (1.2) 6 (2.3) 9 (1.7)
Undetermined 12 (4.7) 21 (8.1) 33 (6.4)
Total 254 (100.0) 261(100.0) 515(100.0)
Since a large proportion of the deaths that occurred in the first one and half year of the study were missed, analysis of cause of death was also made for the deaths excluding the initial one and half year. There were a total of 422 cases found in this category. Communicable diseases accounted for 59.2% of the deaths, non-communicable 25.4%, maternal causes 6.9%, injuries 0.9% and undetermined causes were 7.6% (Table 4).
Table 4 Possible verbal autopsy based causes of death for the last three and half years of the recall period as compared to the total five years in Meskan and Mareko district, Ethiopia. 1995–1999.
Possible cause Year of death
09/01/96-12/31/99 01/01/95-12/31/99
Number (%) Number (%)
I. Communicable Diseases 250 (59.2) 313 (60.8)
Acute Febrile illnesses 102 (24.2) 130 (25.2)
Diarrheal diseases 47 (11.1) 57 (11.1)
All forms of tuberculosis 42 (10.0) 50 (9.7)
HIV/ AIDS 30 (7.1) 38 (7.4)
Pneumonia 22 (5.2) 28 (5.4)
Meningitis 7 (1.7) 10 (1.9)
II. Non-communicable Diseases 95 (25.4) 124 (25.4)
Liver diseases 49 (11.6) 58 (11.3)
Cardio-vascular diseases 29 (6.9) 32 (6.2)
Chronic obstructive lung diseases 23 (5.5) 27 (5.2)
Acute abdomen conditions 6 (1.4) 7 (1.4)
III. Maternal causes 29 (6.9) 36 (7.0)
IV. Injuries 4 (0.9) 9 (1.7)
Undetermined 32 (7.6) 33 (6.4)
Total 422 (100.0) 515 (100.0)
Discussion
This study has attempted to identify the causes of death at a district level based on a simplified verbal autopsy procedure. Although information of this type is recognized to be very important for health planning and decision making it is largely nonexistent in developing countries, especially in rural settings. In the study area the major causes of death were acute febrile illnesses, liver diseases, diarrheal diseases, tuberculosis and HIV/AIDS. Maternity related causes accounted for 14.2% of the total female deaths. The questionnaire was made short and easily understandable to enhance its application on illiterate rural respondents by lay interviewers. The simplification has narrowed the possibilities of making very specific diagnoses that are claimed to be achieved using a more elaborate questionnaire. On the other hand, it would have been impossible to obtain complete data using elaborated questionnaire administered by lay interviewers, especially for the deaths that occurred long ago. In rural areas it is very difficult, if not impossible, to find interviewers with fairly high level of education that can administer and record an elaborate open ended questionnaire. In this study no major problem was detected on the completed questionnaires that can be attributed to the data collector's incompetence.
Information was collected as far back as five years. We observed that the loss of the beloved is unforgettable in the study community and reported without any reservation, especially for adults. The recall period however can affect the quality of detailed information collected for VA use. Baqui utilized a five year recall period for children in Bangladesh and reported that increase recall lapse has no clear adverse effect on data quality [24]. In addition, the questions on events surrounding mortality in this study are very simple and direct. For example, one of the questions was on duration of illness in general, not on duration of specific symptoms. The omission of very specific questions was helpful in minimizing the problem of recall. The major problem associated with the long time span was the unavailability of relatives of the deceased for interview, due to out migration or other reasons. There was a marked decline in the proportion of the missed cases from 1995 to 1999. The high number of missed cases for the deaths during "01/01/95"-"08/31/97" may have affected the results of this study and can be sighted as one of the limitations encountered. This indicates that VA information need to be gathered closer to the event, i.e. within a time span of not more than two years. The simplified algorithm used for diagnosis of cause of death classified illnesses into "broad" categories consisting of 12 causes of death. For example all acute febrile illnesses and all forms of tuberculosis were lumped into one category. Although this "broad" classification has the disadvantage of diagnosing only a limited variety of diseases and leaving out some diseases as undetermined, it makes easier the use of VA and the algorithm in many similar settings once validated. Other studies have demonstrated that simplicity of the questionnaire, the use of lay interviewers, lack of complexity in the algorithm used, and the broad categories of diseases increase the validity of a study and its reproducibility in other settings [6].
Although a validation study was not done on the "expert algorithm" used, the combinations of symptoms and duration of illness used for diagnosing injuries, maternal deaths, acute febrile illnesses, and meningitis are believed to be highly sensitive but not very specific. For example the algorithm of AFI has the combination of "duration of illness less than 15 days", "fever" and "headache". This combination can pick almost all deaths due to AFIs, but it increases the false positivist.
The algorithm used for tuberculosis, AIDS, liver diseases, and pneumonia are less sensitive but more specific than the previously stated diseases categories, because of the sign and symptom combinations used. A validation study for verbal autopsy in adults had shown that the cause specific mortality fraction obtained using "expert algorithms" was within ± 20% of the gold standard for malaria, meningitis, tuberculosis/AIDS, acute abdomen conditions, diarrheal diseases, direct maternal causes, chronic liver diseases and renal disorders [14].
The algorithm used for cardiovascular diseases and chronic obstructive pulmonary diseases may not have performed well because of the unstable nature of the diseases resulting in difficulty of sighting symptoms specific to them [7]. The "expert algorithm" used in this study may not have given the correct diagnosis for some of the diseases at the individual level. However, in the face of absence of information on causes of mortality in Ethiopia, the information generated at the population level using these combinations is very useful in identifying priority health problems that need urgent intervention in rural populations.
Causes of death diagnosed through the expert algorithm showed that one fourth of the deaths were due to acute febrile illnesses. The proportion was much higher in the rural areas than in the town. In contrast, HIV/AIDS accounted for a higher proportion in the town than the rural areas. A previous study in the same area has shown a similar pattern of disease occurrence [16].
We have tried to see if there is any inconsistency in the causes of death as a result of the greater number of missed cases in the first one and half year of the study period. The deaths during the later period of the study have very similar pattern when compared to the total five year deaths. For communicable diseases it was 60.8% vs. 59.2%, for non-communicable 24.1% vs. 25.4% and for maternal causes 7% vs. 6.9%. This consistency can be taken as an indicator of non-differential loss of cases over the specified period of time. This also indicate the robustness of the approach even when data are not complete, which is truly the case in most rural settings of developing countries.
This study has grossly indicated the magnitude of female deaths due to maternal causes, which is very difficult to come by as measuring maternal mortality ratio is very expensive [25-27], such information could be helpful to implement practical interventions locally. Considerations in the line of using the probabilistic approach to interpreting verbal autopsies described by Byass might help to further simplify the methodology and facilitate its application in large surveys [28]. However, it is important to note that the questionnaire in this study was tailored to yield maximum information from mostly illiterate and rural people as administered by lay interviewers. Hence its applicability might be limited to situations similar to the study area both in terms of availability of information and profile of health problems, which is dominated by communicable diseases.
Conclusion
In conclusion, this study provides evidence that interviewing deceased relatives using a simplified VA questionnaire and expert algorithm for diagnosis of causes of death can provide information on priority health problems in resource poor communities of developing countries. The importance of having local information for planning appropriate health interventions at district level is demonstrated in the same area [29]. We encourage more researches to further develop this simplified VA questionnaire and expert algorithm, and to do a validation study in order to enhance its applicability in larger scale health surveys.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
KL participated in the design of the study, field data collection, data analysis, and prepared the draft manuscript. YB participated in the design of the study, field data collection, and data analysis and revised the draft manuscript. Both authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Authors gratefully acknowledge the financial support from the Irish Embassy and Irish Aid, Addis Ababa, Ethiopia. Authors also acknowledge technical and material support obtained from the Department of Community Health, Faculty of Medicine, Addis Ababa University. Special thanks go to the Butajira Rural Health Program and its field staff.
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| 15935096 | PMC1164421 | CC BY | 2021-01-04 16:28:56 | no | BMC Public Health. 2005 Jun 3; 5:58 | utf-8 | BMC Public Health | 2,005 | 10.1186/1471-2458-5-58 | oa_comm |
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BMC Plant BiolBMC Plant Biology1471-2229BioMed Central London 1471-2229-5-91594148410.1186/1471-2229-5-9Methodology ArticleDevelopment and evaluation of a Gal4-mediated LUC/GFP/GUS enhancer trap system in Arabidopsis Engineer Cawas B [email protected] Karen C [email protected] Jon J [email protected] Stan B [email protected] Robert G [email protected] Washington University, Department of Biology Campus Box 1137, 1 Brookings Drive St. Louis, MO 63130, USA2 Monsanto Company, St Louis, MO 63167, USA2005 7 6 2005 5 9 9 15 12 2004 7 6 2005 Copyright © 2005 Engineer et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Gal4 enhancer trap systems driving expression of LacZ and GFP reporters have been characterized and widely used in Drosophila. However, a Gal4 enhancer trap system in Arabidopsis has not been described in the primary literature. In Drosophila, the reporters possess a Gal4 upstream activation sequence (UAS) as five repeats (5XUAS) and lines that express Gal4 from tissue specific enhancers have also been used for the ectopic expression of any transgene (driven by a 5XUAS). While Gal4 transactivation has been demonstrated in Arabidopsis, wide use of a trap has not emerged in part because of the lack of detailed analysis, which is the purpose of the present study.
Results
A key feature of this study is the use of luciferase (LUC) as the primary reporter and rsGFP-GUS as secondary reporters. Reporters driven by a 5XUAS are better suited in Arabidopsis than those containing a 1X or 2X UAS. A 5XUAS-LUC reporter is expressed at high levels in Arabidopsis lines transformed with Gal4 driven by the full, enhanced 35S promoter. In contrast, a minimum 35S (containing the TATA region) upstream of Gal4 acts as an enhancer trap system. Luciferase expression in trap lines of the T1, T2, and T3 generations are generally stable but by the T4 generation approximately 25% of the lines are significantly silenced. This silencing is reversed by growing plants on media containing 5-aza-2'-deoxycytidine. Quantitative multiplex RT-PCR on the Gal4 and LUC mRNA indicate that this silencing can occur at the level of Gal4 or LUC transcription. Production of a 10,000 event library and observations on screening, along with the potential for a Gal4 driver system in other plant species are discussed.
Conclusion
The Gal4 trap system described here uses the 5XUAS-LUC and 5XUAS rsGFP-GUS as reporters and allows for in planta quantitative screening, including the rapid monitoring for silencing. We conclude that in about 75% of the cases silencing is at the level of transcription of the Gal4 transgene and is at an acceptable frequency to make the Gal4 trap system in Arabidopsis of value. This system will be useful for the isolation and comprehensive characterization of specific reporter and driver lines.
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Background
A number of different methods have been developed in Arabidopsis thaliana to discover and analyze promoters that are regulated by tissue specificity or environmental conditions [1-4]. These include the use of reporters GUS, GFP and more recently luciferase (LUC)[5,6] to detect "trapped" DNA elements that confer some type of regulation. Individual reporters and the exact type of trap system (e.g. gene fusion versus enhancer) each have their biases as to the type of expression element which is more frequently identified as well as their own advantages and disadvantages, which have been previously reviewed [7-9]. Of the available technical approaches, in the activator/upstream activating sequence (UAS) bipartite system, a transcriptional activator (factor) is used to trap endogenous enhancers. Transcription factor expression is detected using a reporter gene with the appropriate upstream UAS. This system has the advantage of using trapped lines to subsequently drive the ectopic expression of introduced transgenes. Trapping enhancer elements using the gene for the activator Gal4, which subsequently activates a reporter such as lacZ (possessing a Gal4 UAS), has been very successful in yeast and Drosophila [10]. The consequence for the fruitfly community has been the generation in the last ten years of well-defined Drosophila trap lines that show developmental or tissue specific expression of Gal4 [8,11]. These lines can then be used for expression studies with a reporter, typically lacZ or GFP, but also as driver lines to ectopically express any gene of interest that is placed downstream of the Gal4 UAS [12].
Enhancers are DNA regulatory elements that function over variable distance to alter the level of gene expression. An enhancer is generally not sufficient for gene expression and requires minimal promoter elements for transcription initiation. Enhancer elements provide much of the spatially, temporally and environmentally regulated gene expression in plants. Gal4 trap lines in Arabidopsis which allow rapid and quantitative analysis of gene expression levels will provide a valuable resource for understanding both tissue-specific and environmental regulation of gene expression. As a beginning, Haseloff has described a potential Gal4 trap system that uses a 5XUAS GFP (not red shifted, rs), whereby some Arabidopsis lines showed expression in the roots ([13], p 146–147 of ref. [14] and cited website [15]). The purpose of the present report is to describe the plasmids, silencing properties, and validation of Gal4-specific expression of our trap library in Arabidopsis. To develop an ideal Gal4 trap system, the reporter(s) should be quantitative, sensitive and stable over generations. Because of its long half-life, GFP is rarely used as a reporter for environmental gene regulation, and it is less feasible to perform quantitative expression studies with GFP in planta. We used luciferase as the primary reporter in this study, based on the short half life of luciferase and for its sensitivity and screening capabilities [5,16-18].
It was recently reported that in tobacco the Gal4 system is poorly expressed (i.e. silenced) because the high methylation status of cytosines (28–32%) impairs the binding of Gal4 to the UAS [19]. (Drosophila and yeast DNA is not methylated). The Gal4 binding site (UAS) typically contains GGCN11CCG and it is predicted from crystal structure studies that methylation would prevent binding [20], and methylation has been shown directly to inhibit binding [19]. The study in tobacco employed a 9 or 10X UAS repeat and the authors suggested that with less UAS repeats the affect due to methylation inhibition of Gal4 binding might be less, although this was not tested. A. thaliana possesses only 5% cytosine methylation [21] but the properties of a Gal4 trap system in this organism have not been documented in the primary literature. A previous study in Arabidopsis by Guyer et al used a 10XUAS-GUS reporter with a full 35S promoter driving Gal4 [22]. This study indicated that trans activation was feasible and no problems of silencing or methylation were noted. A question then remains whether Arabidopsis (and plants with methylation frequencies between those of Arabidopsis and tobacco) might be candidates to develop Gal4 trap systems for regulation or ectopic expression studies. The major goals of the present study were to 1) use a 5XUAS-LUC as reporter and determine if Gal4 driven by the full 35S promoter confers constitutive luciferase expression compared to a minimum 35S (thus acting as a trap); 2) determine whether a 5XUAS in Arabidopsis is optimal (compared to a 1X or 2X UAS); 3) determine stability of luciferase trap lines from seedling to seedling and over generations (i.e. silencing rates and basis); 4) produce a trap library with the potential for screening of three reporters (LUC, GUS, rsGFP), each activated by Gal4.
Results and discussion
T-DNA vectors for Gal4 studies using a 5XUAS-LUC reporter
The enhancer trap vectors constructed to examine the feasibility of a Gal4 activator/UAS system in Arabidopsis are diagrammed in Fig 1A and 1B and sequences upstream of the Gal4, LUC+ (pspLuc), and rsGFP-GUS genes are shown in Fig 1C. The first vector, pRGK336, has the full 35S promoter (e35S), including 35S enhancers, directly upstream of the Gal4 orf. (This orf is a hybrid of Gal4 DNA binding domain and the VP16 activation domain). The trap vector, pRGK335, only contains the minimum 35S TATA region. Both vectors possess the T-DNA right border directly upstream of the Gal4 promoter regions (Fig 1C). The bar gene encoding BASTA resistance was chosen for ease in selection of transgenic plants grown in soil. The LUC+ gene was inserted downstream of the minimum 35S and a 5XUAS (Fig 1C).
If the trap system functions in a Gal4-dependent manner, it is expected that the vector with the e35S promoter driving Gal4 expression (pRGK336) should show higher luciferase activities in planta than transformants with pRGK335 (the trap). Initially, approximately 300 seeds from transformed plants were sowed, sprayed with BASTA, and 10 days later sprayed with luciferin and imaged. Fig 2 shows an image of luciferase activity (light emission as shown in red) from these pots. A pot with control plants (i.e. Columbia) not sprayed with BASTA is shown on right. As expected, no Columbia plants exhibited luciferase activity. All BASTA-resistant plants transformed with pRGK336 showed luciferase expression and approximately half of the BASTA-resistant pRGK335 plants showed luciferase expression at the detection level shown. This validated that the luciferase gene can be expressed from both vectors. BASTA-resistant seedlings were transplanted (from BASTA selection media) and 14 days later luciferase activities were measured in planta. Relative levels in each seedling are shown in Fig 3, where zero delineates the Columbia control (i.e. no luciferase). In this experiment, for pRGK336 transformed plants, 32 out of 34 (94%) could be observed as expressors from digitized images of expression (as shown in Fig 2). The remaining two still had significant luciferase expression (200–300 relative units). A profile of the plants transformed with pRGK335 (trap vector) is shown in Fig 3. Approximately 50% were observed on digitized images and had activity above 200 relative units. The average luciferase activity for pRGK336 plants was 2054 units and for pRGK335 plants was 846 units. Thus, the plants appear to depend on the expected Gal4 expression levels for the luciferase expression. Taken together, both the higher percentage of seedlings which express luciferase and the higher level of expression observed with Gal4 expression from pRGK336 compared to pRGK335, suggests that this luciferase activity is dependent on the Gal4 expression.
Similar experiments were performed with seedlings at earlier stages of growth and agar media was used to examine luciferase activities in roots and aerial tissues. Eight day old seedlings were transferred after selection to agar plates containing MS medium with 50 uM luciferin and, after 24 hours, imaged for luciferase activity (Fig 4). Visible images of plants are shown in black on an artificial green background (Fig 4, Left) and the luciferase activities superimposed (Fig 4, Right), such that red or yellow indicate LUC expression. All but two seedlings with pRGK336 showed expression of luciferase in roots and cotyledons. Only 2 out of 21 pRGK335 plants showed expression in roots and 7 out of 21 in cotyledons. The variability in tissue specificity suggests that "trapped" enhancer elements drive Gal4 in the pRGK335 plants. Quantitation of luminescence in each seedling from a similar experiment is shown in Fig 5. Average luciferase activities in plants transformed with pRGK336 were 536 units and with pRGK335 were 151 units, again showing that expression is higher in plants that contain Gal4 driven by the e35S promoter.
We determined inheritance ratios of the BASTA and LUC expression in T2 generations from the trap lines (with pRGK335). In a pool of T2 seeds, 55 out of 220 seedlings (25%) died on BASTA selection, suggesting a 3:1 overall inheritance of BASTA selection, as expected if the majority of transgenics are at a single locus. In a pool of T2 seeds under no selection, 52 out of 100 seedlings had luminescence when sprayed with 1 mM luciferin. Variations from one seedling to another in T3 generations of selected lines were also quantified. Fig 6 shows two examples of lines that were evaluated. T2 seedlings that were not under BASTA selection (called "T2 heterozygous" seedlings in Fig 6) were either heterozygous, homozygous or did not have the BASTA gene due to segregation. As expected, if BASTA resistance was lost, luciferase activity was also absent (e.g. see line 169, "T2 heterozygous" seedlings). Homozygous lines gave consistent expression from seedling to seedling with respect to luciferase levels and tissue specificity. This suggests that lines can be maintained as homozygotes and expected to express consistently within the T3 generation.
Use of a 5XUASrsGFP-GUS secondary reporter and testing whether 5X, 2X, or 1X UAS repeats are better in Arabidopsis
Often, a secondary reporter (or phenotype) is useful for confirmation of a regulatory property or as an additional tool in understanding the expression of a gene. GFP and GUS reporters have their own advantages in use as reporters, so a system was developed that would potentially accommodate the use of all three reporters (LUC, GFP, GUS), each activated by Gal4. A GFP-GUS fusion protein (orf) was used for these studies (see below). Additionally, we wanted to determine whether a 1X, 2X, or 5X repeat would be better in Arabidopsis. A report by Johnstone and colleagues [23] has suggested that in yeast varying the numbers of UAS repeats can affect the "reporter" sensitivity with respect to levels of Gal4. As indicated above [19], it could also be advantageous to have fewer UAS repeats in plants because of the methylation silencing phenomenon.
Arabidopsis was transformed with the three rsGFP-GUS vectors (1X, 2X, 5X UAS) diagrammed in Fig 1B. These are based on pCAMBIA1304, but the GFP was mutated to red-shifted GFP for higher quantum yield [24]. The serine at amino acid position 65 was changed to threonine by PCR based mutagenesis. The indicated UAS regions (with TATA from min35S) were cloned upstream (see sequences in Fig 1C). Columbia plants were transformed and selected with hygromycin. Next, we transformed the Gal4 vectors into various rsGFP-GUS transgenic lines to determine qualitatively which lines (1X, 2X, 5X UAS) expressed more GFP and/or GUS. Table 1 shows a qualitative assessment of their GUS activities in random transgenic plants possessing either a 1X, 2X, or 5X UAS and the Gal4 T-DNA. As expected, plants without T-DNA containing Gal4 did not express GFP, and the large majority did not express GUS or did so at low levels. The only combination that showed a majority of lines with high GUS activities were those with a 5XUAS rsGFP-GUS and the full e35S driving Gal4 (13 out of 22 lines). Some of these latter lines also exhibited GFP expression (see Fig 7 for examples of high GUS and GFP expressors). The 1X and 2X UAS lines showed weaker expression patterns and were therefore unsuitable for our future assays. We selected a single locus (5XUAS-rsGFP-GUS) line, named RGK1, that had high GUS and GFP which lost both activities when the Gal4 (BASTA-resistance) was segregated out in the next generation. A homozygous RGK1 line for this 5XUAS rsGFP-GUS reporter was chosen as the parent strain for producing a library using the pRGK335 trap vector (see below for library construction). The results with different numbers of UAS repeats indicate that in Arabidopsis the 5XUAS is optimal under the experimental methods described. Additionally, these qualitative data are consistent with the hypothesis that Gal4 levels dictate expression from the 5XUAS reporters.
Silencing of the Gal4 system in Arabidopsis
To determine the frequency and properties of silencing in the Gal4 transgenics, we chose random lines of the pRGK336 and pRGK335 that exhibited significant luminescence in T2 seedlings and quantified expression in seedlings from T2, T3, and T4 generations. Initial studies on nine pRGK336 and nine pRGK335 lines indicated that no major drop in LUC expression from generations T2 to T3 occurred (not shown), with some lines decreasing and some increasing, but typically not by more than two to three fold.
A study of pRGK335 (trap) BASTA resistant lines was carried out on T2, T3, and T4 generations, where all three generations were grown on the same plates and assayed for luciferase activity (Table 2). Again, the changes from T2 to T3 were typically less than 3 fold. However, some lines were dramatically silenced when comparing the T2 to T4 generations: 5 out of 18 (28%) showed more than a 10 fold decrease in LUC expression with 4 out of 18 (22%) showing a greater than 100 fold decrease in expression in the T4 generation (relative to T2). Genetic analysis to determine the number of loci in these selected lines was based on T2 plant BASTA resistance/sensitivity ratios. Approximately 25% had an insert at a single locus, 50% had two loci and 25% had more than two loci. There was no correlation between silencing and number of inserts.
To determine whether this dramatic decrease in expression is due to methylation, seedlings from the T4 generation of each silenced line were grown in media with and without 5-aza-2'-deoxycytidine (AZA), an inhibitor of DNA methyltransferase. A silenced pRGK336 (e35S driving Gal4) line was also included in this study. In each of the lines, every seedling was recovered for LUC expression when grown on 7 ug/ml AZA (Fig 8). Quantitation of luciferase activities indicated that the highest recovered expression was with the pRGK336 line, and in general expression was directly proportional to the LUC activity observed in the T2 generation (not shown). This suggests that recovery is proportional to the levels of Gal4 expected in the cell and that silencing is due to methylation, possibly at the level of the 5XUAS (see next paragraph).
Silencing from T2 to T4 (or T3 to T4) generations could occur at the level of Gal4 or the 5XUAS-LUC transcription. To investigate this, mRNA was prepared from seedlings of various generations of selected lines immediately after imaging for luciferase activity. Multiplex Q-RT-PCR was used to determine the levels of Gal4 and LUC mRNA in these samples. Based on the constitutively expressed Ubc-10 gene, mRNA levels were determined. The levels of luciferase activity, Gal4 mRNA, and LUC mRNA are reported in Table 3. Control lines (169 and 213) that show only a minor decrease in luciferase activity also exhibited little change in Gal4 and LUC mRNA. Line 65L-4B is silenced mainly at the level of the 5XUAS LUC, while lines 50-1, 80-2 and 36 are silenced at the level of Gal4 and 5XUAS LUC transcription. Thus, both classes of silencing are observed (see Discussion).
Library construction
With the single locus homozygous line RGK1 (referring to the 5XUASrsGFP-GUS described above) we have generated a library of approximately 10,000 events using the pRGK335 (trap) vector. Data reported in the previous section suggests that about 25% of the silenced lines will be silenced mainly at the 5XUAS LUC); a potential advantage to using the single locus line as recipient is the potential for less silencing in subsequent generations for the 5XUAS rsGFP-GUS (e.g. [25,26]. To ensure uniform representation of events in the library, 66 T1 plants were transplanted to a soil flat after BASTA selection. Each flat was harvested separately and seeds stored in an individual envelope. Equivalent weights of seeds from each individual tray was weighed out and combined with seed from 8 to 9 other trays. If plants had died, amount of seed was adjusted accordingly. Nineteen independent T2 seed pools of approximately 550 events each have been generated. Forty plants were studied further to compare luminescence, GUS and GFP levels and it was determined that the presence of GUS and GFP was related to high luciferase levels. For example, out of 21 plants showing greater than 1200 AU luminescence units, 13 showed GFP expression, whereas none of the plants with less than 1200 AU units exhibited detectable GFP. Screens for lines that exhibit tissue specificity and stable expression in subsequent generations are currently in progress. We have noticed no aberrant properties of transformed plants (e.g. stunted or chlorosis), suggesting that there are no deleterious effects due to the expression of LUC, rsGFP, GUS, Gal4, or bar in the library.
Conclusion
A reliable, quantifiable enhancer trap system based on an activator/UAS approach in Arabidopsis was the principal aim of this research. The Gal4 activator was selected as the trans-activator for our system. While the Gal4 system in Drosophila has been quite useful, previous studies in tobacco revealed technical hurdles, such as methylation-induced silencing. In the tobacco study, a 9 or 10XUAS was used to drive GUS expression and Gal4 was expressed from a full 35S promoter [19]. Tobacco transgenics with this vector showed GUS expression in only 10 out of 60 lines (17%), quite different than the greater than 94% we observed here (with pRGK336). Additionally, specific tobacco transgenic expressor lines showed more than 20 fold variability in GUS expression from seedling to seedling, unlike with Arabidopsis shown here. Results with tobacco were shown to be erratic due to methylation of the UAS repeats, certainly a ramification of the high methylation frequency (32%) in that species [19]. Although we have not compared results using a 10XUAS repeat, a 5XUAS appears to be better suited for expression than a 1X or 2X UAS in Arabidopsis. We have proven that silencing by methylation in Arabidopsis will occur by the 4th generation in approximately 25% of the lines. On the other hand, 75% of lines retain expression through the T4 generation in our study (see below). Moreover, T3 seedlings appear to retain expression and for many applications this may be sufficient.
A general conclusion suggested from our study is that the higher the methylation status of an organism, the less useful a Gal4-type system will be, regardless of the number of UAS repeats employed. A very recent report [27] indicates "that Gal4/VP16-UAS elements provided a useful system for enhancer trap in rice". This is somewhat surprising because rice is approximately 19% methylated [21,28]. Wu et al. used a 6XUAS-GUS reporter and the GUS expression frequency in the initially transformed generation, called T0, was quite impressive (over 70% of transgenic lines) [27]. T1 generation plants had similar expression levels and tissue specificities (as T0), suggesting few effects due to methylation. No generations past the T1 were evaluated. The authors suggest that using a 6XUAS rather than 10XUAS, among a few other technical differences, might explain the lack of problems associated with methylation in rice when compared to that in tobacco [19]. Later generations in the rice system may begin to show silencing properties. Different species may also exhibit silencing effects due to methylation at different generations, and certainly the copy number of inserts will impact the silencing properties. Based on their results with tobacco, Moore and colleagues developed a different UAS bipartite system in which a modified lac repressor DNA-binding domain was combined with the Gal4 activation domain (called LhG4) [29]. This was at least proven technically feasible in tobacco and very recently, this has been applied to Arabidopsis to ectopically drive expression of a selected gene [30]. In this case, the driver lines expressed LhG4 from cloned promoter elements. A bipartite system that uses an ethanol-responsive AlcR activator with the alcA promoter for transactivation has recently been reported [31,32]. This system requires exogenous ethanol and also has been combined with tissue-specific promoters driving AlcR expression. This "ethanol switch" provides certain advantages, as described[31,32], and will have its own unique applications for driver line studies. We conclude from our results that random trapping using the more common Gal4:VP16 results in 25% silencing by the T4 generation but that much of this silencing may be at the level of the Gal4:VP16 transgene expression (Table 3). This suggests that any T-DNA delivered trap library (including LhG4 and AlcR) will result in some silencing in Arabidopsis and that methylation of the 5XUAS is not a major drawback.
Methods
Plasmid construction
The complete DNA sequences of the three plasmids shown in Figures 1A and 1B have been deposited in Gen Bank. Genbank accession numbers are AY739897 (pRGK335); AY739898 (pRGK336); AY739899 (pRGK337).
GFP-GUS vectors
The GFP-GUS fusion vectors are based on pCambia 1304, which has an engineered, fused GFP-GUS orf (Figure 1B). The GFP was changed to redshifted GFP to enhance detection [24]. A PCR product was made using the oligonucleotide primers rsGFP-F (5'CTGTTCCATGGCCAACACTTGTCACTACTTTCACTTATGGTGTT) and rsGFP-reverse (5'AACGATCGGGG AAATTCGAG). The A in bold and underlined was changed from a T to an A to change a serine to a threonine. Both pCambia 1304 and the PCR product were digested with NcoI and BstEII and ligated, resulting in p1304-r-20.
A PCR product containing 5X UAS, m35S and an ER signal was made using the oligonucleotide primers UASCP7F1 (5'TATGGTACCGATTACGCCAAGCTTGCATG) and 5XUAS/ER-REV (`TTGGCCATGGAACAGGTAGTTT) with pBinMGal4-VP16+UASmgfp5-ER (kindly provided by J. Haseloff, Cambridge University, see website [33] as the template. KpnI and NcoI sites are in bold. p1304-r-20 was digested with KpnI and NcoI to remove the LacZ alpha and the full 35S promoter. The PCR product was digested with KpnI and NcoI and ligated to p1304-r-20. The new plasmid with the 5XUAS is designated pRGK337.
To construct derivatives of rsGFP-GUS with 2XUAS and 1XUAS sequences, we took advantage of some natural, spontaneous deletions of the 5XUAS constructs. These contained a 1XUAS or 2XUAS with the sequences shown in Fig 1C. For a 2XUAS, we used the same right and left oligonucleotide primers as described for the pRGK337. Both the PCR product and p1304-r-20 were digested with KpnI and NcoI and ligated. For the 1XUAS version, a natural deletion within the Gal4 vectors described above was used as template. The oligonucleotide primers 5XUAS/ER-FWD (5`CAATAGGTACCTGAACGCGTCGGAGTACTG) and 5XUAS/ER-REV (5'TTGGCCATGGAACAGGTAGTTT) were used. NcoI and KpnI sites are in bold. Both the PCR product and p1304-r-20 were digested with KpnI and NcoI and ligated.
Gal4, LUC vectors
The pRGK336 and pRGK335 plasmids were constructed using pMON51850 as the core vector (containing the gene for bacterial spc/str-resistance), as diagrammed in Figure 1A. The construction of pRGK336 and pRGK335 were the same except for the insertion of the e35S and m35S respectively. These were constructed in the following steps to insert the indicated cassettes (the description begins at the right border as defined in Fig 1A): to insert the m35S and Gal4/VP16, a PCR product containing the m35S and Gal4VP16 from pBinMGal4-VP16+UASmgfp5-ER was used as template and the PmeI and BglII sites were added (sequences shown in Figure 1C); pRGK336 was constructed by replacing PmeI-BglII fragment with the full e35S promoter as a PmeI-BglII fragment (pMON23449); overlapping PCR from BglII to AvrII (in end of NOS 3'UTR) formed a cassette with the Gal4/VP16 orf (pBinMGal4-VP16+UASmgfp5-ER as template) and the NOS 3'UTR (pMON51850); GAL4PME-S (GATCGTTTAAACCTTCGCAAGACCCTTCCTC) GAL4NOS-S (GACGAGTACGGTGGGTAGCCCGATCGTTCAAACATTTGGC) GAL4NOS-R (GCCAAATGTTTGAACGATCGGGCTACCCACCGTACTCGTC) NOS-AVRII (GATCCCTCGGGATCTAGTAACATAGATGAC) an overlapping PCR product from the AvrII (in end of NOS 3'UTR) to MluI site formed a cassette with the BAR gene (pCGN9978) and 7S UTR (pMON51728); 35-AVRII (GATCCCTAQGGCTATCTGTCACTTCATC) BAR7S-S (CCTGCCCGTCACCGAGATTTGACCGTCCTTTGTCTTCAATTTTG) BAR7S-R (CAAAATTGAAGACAAAGGACGGTCAAATCTCGGTGACGGGCAGG) 7S-MLU-R (GATCACGCGTTCAATATTGTGGCAGGAC) for the 5XUAS-LUC-tml region, an MluI-BamHI PCR product (from pRGK337) contained the 5X UAS region; this intermediate plasmid was cut with Kpn1 and BamH1 and to insert the LUC gene, the luciferase vector, psp-luc+Vector, (Promega) was cut with BglII and KpnI and integrated. To leave only the LUC region, these were digested with EcoRI and KpnI and the TML UTR [34,35] was inserted as follows: a TML cassette was made using the oligonucleotide primers TML F1 (5' CGAGAATTCGGGAGGAAATTACACTGAGG) and TML R1 (5' CGGCAGGATATATTCAATTGTAAATTCC); the EcoRI site is in bold while the KpnI site is upstream of the primer [this cassette was derived from a plasmid that contained an overlapping PCR product formed from the TML (pCGN9978); UASTML-S (GATCACGCGTCGGAGTACTGTCCTCCG) GFPTML-S (CAACATGATGAGCTTTAACCCGGGTACCGAGCTC) GFPTML-R (GAGCTCGGTACCCGGGTTAAAGCTCATCATGTTTG) TMLSPE-R (GATCACTAGTTTTCAAATCCTTCAGATGG) and the 5Xtet region (pMON33643) TETSPE-S (GATCACTAGTCGTTAACTGCAGCTGAG) TETMFE-R (GATCCAATTGTAAATTCCTCTCCAAATGAAATGAAC) which contained the indicated KpnI site.] The TML cassette was digested with EcoRI and KpnI and ligated to form pRGK335.
Plants and growth conditions in soil
A. thaliana ecotype Columbia-0 was used for all experiments. Plants in soil were grown in either long day or short day conditions. The long day growth chamber had 16 hours of light (175 uE) at 21°C. The short day chamber had 8 hours of light (175 uE) at 19°C. After seed set, plants were placed in a greenhouse with supplemental lighting to hasten drying. Soil medium was a mixture of Scotts Fafard germination mix, Scotts Rediearth and vermiculite #3, 1:1:1.4 respectively. Plants were fertilized once per week with Peters 15-16-17. Seedlings that were selected for BASTA resistance were sprayed with a 1:200 dilution of Finale concentrate at seven and fourteen days.
Growth conditions of plants on agar
Plants were grown on MS salts and vitamins, 2% sucrose with pH 5.7 and .7% agar. Light regime was 16 hours of light (100 uE) at 22°C and 8 hours of darkness at 20°C. Agar plates were wrapped in Micropore surgical tape (3 M). Selection for plants on agar used hygromycin at 29 ug/ml or BASTA at 25 ug/ml. BASTA (glufosinate ammonium) was from Sigma. Nylon mesh used for seedling transfers is from Sefar America (catalogue # 06-300/34).
Plant transformation
Plants were transformed via Agrobacterium tumefaciens using the floral dipping method [36]. A. tumefaciens ABIwas used for pRGK336 and pRGK335 transformations. A. tumefaciens GV3101 was used for all other transformations. Small scale cultures of 25 mL were grown overnight at 28°C in LB and selected antibiotics. Large scale cultures of 400 mL were inoculated with 1.5 mls of the overnight culture and grown to an OD600 of 1.3. Bacteria were spun down and resuspended to an OD600 of 0.6 in dipping solution consisting of 5% sucrose, 1 mg/ml BAP and 0.03% Silwet. Plants were dipped in the bacterial solution for 5 min, lightly drained, placed on their side and covered for 24 hours.
Plants were started on MS with 2% sucrose on 0.7% agar, with or without selection depending, on the plant background to be transformed. Seedlings were transplanted after 14 days to 2 1/2 inch pots, 4 plants per pot. Plants were moved to a short day growth chamber and covered with a dome for 1 day to aid acclimation. After 4 weeks, plants were moved to long day conditions; bolts were trimmed one time before dipping. Plants were dipped for transformation after 1 week in long day and dipped a second time 6 days later. Three weeks after the final transformation, plants were moved from the long day growth chamber to the greenhouse to hasten the drying process. Seeds were harvested when plants were completely dry.
GFP expression
Seedlings were examined for GFP expression with a Zeiss Stemi SV11 microscope using a 500 nm GFP filter. Images were captured via AxioCam camera and software.
GUS expression
Seedlings were stained for β-Glucuronidase activity as described previously [37]. Whole seedlings were immersed in a solution with 1.5 mM X-Gluc, vacuumed infiltrated 2 times for approximately 1 minute and incubated overnight at 37C in the dark. Tissues were destained via ethanol:acetic acid (3:1, v/v) before viewing. The X-Gluc solution consisted of 1.5 mM X-Gluc, 100 mM NaHPO4 buffer pH 7, 0.5 mM K3 [Fe(CN)6], 0.5 mM K4 [Fe(CN)6] and 10 mM EDTA.
Luciferase imaging and quantitation
Firefly D-Luciferin, potassium salt (synthetic) was from Biosynth International. Luciferin was either sprayed on plants at 1 mM or incorporated into the media at 50 or 100 uM. For imaging, a Fuji LAS-1000 plus CCD Luminescent Image Analyzer from Fujifilm was used. Quantitation of luciferase activity (luminescence) in whole plants, roots, or other tissues used Science Lab 99-Image Gauge Ver. 3.4 software (Fujifilm).
Selection of T3 and T4 plants
6 plants from each T2 line were planted to soil and T3 seed were collected from these plants. This yielded 6 separate pools of T3 seed per line. These T3 seeds were assayed for BASTA resistance and homozygous lines were allowed to set seed (T4 seeds). These lines were used for the generation study in Table 2.
RNA isolation, cDNA synthesis and multiplexed quantitative real-time PCR
Seeds were germinated on MS media and grown for 3 weeks with BASTA selection. Both generations being assayed for each plant line were grown on the same plate. Next, the plants were sprayed with 1 mM luciferin and imaged in the Fuji CCD camera and luciferase activities quantified. Tissue was harvested from whole plants and RNA extracted with the Qiagen RNeasy Plant kit according to the manufacturer's instructions (catalog# 74903). The RNA was treated with Invitrogen Amp grade DNase-I according to manufacturer's instructions (catalog# 18068-015). First-Strand cDNA synthesis was carried out on 2 μg of this RNA for each sample using the Invitrogen SuperScript III Reverse Transcriptase (catalog#18080). The cDNA was diluted 4-fold and used in subsequent Q-RT-PCR experiments. Light upon extension (LUX) primers for the Q-RT-PCR experiments were designed by Invitrogen Corporation's online LUX designer software and the sequences and fluorophore designations were as follows:
Gal4 Forward: CACTTGCCGCCTCAAGAAGCTCAAG [FAM] G
Gal4 Reverse: AGAGTAGCGACACTCCCAGTTGTT
Luciferase Forward: CACCGCTCTTCAATTCTTTATGCCGG [FAM] G
Luciferase Reverse: TGCGAAATGCCCATACTGTTG
UBC-10 Forward: CACTGCCTCGACATCTTGAAGGAGCAG [JOE] G
UBC-10 Reverse: GCTATCTCGGGCACCAAAGG
FAM = 6-carboxy-fluorescein
JOE = 6-carboxy-4', 5'-dichloro-2', 7'-dimethyoxy-fluoresein
Reactions were carried out with the Sigma Jumpstart Taq ReadyMix for quantitative PCR (catalog# D7440) according to manufacturer's instructions with the exception of using a final concentration of 4.5 mM MgCl2 for the Gal4 gene and 6.5 mM MgCl2 for the Luciferase gene. Cycling conditions included 94°C for 120 sec followed by 50 cycles of 94°C for 15 sec, 65°C for 30 sec and 72°C for 30 sec on the Cepheid Smart Cycler System. Every PCR reaction contained the primer pair for the Ubc-10 gene as an internal control in addition to either the Gal4 or luciferase primer pair. For each sample of cDNAs, reactions were carried out in triplicates. For each PCR tube, the ÄCt for the sample gene (either Gal4 or Luciferase) was calculated relative to the UBC-10 gene Ct and then the ÄCt.s were averaged for the triplicates. Fold differences in gene expression between samples were calculated by first determining the ÄÄCt values between samples and then using the formula: Fold Change = 2(ΔΔCt).
Distribution of material
All novel materials described in this study will be available for non-commercial research purposes. Contact RGK: [email protected]
Authors' contributions
CE was responsible for silencing studies, library screening methods and luciferase imaging optimization. KCF was responsible for construction of the three plasmids (as in Figure 1), luciferase imaging and library production and characterization. JS and SD were responsible for construction of plasmids on which some of those in Figure 1 are based and discussion of the studies. RGK is responsible for the design and execution of the study. RGK wrote the first few drafts while all authors participated in writing the paper.
Acknowledgements
We thank Valerie Harlan for technical assistance, Robert Feissner for assistance with the Fuji imaging system, and Rita Varagona, Barry Goldman, Dan Tenesson for discussions, and Eric Richards for reading the manuscript; Craig Pikaard for use of the GFP microscope. This work was supported by the Monsanto/Washington University collaborative agreement.
Figures and Tables
Figure 1 Maps of plasmids and selected DNA sequences of constructs. A, pRGK336 has enhanced 35S upstream of Gal4 while pRGK335 has minimal 35S upstream of Gal4. In both constructs, 5X UAS and m35S are upstream of the luciferase gene (psp-luc). B, There is no Gal4 in pRGK337and the 5XUAS and m35S are upstream of the GFP-GUS fusion protein. 2XUAS and 1XUAS versions are the same map but with the sequence changes noted in Fig 1C and the text. C, Selected DNA sequences of indicated regions. The right border is underlined and the TATA box is boxed; m35S and e35S are in bold. The ATG start site is bolded and underlined with an arrow for the indicated orfs. The UAS sequences are in bold and underlined showing the difference between 1X, 2X and 5X UAS.
Figure 2 Image of pRGK335, pRGK336 and untransformed plants. Image of pRGK335 (left) and pRGK336 (middle) transformed plants and untransformed (right) in soil that have been sprayed with luciferin. Visible (artificially colored green) image has been overlaid with CCD captured luminescence (red).
Figure 3 Quantification of luminescence in individual plants grown in soil. The indicated transformed seedlings were selected with BASTA, transplanted, sprayed with luciferin, and luciferase activity measured. Luciferase activity was normalized for background and area (A-B/mm2).
Figure 4 Images of pRGK335 and pRGK336 plants. Images of pRGK336 (top half of seedlings on each plate), pRGK335 (bottom half of seedlings on each plate) transformed plants on agar media with 50 uM luciferin. Left photo is visible CCD image, plants artificially colored black on a green background. Right photo has luciferase activity superimposed on visible – all yellow and red colors represent luciferase activity.
Figure 5 Quantification of luminescence for seedlings grown on agar. T1 seeds of the indicated transgenics were selected on BASTA, survivors transferred to media with 50 uM luciferin, and imaged. Luciferase activity was normalized for background and area (A-B/mm2).
Figure 6 Luciferase expression to determine variation and stability of homozygous and heterozygous seedlings. Color indicators as shown in Fig 4 (red luciferase overlaid onto black visible: all yellow and red colors represent luciferase activity).
Figure 7 GFP and GUS expression of the 5XUASrsGFP-GUS transgenic. Loss of GFP-GUS expression when Gal4 is removed through segregation ("no GAL4") compared to line possessing GAL4 ("+GAL4"). A, GFP was viewed through a 500 nm GFP filter. B, Whole seedling GUS expression of indicated lines.
Figure 8 Luciferase expression in silenced lines grown with and without AZA. Luciferase expression of rows of the indicated "silenced" AZA (left) compared to media with no AZA (right). Red color indicates LUC activity. All seedlings were the same age. Line 66L 36-2 is a pRGK336 transgenic and all others are with pRGK335.
Table 1 GUS expression in 1X, 2X, and 5X UAS/GFP-GUS plants with different Gal4 backgrounds.
No Gal4
1X 2X 5X
# of Plants a 6 11 8
Gus Expression:
None 2 6 4
Low 4 4 4
High 0 1 0
m35S/Gal4
1X 2X 5X
# of Plants 14 15 17
Gus Expression:
None 8 8 6
Low 3 4 6
High 3 3 5
e35S/Gal4
1X 2X 5X
# of Plants 26 15 22
Gus Expression:
None 12 9 2
Low 7 4 7
High 7 2 13
a T1 seedlings were intially selected on the appropriate chemicals (BASTA and/or hygromycin) and transplanted to soil. Two leaves from each plant were collected after two weeks in soil for Gus assay (as shown in Figure 7).
Table 2 Comparison of luminescence across generations for randomly selected trap lines.
Line # T2a T3 T4 Fold Change Fold Change Fold Change
T2 to T3 T3 to T4 T2 to T4
172 6522 6145 5609 1.1 Same 1.1 Same 1.2 Same
216 2354 2485 2589 0.9 Same 1.0 Same 0.9 Same
25-1 16170 4990 51479 3.2 Decrease 10.3 Increase 3.2 Increase
169 12007 34529 24193 2.9 Increase 1.4 Decrease 2.0 Increase
173 4524 6452 6678 1.4 Increase 1.0 Same 1.5 Increase
212 5157 4150 3318 1.2 Same 1.3 Same 1.6 Decrease
27-4 9859 4283 5129 2.3 Decrease 1.2 Same 1.9 Decrease
47-1 6531 2680 3224 2.4 Decrease 1.2 Same 2.0 Decrease
14-1 3914 4110 1733 1.0 Same 2.4 Decrease 2.3 Decrease
213 2434 5286 895 2.2 Increase 5.9 Decrease 2.7 Decrease
214 3933 2171 1028 1.8 Decrease 2.1 Decrease 3.8 Decrease
211 8475 2423 1251 3.5 Decrease 1.9 Decrease 6.8 Decrease
77-2 1872 3668 200 2.0 Increase 18.0 Decrease 9.4 Decrease
215 11211 5995 479 1.9 Decrease 12.5 Decrease 23.4 Decrease
65L-4B 16022 2987 0 5.4 Decrease 3.0 × 103 Decrease 1.6 × 104 Decrease
18-1 1968 1163 0 1.7 Decrease 1.0 × 103 Decrease 2.0 × 103 Decrease
80-2 12378 6187 19 2.0 Decrease 3.0 × 102 Decrease 6.0 × 102 Decrease
50-1 16746 3867 19 4.3 Decrease 2.0 × 102 Decrease 8.0 × 102 Decrease
a. Luminescence was quantified for pRGK335 (trap) plant lines in multiple generations. Individual 14 day-old seedlings from each generation were grown on the same plates and whole plant luminescence quantified. Luminescence is expressed as Actual minus Background per mm2. 100 μM luciferin was included in the media. For the T2 lines, data is an average of 3 to 5 plants; only luminescent plants were included in the data (As described in the text, luminescence segregated/correlated with BASTA-resistance). For the T3 and T4 generations, all seedlings were imaged, as all were correlated with BASTA-resistance.
Table 3 Quantitative real-time PCR analysis of mRNA for Gal4 and LUC genes in selected linesa.
Line# Fold drop in LUC activity (generations used)b Fold drop in gene expression
GAL4 LUC
169 1.2 (T3/T4) 3 3.5
213 1.8(T3/T4) 2.3 3.3
65L-4B 42 (T2/T4) 1.6 147
50-1 33 (T2/T4) 59 56
36c 82 (T2/T4) 298 71
80-2 20 (T2/T4) 18 35
a All numbers indicate a fold decrease in activity or expression as plant lines progress from the T2 (or T3) to the T4 generation.
b Luciferase activity measured in BASTA-selected 14 day-old seedlings by spraying 1 mM luciferin.
c This is a full 35S-driven Gal4 line as shown in Fig 8.
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| 15941484 | PMC1164422 | CC BY | 2021-01-04 16:03:52 | no | BMC Plant Biol. 2005 Jun 7; 5:9 | utf-8 | BMC Plant Biol | 2,005 | 10.1186/1471-2229-5-9 | oa_comm |
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Cancer Cell IntCancer Cell International1475-2867BioMed Central London 1475-2867-5-181592979110.1186/1475-2867-5-18ReviewPreclinical evaluation of the proteasome inhibitor bortezomib in cancer therapy Boccadoro Mario [email protected] Gareth [email protected] Jamie [email protected] Section of Hematology, University of Torino, Torino, Italy2 Royal Marsden Hospital, Surrey, UK3 St. Bartholomew's Hospital, Department of Haematology, London, UK2005 1 6 2005 5 18 18 22 2 2005 1 6 2005 Copyright © 2005 Boccadoro et al; licensee BioMed Central Ltd.2005Boccadoro et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Bortezomib is a highly selective, reversible inhibitor of the 26S proteasome that is indicated for single-agent use in the treatment of patients with multiple myeloma who have received at least 2 prior therapies and are progressing on their most recent therapy. Clinical investigations have been completed or are under way to evaluate the safety and efficacy of bortezomib alone or in combination with chemotherapy in multiple myeloma, both at relapse and presentation, as well as in other cancer types. The antiproliferative, proapoptotic, antiangiogenic, and antitumor activities of bortezomib result from proteasome inhibition and depend on the altered degradation of a host of regulatory proteins. Exposure to bortezomib has been shown to stabilize p21, p27, and p53, as well as the proapoptotic Bid and Bax proteins, caveolin-1, and inhibitor κB-α, which prevents activation of nuclear factor κB-induced cell survival pathways. Bortezomib also promoted the activation of the proapoptotic c-Jun-NH2 terminal kinase, as well as the endoplasmic reticulum stress response. The anticancer effects of bortezomib as a single agent have been demonstrated in xenograft models of multiple myeloma, adult T-cell leukemia, lung, breast, prostate, pancreatic, head and neck, and colon cancer, and in melanoma. In these preclinical in vivo studies, bortezomib treatment resulted in decreased tumor growth, angiogenesis, and metastasis, as well as increased survival and tumor apoptosis. In several in vitro and/or in vivo cancer models, bortezomib has also been shown to enhance the antitumor properties of several antineoplastic treatments. Importantly, bortezomib was generally well tolerated and did not appear to produce additive toxicities when combined with other therapies in the dosing regimens used in these preclinical in vivo investigations. These findings provide a rationale for further clinical trials using bortezomib alone or in combination regimens with chemotherapy, radiation therapy, immunotherapy, or novel agents in patients with hematologic malignancies or solid tumors.
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Introduction
Bortezomib (VELCADE®, formerly PS-341) was approved for the treatment of patients with relapsed or refractory multiple myeloma in May 2003 by the US Food and Drug Administration [1] and in April 2004 by the Committee for Proprietary Medicinal Products of the European Union. A number of clinical studies evaluating the activity and safety of bortezomib in multiple myeloma, as well as in other types of cancer, have been conducted [2-10] or are ongoing [11]. Therefore, a review of key preclinical studies that have explored the mechanisms of action and provided the rationale for clinical investigation of this novel agent in multiple myeloma and other cancer types is warranted.
Mechanism of action
Bortezomib, a boronic acid dipeptide (Figure 1A) [12], is a highly selective, reversible inhibitor of the 26S proteasome that was first shown to exhibit antitumor properties in a panel of 60 cancer cell lines from the US National Cancer Institute [13]. The proteasome is an enzyme complex that primarily functions in the degradation of misfolded proteins and is essential for the regulation of the cell cycle. Proteasomes are localized in the nucleus and cytosol, where they are largely associated with centrosomes, the cytoskeleton, and the outer endoplasmic reticulum [14]. Damaged intracellular proteins are targeted for elimination by the proteasome through ubiquitination (Figure 1B). Many of the substrates that have been identified are proteins that function in the regulation of transcriptional activation, signal transduction, cell cycle proliferation, and apoptosis (Figure 2, Table 1) [15-29].
Figure 1 Chemical structure of the proteasome inhibitor bortezomib: pyrazylcarbonyl-Phe-Leu-boronate (A). Schematic illustration of the ubiquitin-proteasome pathway. Misfolded proteins (e.g., p53) are targeted for degradation by the 26S proteasome via phosporylation and ubiquitination (B). Following substrate degradation, the ubiquitin tags and peptides are recycled for future use.
Figure 2 Inhibition of the proteasome by bortezomib results in activation of JNK, stabilization of p53, Bid, Bax, p21, p27, caveolin-1, and IκBα, resulting in inhibition of NF-κB. Alteration of the levels of these cellular proteins leads to inhibition of proliferation, migration, and angiogenesis and promotion of apoptosis of cancer cells.
Table 1 Intracellular targets of bortezomib.
Protein Function Effect of bortezomib References
IκBα Regulates the activity of the transcription factor, NF-κB Stabilization Hideshima et al [15], 2001
Russo et al [16], 2001
Sunwoo et al [17], 2001
Hideshima et al [18], 2002
Tan and Waldmann [19], 2002
Ma et al [20], 2003
JNK Phosphorylates and activates the transcription factor c-Jun Activation Hideshima et al [21], 2003
Chauhan et al [22], 2004
Yang et al [23], 2004
p21, p27 CDK inhibitors Stabilization Shah et al [24], 2001
Hideshima et al [15], 2001
Yang et al [23], 2004
p53 Transcription factor and Tumor suppressor Stabilization Williams and McConkey [25], 2003
Hideshima et al [21], 2003
Yang et al [23], 2004
Bid Proapoptotic protein Stabilization Breitschopf et al [26], 2000
Bax Proapoptotic protein Stabilization Li and Dou [27], 2000
Caveolin-1 Promotes cell migration Inhibition of activation Podar et al [28], 2004
gp130 Cytokine signaling receptor Downregulation Hideshima et al [29], 2003
DNA-PKcs DNA repair Cleavage Hideshima et al [21], 2003
ATM DNA repair Cleavage Hideshima et al [21], 2003
IκBα = inhibitor κB-α; JNK = c-Jun-NH2 terminal kinase; CDK = cyclin-dependent kinase; DNA-PKcs = DNA protein kinase catalytic subunit; ATM = ataxia telangiectasia, mutated.
Several independent investigators have found that bortezomib inhibits activation of the transcription factor nuclear factor κB (NF-κB) [15-20,30]. NF-κB is important for cell survival and is activated in response to cell stress, including that induced by cytotoxic agents, radiation, or DNA damage. NF-κB is overexpressed in several tumors and regulates the expression of genes involved in apoptosis (including Bcl-2 and Bcl-xL), cell cycle progression, inflammation, and angiogenesis (including interleukin [IL]-6, IL-8, and vascular endothelial growth factor [VEGF]) [31-33]. NF-κB is normally bound in the cytosol to inhibitor κB-α (IκBα). Phosphorylation, ubiquitination, and degradation of IκBα are required for NF-κB to translocate to the nucleus and activate the transcription of target genes. Bortezomib blocks the activation of NF-κB by preventing proteasomal degradation of IκBα. Through inhibition of NF-κB, bortezomib not only promotes apoptosis of cancer cells but also sensitizes these cells to chemotherapy [15,20,30], radiation [16], or immunotherapy [19]. However, because specific NF-κB inhibition alone via PS-1145 only partially inhibits proliferation of tumor cells [18], the cytotoxic activity of bortezomib must also depend on altered regulation of other signal transduction pathway targets [18].
The intracellular levels of a number of other proteins that regulate gene transcription, apoptosis, and proliferation are significantly affected by bortezomib (Table 1). c-Jun-NH2 terminal kinase (JNK) is a protein that promotes cell death in response to stress and increased levels of misfolded proteins [34]. Bortezomib treatment leads to activation of JNK in multiple myeloma [21,22] and non-small cell lung cancer cells [23]. These studies further showed that specific inhibition of JNK activation, either genetic or pharmacologic, prevented mitochondrial release of cytochrome c and Smac, activation of caspase-8, -9, and -3, and apoptosis.
Proteasome inhibition has also been shown to stabilize the cyclin-dependent kinase inhibitors p21 and p27, the tumor suppressor p53, and the proapoptotic proteins Bid and Bax [15,21,23-27]. The increased levels of activated p21, p27, p53, Bid, and Bax result in inhibition of cell cycle progression and/or promotion of apoptosis in response to bortezomib. Interestingly, sensitivity to proteasome inhibition was partially dependent on the p53 status of breast [35] and lung cancer in vitro [36], but bortezomib-induced apoptosis and/or chemosensitization were p53 independent in prostate [13], multiple myeloma [15], and colon cancer cells [30]. Therefore, the degree of variability in the sensitivity to bortezomib with respect to p53 status appears cell-type dependent.
A recently published study found that bortezomib prevented activation of caveolin-1 in multiple myeloma cells [28]. Activation of caveolin-1, a protein that functions in cell motility or migration in a number of tissues, requires phosphorylation. In this report, bortezomib was shown to prevent phosphorylation of caveolin-1 by VEGF, a proangiogenic cytokine and transcriptional target of NF-κB [37]. Bortezomib also inhibited VEGF secretion by the bone marrow. Together, these findings demonstrate important mechanisms by which bortezomib may inhibit migration of cancer cells as well as tumor angiogenesis.
The specific proteins mentioned have all been shown to be at least partially responsible in various models for the antiproliferative, proapoptotic, antiangiogenic, and antitumor effects of bortezomib. However, recent studies have found that bortezomib results in cytotoxic activity through activation of the endoplasmic reticulum stress response [38-41]. The mechanism appears to involve blockade of retrograde transportation and degradation of damaged endoplasmic reticulum proteins by proteasome inhibition [42]. Further studies are necessary to link these new findings with the specific intracellular signals that have been previously implicated in the anticancer activities of bortezomib.
Bortezomib alone
Bortezomib has shown promising antitumor activity in a number of preclinical cancer murine models in vivo (Table 2) [13,17,19,24,30,43-49]. In a xenograft model of multiple myeloma, bortezomib treatment resulted in significant inhibition of tumor growth, an increase in overall survival, and a decrease in tumor angiogenesis [43]. The proteasome inhibitor was well tolerated up to 0.5 mg/kg intravenously (IV) twice weekly for 4 weeks, with dose-limiting toxicities, including weight loss, at 1 mg/kg. Two recent reports evaluating the efficacy of bortezomib in murine xenograft models of adult T-cell leukemia have reached contradictory conclusions. Tan and Waldman found that bortezomib treatment alone, 0.06 mg/kg intraperitoneally (IP), daily for 3 weeks, did not produce significant antitumor effects [19]. However, a second group reported that bortezomib 1.0 mg/kg IP twice weekly for 2 weeks resulted in antitumor activity [44]. Whereas Tan and Waldman reported lethality at a more aggressive dosing regimen of bortezomib 0.1 mg/kg IP twice daily for 2 weeks, slight, temporary weight loss was the only adverse effect described by the second group of investigators [44]. It seems plausible that the discrepancy in activity and toxicity may be due to the differences in the doses and dosing regimens. Based on the finding that proteasome inhibition by bortezomib lasts for up to 72 hours [8], more recent preclinical studies have used twice-weekly, rather than daily or twice-daily dosing, and have shown overall greater activity with less toxicity.
Table 2 Activity of bortezomib in tumor models in vivo.
Cancer Activity MTD References
Multiple myeloma Decreased tumor growth and angiogenesis; increased survival 0.5 mg/kg IV twice weekly for 4 weeks LeBlanc et al [43], 2002
Adult T-cell leukemia Decreased or no effect on tumor growth 1.0 mg/kg IP twice weekly for 2 weeks Tan and Waldmann [19], 2002
Satou et al [44], 2004
Lung Tumor growth delay and decreased lung metastases 1.0 mg/kg PO once daily for 18 days Teicher et al [45], 1999
Breast Decreased surviving fraction of tumor cells 5.0 mg/kg IP once Teicher et al [45], 1999
Prostate Decreased tumor growth; decrease or no effect on angiogenesis 1.0 mg/kg IV weekly for 4 weeks, or q 72 hrs for 15 days Adams et al [13], 1999
Williams et al [46], 2003
Pancreatic Decreased or no effect on tumor growth and angiogenesis; increased or no effect on tumor apoptosis 1.0 mg/kg IV biweekly for 2 to 3 weeks, or weekly for 4 weeks or 0.25 mg/kg IP biweekly for 4 weeks Shah et al [24], 2001
Nawrocki et al [47], 2002
Bold et al [48], 2001
Head and neck Decreased tumor growth and angiogenesis 1.5 mg/kg IP 3 times per week for 3 weeks Sunwoo et al [17], 2001
Colon Decreased tumor growth and increased tumor apoptosis 1.0 mg/kg IV twice weekly Cusack et al [30], 2001
Melanoma Decreased tumor growth and angiogenesis; increased tumor apoptosis 1.25 mg/kg SC twice weekly for 5 weeks Amiri et al [49], 2004
MTD = maximum tolerated dose; IV = intravenous; IP = intraperitoneal; PO = by mouth; SC = subcutaneous.
An evaluation of the effects of bortezomib in murine xenograft models of both lung and breast cancer was also conducted [45]. Treatment with oral bortezomib 1.0 mg/kg daily for 18 days caused tumor growth delays, as well as a decrease in the number of metastases in the Lewis lung cancer model. Furthermore, in a murine model, bortezomib at a single IP dose of up to 5 mg/kg significantly decreased the surviving fraction of breast tumor cells. A decrease in the level of colony-forming-unit granulocyte macrophages was the only toxicity noted in these experiments.
Two groups of investigators have evaluated the efficacy of bortezomib in murine xenograft models of prostate cancer. The first study concluded that bortezomib 1.0 mg/kg IV weekly for 4 weeks reduced tumor growth by 60% [13]. The second study, in which bortezomib 1.0 mg/kg IV every 72 hours for 15 days was administered, produced similar results, with 50% and 80% inhibition in tumor growth in two xenograft models [46]. This report further showed that bortezomib significantly inhibited tumor angiogenesis in one of the models, as measured by a decrease in the number of CD31+ vessels in tumor sections. No toxicities were detected in either of these studies.
In a study of pancreatic cancer murine xenografts, treatment with bortezomib 1.0 mg/kg IV or IP weekly for 4 weeks resulted in a 72% or 84% reduction in tumor growth, as well as an increase in tumor cell apoptosis, with no evidence of toxicity [24]. Another group found that bortezomib 1.0 mg/kg IV biweekly for 2 to 3 weeks significantly inhibited tumor growth and angiogenesis and promoted apoptosis in 1 of 2 pancreatic cancer xenograft murine models [47]. These investigators also reported adverse events, including decreased body weight, diarrhea, and gastrointestinal inflammation at doses above 1.0 mg/kg and lethality at doses above 1.5 mg/kg. Finally, bortezomib produced significant antitumor, proapoptotic, and/or antiangiogenic effects in murine xenograft models of head and neck [17] and colon cancer [30], as well as melanoma [49]. Adverse events, including dehydration, lethargy, weight loss, and death, were noted at doses of 2.0 mg/kg IP 3 times weekly for 3 weeks in one of these studies [17], whereas the other studies did not report any toxicities at lower doses (Table 2).
These preclinical investigations have collectively demonstrated the antitumor activity of bortezomib as a single agent at tolerable levels in a variety of murine cancer models. However, because the standard approach to antineoplastic therapy generally involves the administration of more than one agent or modality in an effort to prevent the development of chemoresistance and increase tumor cell kill, the effects of bortezomib in combination with chemotherapy, radiation, immunotherapy, or novel agents have been investigated in vitro and/or in vivo.
Combination therapy
A number of preclinical studies have evaluated the activity of bortezomib in combination with other therapies (Table 3) [15,16,19,20,24,30,39,40,45,48-61]. The finding that several cytotoxic agents as well as radiation [62,63] activate NF-κB is a major rationale for combining proteasome inhibitors with these therapies in the treatment of cancer. Activated NF-κB is free to translocate to the nucleus and induce the expression of proinflammatory and antiapoptotic genes, such as Bcl-2 and Bcl-xL, which promote tumor cell survival [31-33]. Furthermore, inhibition of NF-κB has been implicated as an important mechanism by which bortezomib sensitizes tumor cells to various drugs or radiation [15,16,20,24,30,48-50,55,57,59,60].
Table 3 Evaluation of bortezomib in combination with other therapies.
Type of therapy Drug or agent Cancer References
Chemotherapy 5-Fluorouracil, cisplatin, paclitaxel, doxorubicin, cyclophosphamide Breast, lung Teicher et al [45], 1999
Melphalan, doxorubicin, dexamethasone Multiple myeloma Hideshima et al [15], 2001
Mitsiades et al [50], 2003
Ma et al [20], 2003
Topoisomerase inhibitor: irinotecan Colon Pancreatic Cusack et al [30], 2001
Shah et al [24], 2001
Gemcitabine Pancreatic Bladder Bold et al [48], 2001
Kamat et al [51], 2004
Pegylated liposomal doxorubicin Breast Small et al [52], 2004
Docetaxel Pancreatic Nawrocki et al [53], 2004
Temozolomide Melanoma Amiri et al [49], 2004
Radiation therapy Breast Colon Prostate Teicher et al [45], 1999
Russo et al [16], 2001
Pervan et al [54], 2001
Immunotherapy Daclizumab Adult T-cell leukemia Tan and Waldmann [19], 2002
Novel agents TRAIL/Apo2L Multiple myeloma, myeloid leukemia, renal Mitsiades et al [55], 2001
Sayers et al [56], 2003
HSP90 inhibitor: 17-AAG Breast Mimnaugh et al [39], 2004
HDAC inhibitors: SAHA, sodium butyrate CML, multiple myeloma, lung Denlinger et al [40], 2004
Yu et al [57], 2003
Mitsiades et al [58], 2004
Denlinger et al [59], 2004
Pei et al [60], 2004
Transplantation Allogeneic BMT Leukemia Sun et al [61], 2004
HSP = heat shock protein; HDAC = histone deacetylase; CML = chronic myelogenous leukemia; BMT = bone marrow transplantation.
Bortezomib with chemotherapy
In an investigation of lung cancer, bortezomib in combination with chemotherapeutic agents, including 5-fluorouracil, cisplatin, paclitaxel, or doxorubicin, produced additive antitumor and antimetastatic effects [45]. In this same study, bortezomib also increased tumor-cell killing by cyclophosphamide and cisplatin, as well as tumor-cell killing by radiation, in an in vitro-in vivo model of breast cancer. Although toxicity is difficult to assess in preclinical models, no added toxicities were observed when bortezomib was added to the other therapies, and bortezomib dose modifications were not required.
Several studies evaluating the effects of bortezomib in combination with other therapies have been conducted in multiple myeloma. These in vitro experiments collectively confirmed that bortezomib enhanced the antiproliferative and proapoptotic effects of conventional antimyeloma agents, including melphalan, doxorubicin, and dexamethasone [15,20,50]. These investigators also reported that multiple myeloma cell lines that were previously resistant to melphalan, doxorubicin, dexamethasone, or mitoxantrone were sensitized up to 1,000,000-fold by prior exposure to subtoxic concentrations of bortezomib. Finally, the researchers showed that bortezomib was not only directly cytotoxic to the multiple myeloma cells but that it also altered the microenvironment through inhibition of IL-6 to prevent the growth of tumor cells in proximity to the bone marrow [15].
Two groups of researchers examined the effects of bortezomib in combination with the topoisomerase inhibitor irinotecan in murine xenograft models of colon [30] and pancreatic [24] cancer. Both these studies concluded that combined inhibition of the proteasome and topoisomerase resulted in enhanced antiproliferative and proapoptotic effects. The combination of bortezomib and irinotecan therapy further resulted in a 94% [30] or 89% [24] decrease in tumor size compared with controls. These tumors were also significantly smaller than those of the mice receiving either bortezomib (26% or 65% decrease in tumor size relative to controls) or irinotecan (48% or 43% decrease in tumor size relative to controls) as single agents.
Xenograft models of pancreatic cancer were also used to evaluate the activity of bortezomib in combination with other standard chemotherapies [48,53]. Inhibition of the proteasome increased the sensitivity of tumors to both gemcitabine [48] and docetaxel [53], because bortezomib in combination with these agents resulted in significant enhancement of antiproliferative, proapoptotic, antitumor, and/or antiangiogenic activities.
The combination of bortezomib with temozolomide was studied in models of malignant melanoma [49]. Bortezomib enhanced the antiproliferative and cytotoxic effects of temozolomide in melanoma cells in vitro, and this combined therapy produced complete remission of tumors lasting more than 200 days in murine xenografts in vivo. In contrast, tumors eventually progressed in mice receiving either drug alone. Although specific toxicity evaluations were not performed, no toxicities were observed in any of these investigations.
Bortezomib with radiation, immunotherapy, or novel agents
A number of studies have reported on the radiosensitizing properties of bortezomib. In addition to the previously mentioned study in breast cancer [45], other laboratories have demonstrated that bortezomib sensitizes colon [16] and prostate [54] cancers to radiation-induced cytotoxicity. In both clonogenic cell survival assays and murine xenograft tumor models, pretreatment with bortezomib enhanced the anticancer effects of irradiation with no observed toxicity.
Another murine xenograft model was used to investigate the effects of bortezomib in combination with daclizumab, a humanized anti-IL-2Rα antibody in adult T-cell leukemia [19]. Although either agent alone resulted in partial or no responses, bortezomib plus daclizumab resulted in prolongation of the survival of tumor-bearing mice. The only adverse effect of the combined therapy was a slight temporary weight loss during the treatment regimen.
Bortezomib has also demonstrated enhanced in vitro and/or in vivo anticancer effects when combined with novel agents such as TRAIL/Apo2L, a cell death-inducing ligand [55,56], 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat shock protein 90 [39], and suberoylanilide hydroxamic acid (SAHA) or sodium butyrate, both histone deactylase inhibitors [40,57,59,60]. Pretreatment with bortezomib sensitized multiple myeloma, myeloid leukemia, and renal cancer cells but not normal B lymphocytes to TRAIL/Apo2L-induced apoptosis [55,56]. In an in vivo experiment, bone marrow and renal cancer cell mixtures, with or without bortezomib and/or TRAIL/Apo2L, were transplanted into the bone marrow of mice. Whereas all the mice receiving cells treated with TRAIL/Apo2L died of leukemia within 35 days, 50% of those receiving cells treated with bortezomib and 90% of those receiving cells treated with both TRAIL/Apo2L and bortezomib survived more than 100 days [56]. A mild transient thrombocytopenia was the only toxicity observed in this study. Finally, while the combination of bortezomib and 17-AAG, SAHA, or sodium butyrate resulted in synergistic antiproliferative and proapoptotic effects in vitro [39,40,57-60], these combinations have yet to be evaluated in tumor xenograft models in vivo.
Sequence of administration
One of the areas of controversy has been the appropriate timing of therapy with bortezomib in relation to other antineoplastic agents. A brief discussion of some of the conflicting results is warranted as these preclinical studies may ultimately provide the rationale for clinical trials. In vitro experiments conducted by Mitsiades and colleagues revealed that optimal antimyeloma activity was achieved when bortezomib was administered 24 hours after doxorubicin [50]. The sequence of chemotherapy (gemcitabine) followed by bortezomib was also found to be the most effective in a model of pancreatic cancer [64]. In contrast, Pei et al showed that bortezomib followed by a second antineoplastic agent (SAHA) yielded the highest level of apoptosis in a model of myeloma [60], and Mimnaugh and colleagues found that maximal antiproliferative effects were observed upon simultaneous administration of bortezomib and a heat shock protein 90 inhibitor in a model of breast cancer [39]. It is important to note that these studies utilized bortezomib in combination with different antineoplastic agents in unique cancer models. Further studies may elucidate the reasons for the inconsistent findings.
Conclusion
The proteasome inhibitor bortezomib exhibits antiproliferative, proapoptotic, antiangiogenic, and antitumor activities in several cancer models. The mechanism of action of bortezomib involves stabilization of NF-κB, p21, p27, p53, Bid, and Bax, inhibition of caveolin-1 activation, and activation of JNK as well as the endoplasmic reticulum stress response. These preclinical evaluations have found that bortezomib is well tolerated at doses that demonstrated antitumor activity in xenograft models of multiple myeloma, adult T-cell leukemia, cancer of the lung, breast, prostate, pancreas, head and neck, and colon, as well as melanoma. Bortezomib also enhances the anticancer effects of chemotherapy, radiation therapy, immunotherapy, or novel agents, without added toxicities requiring dose modifications. The studies provide a rationale for clinical trials of bortezomib alone or in combination with other therapies in patients with solid tumors or hematologic malignancies.
List of abbreviations
NF-κB = nuclear factor-κB; IκBα = inhibitor κB-α; VEGF = vascular endothelial growth factor; ER = endoplasmic reticulum.
Competing interests
M.B. has received consulting and lecture fees from Millennium Pharmaceuticals, Inc. G.M. declares that he has no competing interests; he is supported by the Leukaemia Research Fund and the International Myeloma Foundation. J.C. has received advisory board and speakers' bureau fees from Millennium Pharmaceuticals, Inc. and Ortho Biotech.
Authors' contributions
M.B. reviewed the literature and drafted the manuscript. G.M. and J.C. reviewed and revised the manuscript. All authors read and approved the final version.
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| 15929791 | PMC1164423 | CC BY | 2021-01-04 16:24:16 | no | Cancer Cell Int. 2005 Jun 1; 5:18 | utf-8 | Cancer Cell Int | 2,005 | 10.1186/1475-2867-5-18 | oa_comm |
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Epidemiol Perspect InnovEpidemiologic perspectives & innovations : EP+I1742-5573BioMed Central London 1742-5573-2-51594148610.1186/1742-5573-2-5EditorialIntroducing article-processing charges and inviting "detailed methods sections" articles Phillips Carl V [email protected] Department of Public Health Sciences, University of Alberta, 13–103 Clinical Sciences Building, Edmonton, Alberta T6G 2G3, Canada2005 7 6 2005 2 5 5 13 4 2005 7 6 2005 Copyright © 2005 Phillips; licensee BioMed Central Ltd.2005Phillips; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This editorial introduces the use of article-processing charges at Epidemiologic Perspectives & Innovations and reviews that advantages of publishing in an Open Access journal. In addition, it introduces a new type of article the journal hopes to publish, detailed reports of study design or data analysis methods that have been used in health science research. The new type of article is intended to supplement the woefully constrained methods sections in standard research report articles, providing information that better fulfills the goals of scientific publishing.
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Introduction
Epidemiologic Perspectives & Innovations (EP&I) is published by BioMed Central (BMC), an independent publisher committed to ensuring peer-reviewed research is Open Access – that it is universally and freely available online to everyone and its authors retain copyright. In order to fund the publication of the journal while fulfilling our commitment to make all content of EP&I free to readers, the publisher and editors of the journal have introduced an article-processing charge. Starting April 2005, authors of articles accepted for publication will be asked to pay a charge of £330 (currently approximately US$625 or €480). There is no charge if a submission is rejected. Authors at the many BMC member institutions receive an exemption from the fee, and we encourage authors to help persuade their institutions to become members if they are not already (see endnote 1). Waiver requests, particularly from authors with financial hardship, will be considered on a case-by-case basis.
For paper journals, the standard subscription model is that readers pay, either through personal or institutional subscriptions, and this is typically extended to online versions. The result is that many potential readers are discouraged from viewing articles. Libraries, faced with escalating journal costs, have had to cut back on their commitment to providing access. Moreover, except for the shortest articles, authors at many paper journals have to pay a per-page fee. Unlike these page charges, the BMC article processing charge is a flat fee, regardless of the length of the article, accompanying graphics, or attached files, and thus is relatively modest.
Advantages of Open Access
EP&I's policy of Open Access changes the way in which articles are published. All articles are available for free to anyone with internet access. Readers are not limited by what their library can afford, and can easily access articles via web-based searches (using research databases or general web search engines), increasingly the most popular method for finding publications. This easier availability has been shown to make articles more highly cited [1]. It also fulfills the requirements that are increasingly being imposed by funders to make the products of their funding publicly available.
Under the Open Access model, authors retain copyrights to their work (beyond granting the journal the right to publish and archive the article, and readers the right to make appropriate use of the published material), allowing the authors to freely distribute, anthologize, and repost their work. This helps avoid such absurdities of the standard publishing model as authors having to pay substantial reprint fees (or violate copyright) to hand copies of articles to their students, often after already paying the journal to publish the article (see endnote 2).
Articles published at EP&I are available at the journal's website in addition to being archived in PubMed Central [2], the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam [3] in Germany, at INIST [4] in France and in e-Depot [5], the National Library of the Netherlands' digital archive of all electronic publications. Authors can easily check the number of times their article has been accessed via the EP&I website (though not the other archives), providing an estimate of the article's readership, another feature not possible with paper journal articles.
Increasingly, traditional paper journals are adopting certain Open Access policies piecemeal, including free online access (though often only part of their archives), online supplemental material, and article-processing charges. But by publishing at EP&I and other fully Open Access journals, authors can be assured of having all the advantages of Open Access: free access to all readers from the day of publication, retained copyright, and seamless online links to their articles and accompanying files, while retaining all the advantages of peer-review and appearance in scientific indexes.
Publishing "better methods sections" articles
The online publishing model, combined with the mission of EP&I, allows us to provide a forum for articles that are unlikely to be published in paper journals [6]. We are pleased to have made a good start on our mission of broadening what is published as legitimate health science research. We have published or expect to publish soon articles on quantitative methodology, decision making, new software, teaching methods, historical perspectives, and re-analyses of previous results. Several of these, despite their very high quality, were unlikely to have been published in other health science journals. We would now like to further take advantage of online publishing and invite submissions of a new kind of article.
A raison d'etre of scientific publishing is to allow other researchers to assess the validity of a study and to replicate it by following the recipe themselves, goals that are not compatible with squeezing the methods into a thousand words, as is typically required. Sufficiently describing a field study's data collection methods seems to require at least three or four thousand words, as does all but the simplest data analysis (see endnote 3). A recent editorial in the American Journal of Epidemiology [8] went so far as to praise the authors of that issue's lead research article [9] for getting the article down to 2,164 words. We will resist the temptation to itemize what was noticeably missing from the 548-word methods section in the praised article (let alone speculate about important information that a reader might never even realize was omitted), and instead take the high road, offering a new outlet for researchers who are pressured to reduce the length of their articles in paper journals (see endnote 4).
EP&I will publish methods articles that are primarily descriptive, presenting the detailed workings of the design and implementation of a study, how the data was analyzed, or both. We welcome submissions that report in detail the methods that produced previously published results, or details of methods in advance of the publication of results. Such submissions should use the Methods article type (see our instructions to authors for details) and the title should either lead with "Detailed Methods:..." or otherwise include the phrase "detailed methods". Authors are welcome to report some study results to provide context, but the article's focus should be the methods, not the results. We also encourage investigative reporting – submissions by authors who have been able to determine some of the not-publicly-available details of the methodology used in influential research studies published by other authors (see endnote 5).
Since the purpose of this type of article is to provide enough detail to scrutinize or replicate a method, authors should include any detail that required a decision about study protocol or analysis method. Though we have no set limit, we would expect any submission that contained enough detail to be more than 4000 words long. Details that should be reported include:
• What previously established study designs were used as a template for the study and why? What variations on that template were introduced, and why?
• What data analysis methods (specific statistical methods, choices of functional forms, subgroup analyses, etc.) were tried and what results did they produce? Why was a certain one chosen to produce "the" results of the study? If results have been published, what other data analyses were run but not reported, and why?
• Why were certain cutpoints chosen to categorize variables, define exclusion criteria, etc.?
• What design decisions, in retrospect, were suboptimal or led to notable problems? What changes in the originally conceived protocol were introduced and why?
• What, if anything, in the study design or analysis method is particularly noteworthy and recommended for use to future researchers?
To better conceptualize the goals of such publications, authors should think of themselves as trying to fulfill one or more of the following goals:
• Providing enough detail about data sources and analysis to allow readers to make better use of the study results. Such uses could include quantifying uncertainty about the study results (see, e.g., [10-13]), incorporating the results into meta-analysis or other summary analysis, or assessing the basic validity of the conclusions and the degree of "publication bias in situ" [7]. Typically, readers must guess at such details, dramatically diminishing the value of the study to the science.
• Presenting a case-study lesson for researchers who want to learn more about data gathering and analysis methods. Typically junior health researchers learn methods from one mentor or the overly-idealized presentations in textbooks, and even senior researchers have limited information about how others do things. There is a lot of room for better open exchange.
• Telling the story of how health science actually happens, as might be of interest to such chroniclers as historians of science, sociologists, and popular science writers.
Some partial solutions to the problem of diminutive methods sections already exist, including online methodology appendices and the reporting of a study's methods across multiple published articles. But these options have limitations and do not go as far as our proposed article type. Moreover, they do not properly reward authors with credit for a publication for the extra effort required to report their methods in a scientifically complete manner.
Conclusion
We are delighted to be able to provide this forum for scientific publishing and to be able to participate in the Open Access movement. We hope you will support this effort by submitting a paper to EP&I and submitting your reports of study results to other Open Access journals.
Endnotes
1. For a list of member institutions, see .
2. For more details on copyright and other policies, see the BioMed Central Open Access Charter, .
3. The value of carefully reported methods, rather than broad sketches, is exemplified by the huge number of choices that must be made about how data is analyzed and reported, and the dramatic effect those choices can have on the reported results [7].
4. We cannot resist pointing out a few observations: The length of the methods section of the praised article, which describes a field study and data analysis methods, comes out to about 70 words per author. The article's discussion section is almost twice as long as the methods, and though our journal encourages articles that are entirely "discussion" [6], this seems imbalanced for a report of study results. The editorial also went so far as to suggest that articles over 3000 words are "often boring." Regarding the latter point, we believe that readers of EP&I will find ample evidence to the contrary.
5. Such information might be inferred by replicating the results, examining regulatory or litigation testimony, or other methods. We encourage authors of such analyses to contact the original study authors to confirm or add to the analysis, and to invite them to be coauthors if they are willing to contribute substantially.
Competing interests
Parts of this editorial were adapted from a previous article (Slade E et al. Critical Care 2003, 7:331–332) that was suggested as a template by the publisher (used by permission).
Some critics of the author-pays publishing model have suggested that journals have a financial incentive to accept more articles to increase revenue, allowing authors to buy their way into publications. We believe that the quality of articles in EP&I speaks for itself, and makes it clear that our decisions to publish are based on the quality of the work. At this point, the entire article-publishing charge goes to the publisher, not to the editorial side of the journal (which makes decisions about what to publish). All scientific researchers and journal editors have motives outside some Platonic ideal of scientific publishing. These include the oft-cited financial interests, but perhaps more importantly also the bolstering of careers (publishing more articles thickens the CVs of authors, and increases the credit we get for being editors) and scientific politics (health researchers are very often trying to confirm something they believe to be true – regardless of who funded the study! – and the editors of this journal are trying to change how health research is done and reported). We continue to encourage authors to disclose competing interests, and to not mistake that phrase for "funding source".
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Int J Equity HealthInternational Journal for Equity in Health1475-9276BioMed Central London 1475-9276-4-71595525510.1186/1475-9276-4-7ResearchPerceptions of substance use, treatment options and training needs among Iranian primary care physicians Shakeshaft Anthony [email protected] Bijan [email protected] Carolyn [email protected] Kate A [email protected] Program of International Research and Training (PIRT), National Drug and Alcohol Research Centre (NDARC), University of New South Wales, Sydney, Australia2 3rd Floor, Building 19, Soori Alley, Moradi Alley, Bani-Hashemi St, Resalat Boulevard, Tehran, Iran3 National Health and Medical Research Centre Fellow, The National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, Australia2005 15 6 2005 4 7 7 11 10 2004 15 6 2005 Copyright © 2005 Shakeshaft et al; licensee BioMed Central Ltd.2005Shakeshaft et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In order to be optimally effective, continuing training programmes for health-care professionals need to be tailored so that they target specific knowledge deficits, both in terms of topic content and appropriate intervention strategies. A first step in designing tailored treatment programmes is to identify the characteristics of the relevant health-care professional group, their current levels of content and treatment knowledge, the estimated prevalence of drug and alcohol problems among their patients and their preferred options for receiving continuing education and training. This study reports the results of a survey of 53 primary care physicians working in Iran. The majority were male, had a mean age of 44 years and saw approximately 94 patients per week. In terms of their patients' drug use, primary care physicians thought most patients with a substance use problem were male, women were most likely to use tobacco (52%), opium (32%) and marijuana/hashish and young people were most likely to use tobacco, alcohol, marijuana and heroin. Counselling and nicotine patches were the treatments most commonly provided. Although the majority (55%) reported referring patients to other services, more than a third did not. Most primary care physicians reported being interested in attending further training on substance abuse issues. The implications of these data for ongoing education and training of primary care physicians in Iran are discussed.
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Background
The collection and interpretation of accurate descriptive data is an essential step in ultimately reducing the burden of harm associated with substance use [1,2]. Traditionally, these data have been sought to identify the characteristics of at-risk individuals or groups, quantify the size of the problem (incidence and prevalence) and, when repeated on the same population and using the same measures, monitor trends over time and assess the effects of interventions. More recently, these same principles have been applied to health care providers, as a mechanism for improving their quality of care [3]. As for at-risk individuals, accurate descriptive data identify the characteristics of health care providers and their knowledge gaps, and facilitate quality assurance monitoring of their practice over time.
Descriptive data also help optimise the cost-effectiveness of interventions in two ways. Firstly, at an individual level, they enable greater accuracy in tailoring interventions and treatment goals to specific problem behaviours [4]. Secondly, at a systems level, they provide an empirical rationale for the equitable distribution of intervention resources, a notion espoused by the World Health Organisation (WHO) as a key principle of accessibility to primary health care, in which core services available to all are complemented by interventions targeting those at increased risk [5].
As is typically the case internationally, the centrality of primary care in Iran is reflected in its role as the usual first point of contact with the formal health-care system. It is the principal juncture at which a range of health services can be mobilised and co-ordinated in order to manage complex health problems, such as substance abuse.
In assessing the potential to improve the quality and equity of primary health care for substance abuse problems in Iran, an initial step is to determine the characteristics of primary care practitioners, including their knowledge and perceptions of substance use, as well as identify their requirements for on-going education and training.
Therefore, this study has three aims. Firstly, to describe the demographic and practice characteristics of a sample of General Practitioners (GPs) working in Iran. Secondly, to identify their perceptions of substance use among their patients and their current practice regarding treatment. Thirdly, to identify Iranian GPs' interest in on-going education and training, their knowledge gaps and their preferences for the format of such training.
Method
Sample
GPs attending one of two mandatory training workshops in Tehran, Iran, were asked to complete a written questionnaire. Both workshops were held in the winter of 2003 and were attended by 50 GPs each. The topic of one workshop was an examination of psychiatry and medicine and the other focused on surgery. GPs throughout Iran travel to Tehran to attend these workshops, because they require ongoing accreditation to renew their licence to practice.
Measures
The pen-and-paper questionnaire was devised in English and translated into Farsi. Specific questions were asked in a number of domains, covering primary care practitioners' demographic and practice characteristics, and their perceptions of: substance use among their patients; substance use treatment characteristics; their current practice in regard to treatment for substance use; and their on-going training and education preferences. The full survey is available at the PIRT website .
Procedure
The questionnaire items were devised in conjunction with an Iranian GP (BN), who also distributed the questionnaires at the GP workshops. The completed questionnaires were photocopied and couriered to the research centre in Sydney, Australia. GPs' responses were translated back from Farsi to English, entered into an Excel spreadsheet and transferred to SPSS for Windows, Version 12, for analyses. Descriptive statistics report means, percentages and ranges as appropriate.
Results
Sample
Of the 100 GPs who attended one of the workshops, 53 completed the questionnaire.
GP demographic and practice characteristics
The majority of GPs were male (84%) with a mean age of 44 years (range 26 to 74 years). Their year of graduation from medical school ranged from 1950 to 2001 and the mean number of years practicing as a primary care physician was 16. The estimated mean number of patients seen per week was 94, of whom an estimated mean of 12 were substance users. Thirty eight percent of GPs reported seeing more than five substance using patients per week, while 2% said they have no substance using patients. GPs visited an estimated mean of 15 substance users per week, as opposed to seeing them in their primary care practice.
GPs' perceptions of substance use characteristics of their patients
Table 1 reports GPs' perceptions of substance use characteristics among their patients.
Table 1 GPs' perceptions of substance use characteristics of their patients (N = 53)
Characteristica % or n
Of your patients who have drug problems, what % are:
Male 81%
What is the cause of addiction?b
Emotional 49%
Social 45%
Family 35%
Economic 31%
Leisure 31%
Wrong role models 26%
In the last 5 years, have drug addiction trends increased, decreased, unchanged:
Increased 94%
In the last 5 years, drug abuse increased more for males or femalesc:
Females 42%
Males 38%
Same 18%
If increased, why?d
Social/family issues 22
Economic reasons 20
Unemployment 17
Easy access to drugs 17
Lack of recreation/sport facilities 10
Youth-related issues 9
Ineffective law enforcement/government action 6
Inadequate education 2
Other 10
What is your view of drug addicts?b
Psychologically ill 82%
Physically ill 22%
Burden on society 22%
Criminals 2%
Level of awareness about substance abusee
Moderate 54%
High 27%
aData missing: n = 2–5
bDoes not sum to 100%, because some GPs gave more than one answer and some gave no answer
cDon't know = 2%
dDoes not sum to 53, because some GPs gave more than one answer and same gave no answer
eAwareness rated on Likert scale from 1 (very unaware) – 10 (very aware): low = 1–3; moderate = 4–8; high = 8–10
Most GPs viewed substance use problems as a psychological issue (82%) and that most patients with a substance use problem are male (81%). A substantial percentage of GPs reported moderate (54%) or high (27%) awareness of substance abuse issues. Some GPs thought emotional (49%) or social (45%) factors were the cause of addiction. Almost all GPs (94%) thought drug addiction trends in Iran have increased in the previous five years and a comparable percentage thought the increase had been greater for females (42%) compared to males (38%). The most commonly cited reasons for increased drug addiction were social or family issues (n = 22), followed by economic factors (n = 20).
GPs' perceptions of substance use characteristics by sex and age
The majority of GPs perceived that women were most likely to use tobacco (52%), opium (32%), marijuana/hashish (32%) and heroin (14%). The majority of GPs thought young people (aged less than 25 years) were willing to use tobacco (97%), alcohol (63%), marijuana (56%) and heroin (53%), but were relatively unlikely to use black water (the dissolved residue from an opium pipe) (7%) and barbiturates (7%). The majority of GPs thought people aged 25 to 44 years were willing to use opium (84%), alcohol (61%) and heroin (52%), while those aged at least 45 years, were most willing to use opium (60%), followed by black water (28%) and alcohol (22%).
GPs' perceptions of patient and treatment characteristics associated with interventions for substance use
Table 2 reports GPs' perceptions of patient and treatment characteristics associated with interventions for substance use.
Table 2 GPs' perceptions of patient and treatment characteristics associated with interventions for substance use (N = 53)
Characteristica % or n
Who is more willing to quit?
Males 62%
Don't know 11%
Target groups for campaigns against abuse?
Youth ≤ 25 years 68%
Urban 5%
Users and drug and alcohol organisations 8%
Jails 3%
Other 16%
What is the most important motive for quitting?b
Family or marital pressure 60%
Economic pressure 44%
Tired of addiction 42%
Legal issues 22%
Employment issues 12%
Physical illness 4%
Other 4%
What is a suitable substitute for substance use?c
Drug or other treatment 18
Societal, family or community factors 16
Recreational/sporting activities 11
Employment 10
Restricted access or punitive (eg. jail) 3
Nothing or don't know 7
Most effective methods for quitting nicotine?c
Counselling or psycho-social interventions 22
Recreational or sporting activities 9
Pharmacotherapy 7
Combination of therapies 3
Alternative therapies 1
Individual's determination to quit 2
Don't know 3
Important side-effects of quitting nicotine?c
Restlessness/irritability 18
Physical symptoms/effects 13
Emotional symptoms/effects 7
Non-specified withdrawal symptoms 4
None 7
Effectiveness of complementary medicines for nicotine dependence?c
Not effective/minimal 10
Somewhat effective 12
Effective 6
Other 6
Don't know 12
Suitability of GP to provide drug services
Suitable 71%
aData missing: n = 3–12
bDoes not sum to 100%, because these questions asked separately
cDoes not sum to 53, because some GPs gave more than one answer and some gave no answer
The majority of GPs thought males were more willing to quit substance use (62%) and that those aged 25 years or less were the most appropriate group at which to target campaigns against substance misuse (68%). The majority of GPs thought family or marital pressure was the main motive for quitting substance use (60%), followed by economic pressure (44%), being tired of addiction (42%) and legal problems (22%). The most commonly cited suitable substitute for drug use was some type of treatment (n = 18). With regard to nicotine, the most commonly cited effective method for quitting use was counselling or psycho-social intervention (n = 22), while the most commonly cited important side-effect was restlessness or irritability (n = 18) and relatively few (n = 6) regarded complementary approaches, such as hypnotherapy and chiropractic therapy, as effective. The majority of GPs (71%) thought primary care was a suitable setting for delivery of services to patients with substance use problems.
GPs' current practice in regard to treatment for substance misuse
Table 3 reports GPs' current practice.
Table 3 GPs' current practice in regard to treatment for substance misuse (N = 53)
Current treatment practicesa % or n
I conduct diagnostic physiological assessments
No 75%
Blood, heart, lung test 18%
Blood test only 4%
Spirometry 4%
I prescribe medications for detoxification
No 61%
Treatments I commonly use areb:
Counselling or psychotherapy 20
Nicotine patch 11
Non-specified medication 8
Personal/family responsibility 4
Alternative therapy 3
Other 7
Don't know 4
I provide the following relapse informationb:
Avoid or reduce contact with drug users 8
Psychological/personal determination factors most important 7
Family/friends/social support important 6
Recommend counselling or pharmacotherapy 6
No relapse problems 2
Don't know 2
Other 10
I refer to social workers or detoxification units
No 39%
Yes, some patients 43%
Yes, all patients 7%
Yes, if patient is willing 5%
Not applicable 7%
aData missing: n = 7–25
bDoes not sum to 53, because some GPs gave more than one answer and some gave no answer
The majority of GPs reported that they did not conduct diagnostic physiological assessments (75%) and that they did not prescribe medications for detoxification (61%). The most commonly provided treatments reported were counselling or psychotherapy (n = 20), followed by nicotine patches (n = 11). The most commonly provided relapse information was to avoid or reduce contact with other drug users (n = 8). The majority of GPs said that they referred patients to a social worker or detoxification unit (55%), while a substantial percentage reported not referring at all (39%).
GPs' on-going training and education preferences
The majority of GPs reported that they were interested in attending seminars and further training related to substance abuse issues (89%) and the most preferred format for such training was two days over a two week period (54%), followed by two days in one week (25%) and two consecutive days (13%). Six per cent reported no preference.
Discussion
The potential to improve the quality and equity of primary health care for substance abuse problems in Iran, is clearly evidenced by the low proportion of GPs (27%) who currently rate their level of awareness of substance abuse issues as high, despite having been practicing as GPs for a mean of 16 years. This apparent lack of knowledge and skills regarding substance abuse issues is consistent with views of GPs in Western countries [6,7]. The appropriateness of on-going education and training workshops as a mechanism to realise this potential for improvement is reflected by the high proportion of GPs (89%) who indicated an interest in attending such seminars.
The results of this survey also identify potential topics for these workshops that are most likely to maximise their impact. That a minority of GPs (22%) consider patients with substance use problems as being physically ill (Table 1) and do not regard physical illness as an important motive for quitting substance use (4%), suggests a teaching focus on the development of physiological dependence following initiation to drug use would improve GPs' understanding of the nature and impact of physiological dependence. In turn, this may encourage the high proportion of GPs who do not currently prescribe medication for detoxification (61%) or conduct physiological assessments (75%), to review their practice (Table 3).
Similarly, a greater number of GPs may be more inclined to discuss problems associated with relapse to drug use, complemented by monitored pharmacotherapy, or on-going counselling (Table 3). Although a lack of resources may help explain a reluctance to prescribe medication and conduct physiological assessments, GP training could emphasise the need to address issues around physical dependence, as well as the social/familial/economic impacts, along with basic physiological tests that could be conducted periodically. GPs would likely find this approach acceptable, given some cited drug substitution as a suitable treatment and reported currently prescribing nicotine patches (Table 3). Such a change in Iranian GPs' practice would also appropriately reflect the apparent improved rates of quitting smoking, compared to monotherapy or unassisted attempts to quit, in the international literature [8-11].
GPs are clearly aware of the importance of prevention, as evidenced by two-thirds thinking that campaigns against drug abuse would most effectively target young people, presumably prior to the onset of significant dependence. This suggests GPs would be prepared to become involved in preventive activities, in addition to their more traditional role of providing treatment. Therefore, training programmes might also usefully provide GPs with resources and ideas that they can implement to facilitate effective prevention. For example, cost-effective GP prevention efforts would target defined groups with high prevalence rates of use for particular substances: young people aged less than 25 years could be targeted with programs aimed at minimising harm associated with smoking, alcohol and marijuana use, given the majority of GPs perceive that young people are relatively willing to use tobacco (97%), alcohol (63%) and marijuana or hashish (56%). A particular area of concern, given disproportionately greater probability of substantial negative social and health outcomes, is that 53% of GPs perceive young people are willing to use heroin.
Another example may be the apparent importance of targeting womens' use of tobacco, opium and marijuana or hashish, given 52%, 32% and 32% of GPs' respectively, perceive that these are the substances most likely to be used by women. As such, GPs' consultations with their female patients could emphasise the importance of not smoking any substances to reduce the possibility of deleterious health outcomes.
In addition to the potential to improve the quality and equity of primary health care for substance abuse problems in Iran at the GP level, there also appear to be opportunities at the administrative level. For example, there may be value in the Health Department exploring opportunities for formalising referral networks, in order to achieve clarity around pathways from primary care settings to more specialist clinical settings, given only about one-third of GPs (39%) report referring to social workers or detoxification units. There may also be scope to make pharmacotherapies of demonstrated cost-effectiveness more readily available to GPs to prescribe, along with greater access to relevant physiological tests. Clearly, the feasibility of these options needs to be considered within the context of the Iranian health care system.
The generalisability of these results to the population of primary care practitioners in Iran is likely to be limited by two main factors. Firstly, only a small proportion of GPs in Iran participated in this study. However, an attempt was made to minimise this limitation by ensuring that the workshops from which these practitioners were recruited was unrelated to substance use and, as such, the views of these practitioners are likely to represent typical levels of interest and knowledge in drug- and alcohol-related issues. The likelihood that these GPs are typical of Iranian GPs is further indicated by the mandatory nature of the workshops: that they are compulsory for ongoing professional accreditation in Iran decreases the possibility that workshops are only attended by a self-selected sub-group of GPs.
Secondly, since only 53% of those who attended a workshop completed a questionnaire, it may be that those with an interest in the drug and alcohol field, or those who see a relatively large proportion of patients with substance misuse problems, were more likely to complete the survey. The implication of that scenario is that these results are likely to represent a bias towards GPs with greater knowledge or experience in providing health care services to patients with drug and alcohol problems, in which case identified knowledge deficits and training needs would actually be greater among the general population of GPs in Iran.
Future international collaborative research efforts could be improved in a number of ways. Firstly, the process of translation and back translation is susceptible to error and is difficult to monitor accurately. A possible method of reducing translation errors could be to employ translators with substantive knowledge in the drug and alcohol field. Secondly, working with one local researcher to apply a standardised and agreed protocol for distributing the survey, would minimise the possibility of bias resulting from different people administering the survey in different ways. Thirdly, clarity regarding definitions of key terms during the development of survey items and protocols is crucial. Using the example of this study, terms such as 'drug addict' and 'complementary medicine' should be clearly defined.
Competing interests
The author(s) declare that they have no competing interests.
Acknowledgements
This study was primarily supported by a grant from the Commonwealth Department of Health and Aged Care, through the Program of International Research and Training (PIRT) at the National Drug and Alcohol Research Centre (NDARC), University of NSW. The authors acknowledge the valuable assistance provided by the Iranian GPs in responding to the survey and Ms Navid Nabipour for translations and data entry.
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| 15955255 | PMC1164425 | CC BY | 2021-01-04 16:39:31 | no | Int J Equity Health. 2005 Jun 15; 4:7 | utf-8 | Int J Equity Health | 2,005 | 10.1186/1475-9276-4-7 | oa_comm |
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Harm Reduct JHarm Reduction Journal1477-7517BioMed Central London 1477-7517-2-71593510310.1186/1477-7517-2-7ResearchDrug use and risk behaviours among injecting drug users: a comparison between sex workers and non-sex workers in Sydney, Australia Roxburgh Amanda [email protected] Louisa [email protected] Courtney [email protected] National Drug and Alcohol Research Centre University of New South Wales, Sydney, NSW 2052, Australia2005 6 6 2005 2 7 7 4 2 2005 6 6 2005 Copyright © 2005 Roxburgh et al; licensee BioMed Central Ltd.2005Roxburgh et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
This paper examines the differences in demographics, drug use patterns and self reported risk behaviours between regular injecting drug users (IDU) who report engaging in sex work for money or drugs and regular injecting drug users who do not.
Methods
Cross sectional data collected from regular IDU interviewed as part of the New South Wales (NSW) Illicit Drug Reporting System (IDRS) in 2003 were analysed.
Results
IDU who reported engaging in sex work were more likely to be female, and identify as being of Aboriginal and/or Torres Strait Islander descent. They initiated injecting drug use at a significantly younger age and were more likely to report injection related problems than IDU who had not engaged in sex work. There were no differences in the drug classes used, but findings suggested that the sex workers tended to be more frequent users of crystalline methamphetamine (ice) and benzodiazepines.
Conclusion
The similarities between these groups were more striking than the differences. Further research, examining a larger sample is needed to clarify whether injecting drug users who are sex workers have heavier use patterns.
sex workers/prostitutespatterns of drug userisk taking behaviourinjecting drug users
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Background
The last two decades have seen an increasing interest in the study of sex workers as a marginalised group at increased risk for poorer mental and physical health outcomes, inequitable access to housing and the problematic use of illicit drugs [1]. Previous research has documented the risks of blood borne virus (BBV) transmission and sexually transmitted infections among sex workers due to unprotected sex with clients [2], the relatively high rates of HIV among sex workers in some countries, and the potential risks posed to the broader community via BBV transmission through clients to the general population [3]. It should be noted that HIV prevalence among sex workers differs in Asian countries compared to North America and Europe. In the latter countries research has shown that HIV prevalence is no different among IDU and sex workers who are IDU [4], and that HIV is more prevalent among sex workers who are IDU compared to sex workers who are not IDU [5], indicative that it is injecting drug use that puts these groups at risk of HIV. In countries such as Africa, HIV infection is largely associated with heterosexual activities [6].
The literature suggests that sex workers are disadvantaged across a number of domains. One study that examined mental health status among a group of Puerto Rican sex workers [7] found that the overwhelming majority (91%) reported a high rate of depressive symptoms (measured using the Centre for Epidemiologic Studies Depression Scale). The authors also found that street sex workers reached significantly higher levels of depressive symptoms (86%) than brothel workers (45%). Approximately half of the sample (47%) reported injecting drug use, and a significantly higher proportion of those injecting drugs (90%) reported high levels of depressive symptoms compared to non injecting drug users (52%).
Another study comparing street sex workers and non street sex workers in Sydney, Australia [8] found that street sex workers were; predominantly female, significantly more likely to identify as being of Aboriginal and/or Torres Strait Islander descent (20%), and to be currently injecting drugs (77%) than those working `indoors' (7%). Street sex workers also had high rates of Hepatitis C (71%), possibly indicative of their injecting drug use. Interestingly, a significantly higher proportion of `indoor' sex workers (48%) reported alcohol use than street sex workers (29%). Approximately one quarter of the street sex workers had no permanent accommodation, and a similar proportion (27%) reported no supportive relationships.
In contrast to these findings, a study comparing sex workers and non sex workers in New Zealand [9] found no differences between the groups across domains such as access to accommodation, level of social support, and mental and physical health. However a significantly higher proportion of sex workers (76%) reported tobacco use than non sex workers (29%) and sex workers also reported higher consumption of alcohol (58% reported drinking more than 5 standard drinks per occasion of use compared to 23% of non sex workers). The absence of differences between sex workers and non sex workers in this study may be attributed to the fact that only 2 of the sex workers sampled were street workers. Previous studies suggest that street sex workers are a more marginalised group than non street sex workers, and if the sample contained a greater number of street sex workers, the authors may have found more significant differences on a range of variables.
These patterns are also evident among male sex workers. An Australian study sampled male sex workers in three cities (Melbourne, Sydney and Brisbane) to document their characteristics [10]. When street sex workers were compared with non street sex workers, they were less educated, more likely to report financial problems, less likely to be tested for blood borne viruses and sexually transmitted infections, and were higher drug users than non street sex workers.
These differences are also evident in the U.K, with studies showing a higher prevalence of injecting drug use and more problematic drug use, among street sex workers compared to non street sex workers. There is also evidence in the U.K. literature of women moving to street based sex work from indoor markets due to problematic drug use [11].
Research reviewed (e.g. [7]) indicates that drug use is an important predictor for poorer outcomes for sex workers, which has generated an interest in the role of drug use, and drug use patterns among this group. An ethnographic study of women in New York who engaged in sex work [12] found that drug use played a substantial role in the way these women conducted their sex work. Crack cocaine had a particularly deleterious effect on sex workers as it was thought to lead to lowering of the price of sex work exchanges, engendering a more hostile environment among sex workers and more violent exchanges with clients, and an increased potential for high risk sexual encounters. Many of the women Maher interviewed also used crack in order to facilitate their engagement in sex work.
One study of a group of cocaine `dependent' sex workers in the United States [13] found that two thirds of the sex workers came from ethnic minority groups, two thirds had completed less than 12 years of education, and a fifth were homeless. Another U.S. study, investigating "crack" cocaine smoking sex workers [14] mirrored these results, as did an Australian study [8] in which 20% of street sex workers identified as Aboriginal and/or Torres Strait Islanders and a fifth had no permanent accommodation.
The risks faced by sex workers are further compounded by drug use, with studies documenting associations between sex workers' drug use and the poorer safety outcome of the sex encounter (e.g. [15]), and risk of BBV transmission due to injecting drug use and sharing of needles [2]. Sex workers are a group characterised by high levels of drug dependence and those who inject drugs may be at greater risk on a multitude of factors than sex workers who do not. A study of 51 female sex workers in London who were current drug users [2] found that the majority of women using heroin (88%) were daily users, and many reported high levels of dependence in accordance with the Severity of Dependence Scale (SDS). The majority of IDU sex workers (75%) had used injecting equipment after someone else. However, the sharing of injecting equipment was related to severity of dependence on heroin rather than sex worker status per se. This finding suggests that dependent drug use may be a key factor for engaging in risk behaviours rather than sex work.
Comparative research has been conducted in Australia examining drug use among sex workers and various other groups, including women from community health services [16] and women from general population surveys [17]. Findings suggest that female sex workers have higher rates of illicit drug use [16], heavier use of alcohol and tobacco [9,16,17] and higher rates of sharing injecting equipment [17] compared to women from the general community. While these findings provide some insight into drug use patterns among each group, they have tended to sample non sex workers from populations that are likely to be quite different across a number of domains than sex workers, therefore limiting the validity of comparisons and conclusions about the risks that involvement in sex work may carry.
One U.S. study has examined similar groups. Logan, Leukefeld and Farabee [18] investigated the differences between female crack users according to whether or not they engaged in sex work, and found that both groups were just as likely to be African American, to be unemployed, to have similar educational backgrounds, and similar drug use patterns. However, women engaging in sex work were likely to have less access to accommodation, more frequent contact with the criminal justice system, earlier initiation of alcohol and cocaine use and higher rates of injecting drug use than non sex workers. In summary, while these groups were similar in some respects, there were also important differences, indicating that among an already marginalised group (i.e. crack users) sex workers are more likely to be even more marginalised than their non sex worker counterparts.
Investigating differences between sex workers and non sex workers among injecting drug users (IDU) may provide new insight into whether sex work status is likely to increase the risks among an already marginalised group. To the best of the authors' knowledge there has been no research conducted in Australia among IDU to determine whether there are differences between sex workers and non sex workers with regard to drug use patterns and risk behaviours. Previous research in Australia has focused on sex workers and their drug use patterns without comparison data. Australian findings are likely to differ from studies conducted in America, as there is little, if any, crack use in this country [19], and heroin is the most commonly injected drug among sentinel groups of regular IDU, particularly in Sydney [20,21].
The current study aims to examine whether regular injecting drug users who engage in sex work are at greater risk for adverse outcomes (such as homelessness and poor mental health), are more likely to engage in risky behaviours (needle sharing, criminal activity), and have different drug use patterns than injecting drug users who do not engage in sex work. Data are drawn from the Illicit Drug Reporting System (IDRS), in which sentinel groups of regular IDU are sampled annually.
Aims
1. to document the proportion among a sentinel group of regular IDU who report engaging in sex work for money and/or drugs;
2. to compare the demographics of this group with regular IDU who do not report sex work;
3. to examine and compare the drug use patterns of these groups;
4. to consider and compare self-reported risk behaviours in these groups.
Method
This paper used cross-sectional survey data collected in 2003 on a sentinel population of regular IDU regarding their drug use history, patterns of use, risk taking behaviours and drug-related harms. Data were from the NSW Illicit Drug Reporting System (IDRS). The IDRS is conducted annually in June using the same methodology, and provides sensitive data on trends and changes in drug use over time [22].
Participants were recruited through Needle and Syringe Programs (NSPs) in Sydney, NSW. NSP sites were chosen due to their proximity to street based illicit drug markets, as these markets are likely to attract regular IDU. Interviewers were positioned in the waiting area of the NSP, and clients were asked if they were interested in participating in a confidential survey being conducted by the University of New South Wales. Although some clients declined to be interviewed, refusal to participate did not present as a major issue. Participants received reimbursement of $30 for travelling expenses. Eligibility criteria for entry into the study were: (i) at least monthly injection in the six months preceding the interview; and (ii) residence in Sydney for twelve months preceding the interview, with no significant periods of incarceration or residence in inpatient rehabilitation programs. One hundred and fifty four regular IDU were eligible to participate in the New South Wales IDRS in 2003. Prior to commencing the interview, each participant was provided with information about the study as well as an assurance of confidentiality. Once the participant provided written consent for involvement in the study, a structured interview of approximately 45 minutes duration was conducted. No identifying data was collected at any time throughout the interview or recorded on the questionnaire. Responses were coded according to closed data fields on the interview schedule. IDU sampled for the IDRS are not intended to be representative of all IDU, but do provide important information about patterns of illicit drug use among IDU who are actively engaged in illicit drug markets, a group that we wished to examine in this study.
IDU who reported current engagement in sex work for money and or drugs are classified as sex workers for the purpose of this paper.
Statistical Analyses
Differences in demographics and drug preference were analysed using chi square statistics. Differences in age of initiation into injecting drug use were analysed using the t test statistic. Mann Whitney tests were employed to analyse differences in drug use patterns (i.e. median days of use in the preceding six months) and expenditure on drugs.
Limitations
The results of the current study should be interpreted as indicative of certain trends, given the relatively small number of sex workers sampled. Further research in Australia, examining issues raised in this study, needs to be conducted among larger groups of sex workers for more definitive results. Findings should also be interpreted within the context of street based sex workers, who differ from commercial sex workers in several domains [7,8,10]. While this limits the generalisability of findings to other sex workers, sampling street based sex workers serves the aims of this study well; many street based sex work markets function as an adjunct to illicit drug markets [12], with street based sex workers operating within close proximity to street based drug markets.
Results
Demographic Characteristics
The demographic characteristics of IDU according to sex work status are presented in Table 1. A total of 22 participants identified as having performed sex work for money and/or drugs in the month preceding interview. This represented 14% of the total IDU sample interviewed. This proportion was similar to those in previous years: 15% in 2002 and 7% in 2001 reported sex work as their main source of employment in the month preceding interview.
Table 1 Demographics of IDU by sex work status
Sex workers % (n = 22) Non-sex workers % (n = 132)
female 77 23
transgender 0 0
Age (M) 32 33
Years education (M) 8.9 9.7
ATSI 59** 28
not engaged in other employment 96 85
homeless 5 12
prison history 64 68
in drug treatment past 6 months 68 66
currently in drug treatment 45 47
criminal activity main income past month 0 28
**significant at p < 0.01
Among those in the 2003 sample who identified as engaging in sex work, 5 were male. The average age of sex workers (SW) was 32 years old (comparable to non sex workers (N-SW) who were, on average, 33 years old), and SW had completed an average of 8.9 years of education (compared to 9.7 years for N-SW). Sex workers were significantly more likely to identify as being of Aboriginal and/or Torres Strait Islander descent compared to N-SW (Table 1) (χ2 = 6.94, df = 1, p < 0.01).
Ninety six percent of SW reported that they were not engaged in any other form of employment compared to 85% of N-SW. There were no significant differences between the two groups in the likelihood of reporting a prison history, participation in drug treatment or homelessness.
Drug use history
The mean age of initiation into injecting drug use was significantly younger for SW (17.6 years) than N-SW (20.3 years) (t = 2.035, df = 152, p < 0.05). There were similarities between the groups with regard to the first drug they injected: heroin was the most common first drug injected, followed by methamphetamine. There was no difference in the mean number of drug classes SW and N-SW reported ever using (Table 2).
Table 2 Drug use history & current drug use of IDU by sex work status
Sex workers % (n = 22) Non-sex workers % (n = 132)
Age first injected (M) 17.6* 20.3
% heroin first injected 59 63
% amphetamines first injected 41 33
No. drug classes ever used (M) 10.6 10.1
% heroin drug of choice 96 83
% heroin injected most in past month 87 83
Daily or more injecting past month 82 65
Heroin
injected last 6 months 100 96
Median days injected 175 170
Cocaine
injected last 6 months 50 48
Median days injected 6 5
Methamphetamine (ice)
injected last 6 months 36 35
Median days injected 36 5
Methamphetamine (speed)
injected last 6 months 36 29
Median days injected 2 3.5
Benzodiazepines
injected last 6 months 18 19
Median days injected 90 12
Alcohol
used last 6 months 68 68
Median days used 24 18
No. drug classes used last 6 months (M) 7 6.5
Spent money on drugs yesterday 100* 77
Median amount spent on drugs yesterday $145** $100
*significant at p < 0.05
**significant at p < 0.01
Current drug use
All but one of the SW reported heroin as their drug of choice (the remaining sex worker nominated benzodiazepines); among N-SW the majority reported heroin as their drug of choice, with smaller proportions nominating methamphetamine, cocaine, cannabis, and benzodiazepines. Heroin was reported as the drug most frequently injected in the month preceding interview among both SW and N-SW. Heroin was also most commonly reported as the last drug injected by both groups. There were no significant differences between proportions reporting injecting at least daily in the preceding month (Table 2).
Table 2 shows the classes of drugs used in the six months preceding interview and frequency of use during this period. Patterns of drug use among SW and N-SW were similar for most drugs with a few notable exceptions. Similar proportions reported using crystalline methamphetamine (ice) in the preceding six months, however SW reported using it on a median of 36 days compared with 5 days among N-SW. Likewise, although similar proportions reported intravenous benzodiazepine use in the preceding six months, SW reported a median of 90 days injecting compared to 12 days among N-SW. There was no difference in the mean number of drug classes SW and N-SW reported using in the preceding six months.
Sex workers were significantly more likely than N-SW to have spent money on drugs on the day preceding interview (χ2 = 4.84, df = 1, p < 0.05), and to have spent significantly more on that day than N-SW (Table 2) (Mann-Whitney = 800, p < 0.01).
Risk behaviours
Larger proportions of SW than N-SW reported borrowing used needles after someone else had already used them in the month preceding interview, and larger proportions had lent needles to others after they had used them. These differences were not significant. There was no difference in proportions sharing other injecting equipment in the preceding month. Likewise, there were no differences between proportions reporting last injecting in a public place, and usually injecting in a public place in the month preceding interview (Table 3).
Table 3 Self-reported risk behaviours & problems among IDU by sex work status
Sex workers % (n = 22) Non-sex workers % (n = 132)
borrowed needles in past month 14 5
lent needles in past month 23 11
last injected in public place in past month 36 26
usually injected in public place in past month 23 28
injection related problems past month 86* 55
attended mental health professional past 6 months 32 25
property crime in past month 32 31
drug dealing in past month 36 36
arrested in past 12 months 50 49
*significant at p < 0.05
Sex workers were significantly more likely to report injection related problems than N-SW (χ2 = 6.32, df = 1, p < 0.05), with the most common injection related problems reported among SW being prominent scarring and bruising and difficulty injecting.
There were no differences between proportions of SW and N-SW reporting attending a mental health professional for mental health problems other than drug dependence in the preceding six months. Nor were there differences in proportions reporting engaging in property crime or drug dealing in the preceding month, or being arrested in the previous twelve months (Table 3).
Discussion
This paper examined whether regular IDU who reported engaging in sex work were different from those who did not. Sex workers were more likely to identify as being of Aboriginal and/or Torres Strait Islander descent than non-sex workers, and this is consistent with previous research that has found that women who come from socially and economically disadvantaged ethnic minorities are over represented among sex workers [8,13,14], and also over represented among Australian injecting drug users [21,23-25]. These findings raise several implications for both health and drug treatment agencies, as well as for future research. Firstly, agencies providing health services for SW may need to consider tailoring programs to the needs of individuals who identify as ATSI, which could involve ATSI liaison personnel as part of outreach teams and services provided on site. Drug treatment programs also need to be more relevant for this population, as while ATSI are over represented among Australian IDU, they are under represented among IDU accessing drug treatment services [26]. There is general agreement among researchers that there is remarkably little published information available on ATSI IDU [27], and a paucity of research on what constitutes "culturally appropriate" treatment interventions for these populations [28]. Further research, establishing why so few ATSI IDU utilise available treatment programs, and identification of potential barriers for this group, is warranted in order to develop relevant programs that would encourage attendance.
There were no significant differences in drug types used by SW and N-SW, however SW initiated injecting drug use at a younger age. Again, these results are consistent with the literature that suggests that earlier age of initiation has been associated with a range of adverse outcomes later in life. Evidence suggests that those who have begun substance use by an early age are more likely to develop problematic substance use [29-32], engage in risky sexual behaviour [31,33], become involved in criminal activity [31], and complete fewer years of education [34]. Earlier initiates to substance use are also more likely to become more dependent, use for a longer time and have more drug-related problems. [35-40]. In the current study, among an already marginalised group of regular IDU, earlier initiation to drug use appeared to be associated with an additional risky behaviour – sex work. Research in Australia has illustrated these risks, with sex workers (particularly those who are street based) being more vulnerable to adverse contact with law enforcement, subject to physical assault, rape, kidnap, and being threatened with a weapon ([8,10,41,42]).
Earlier initiation of injecting drug use among this group indicates the need for greater emphasis on early intervention, in order to reduce the likelihood of young people entering sex work and/or developing problematic drug use [11]. For maximum effect, interventions should target several groups at different stages. Considerable research and public interest has been focused upon ways in which substance use among young people may be reduced, and to encourage those who have begun use at an early age to cease or moderate their use. Interventions have involved primary prevention (for example, drug education in schools or general population campaigns) ([43-45]); secondary interventions (such as targeted programs aimed at "at-risk" children) ([46]); and tertiary interventions (most often involving treatment for young persons who have developed problematic use, or interventions designed to reduce the initiation of injecting) ([47-50]).
Patterns of drug use were similar among both SW and N-SW, however results were indicative of heavier use of particular drugs among SW (i.e. ice and benzodiazepines). Due to small numbers of SW reporting recent benzodiazepine injection (n = 4) and ice use (n = 8), findings did not reach significance, and a larger sample size may highlight these trends more clearly. Trends of heavier drug use among SW are consistent with the literature documenting high levels of drug dependence in these groups [2]. The authors (AR and LD) are currently undertaking a study investigating a range of issues (including drug use patterns) among female street based sex workers and results should provide more definitive trends with regard to drug use in this group
Sex workers were more likely to have spent money, and to have spent more money on drugs on the previous day than N-SW, and this is most likely to be due to SW having more disposable income available to them than N-SW. However, it may also be an indicator of heavier drug use.
There were no differences between proportions of SW and N-SW reporting borrowing and lending used needles. Consistent with previous research [2,51], what seemed to be more indicative of the likelihood of borrowing needles was frequency of heroin use; 70% of IDU in the current study (regardless of sex work status) who reported borrowing needles were daily heroin users. People who had used the needle before them were reported as partners or close friends. Likewise, 74% of IDU (regardless of sex work status) who reported lending needles had used heroin on every third day or more (range 72–180 days) in the preceding six months (47% were daily users). These findings suggest that high levels of drug use may play a more important role in decisions to engage in risk taking behaviours than sex work does.
Sex workers were more likely to report injection related problems than N-SW however, given that the majority of SW were female, this finding may be more indicative of gender differences than sex work status per se. A paper describing the characteristics of clients attending the Medically Supervised Injecting Centre (MSIC) in Sydney [52] reported that females were twice as likely to report injection related problems than males, a finding that is consistent with other studies [1,8]. This finding is indicative of the need to ensure that these women have access to primary treatment services, and that there are no barriers to such services. Continued education campaigns, outlining strategies to minimise injection related harms also remain a priority.
Overall, these results suggest that the differences among injecting drug users who are sex workers and those who are not, are less striking than the similarities. Drug patterns were generally no different between these groups however, there was some indication of heavier use of crystalline methamphetamine and benzodiazepines among sex workers, and future research examining a larger sample is needed. Risk behaviours and poorer injection related outcomes appeared to be associated with factors other than sex work status (such as frequency of drug use and gender), perhaps suggesting that overall, injecting drug users who are sex workers may be at no greater risk of adverse outcomes (with the exception of the risks involved in street based sex work) than those who are not sex workers. It should be noted however, that this study did not examine condom use among injecting drug users, or the relationship between drug use and the safe outcome of sexual encounters, and these are undoubtedly issues of relevance for injecting drug users who engage in sex work.
Conclusion
Few differences were found in the current sample of regular IDU who engaged in sex work compared with those who did not. There are however, several policy implications arising from differences that were apparent. Firstly, there needs to be an increased focus on more specific programs targeting SW who identify as ATSI, as well as further research into more culturally appropriate drug treatment services for this group. Second, greater emphasis needs to be placed on the continued development of primary, secondary and tertiary intervention programs targeting young people in a range of settings. There are some excellent programs currently available in Australia, and inclusion of more specific education regarding the risks involved in sex work as well as exploration of alternative employment opportunities would prove useful. Access to primary treatment settings for SW who are IDU is also important given the range of injection related problems they experience. Finally, due to relatively small numbers of IDU in this sample engaging in sex work, further research is required. In response, the National Drug and Alcohol Research Centre is currently undertaking research to assess a range of issues among female street based sex workers in Sydney. Education that targets safe sex practices among sex workers should remain a priority, given the high rate of problems encountered among this group, and the risks they face due to contact with multiple sex partners.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
AR was involved in collecting the data, performing statistical analysis, conducting a detailed literature review and drafting the manuscript. LD suggested the idea for the study and provided detailed structural comment on, and assistance with drafting the manuscript. CB was involved in collecting the data, providing detailed comment on the content, and assistance with drafting the manuscript. All authors read and approved the final manuscript.
Acknowledgements
The Illicit Drug Reporting System is funded by the Australian Government Department of Health and Ageing and the National Drug Law Enforcement Research Fund (NDLERF).
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| 15935103 | PMC1164426 | CC BY | 2021-01-04 16:36:50 | no | Harm Reduct J. 2005 Jun 6; 2:7 | utf-8 | Harm Reduct J | 2,005 | 10.1186/1477-7517-2-7 | oa_comm |
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Int Semin Surg OncolInternational seminars in surgical oncology : ISSO1477-7800BioMed Central London 1477-7800-2-111590452910.1186/1477-7800-2-11ResearchBreast weight and hormone receptor status in women with breast cancer Salhab M [email protected] Sarakbi W [email protected] K [email protected] St George's and The Princess Grace Hospitals, London, United Kingdom2005 16 5 2005 2 11 11 3 5 2005 16 5 2005 Copyright © 2005 Salhab et al; licensee BioMed Central Ltd.2005Salhab et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Breast cancerpostmenopausalreceptorsbreast weight and aromatase
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Abstract
Introduction
Aromatase activity in peripheral tissues including the breast is the main source of estrogen in postmenopausal women. There is evidence that local estrogen synthesis by breast aromatase contributes to mammary carcinogenesis. Therefore, we have postulated that high breast weight is associated with ER+ tumours.
Patients and methods
The mastectomy specimen weight, ER and PgR status for 62 consecutive patients who had a total mastectomy for operable breast cancer were retrospectively reviewed. The ER/PgR positivity was assessed using immunohistochemistry (Quickscore system 0–8) by a breast pathologist. ER/PgR status was considered positive if the score was 4 – 8.
Results
Overall the breast weight was higher in patients with ER+ disease. The weight was found to be significantly higher in women aged 50 years or older with ER+ tumours (669 vs. 220 grams, p = 0.015). There was no significant difference in breast weight between ER+ and ER- tumours in women aged less than 50 years (median weight: 440 vs.408 grams, p = 0.379). We observed a non-significant association between higher breast weight and PgR positivity (809 vs. 510 grams, p = 0.084) and absence of c-erb B2 (p = 0.088).
Conclusion
In women aged 50 years or older with breast cancer, high breast weight is significantly associated with ER+ tumours. If this is confirmed in larger prospective studies, our findings may have implications regarding breast cancer prevention with anti-estrogens.
Introduction
Breast cancer remains the most common cancer in females. It has been estimated that one in eight women will develop breast cancer during their lifetime in the USA [1]
Approximately two thirds of postmenopausal breast cancer patients have hormone dependent breast cancer that requires estrogen for tumour growth. It is well established that estrogens enhance growth and proliferation of certain target cells such as breast epithelial cells and estrogen dependent mammary carcinoma cells [2].
In postmenopausal women, estradiol does not appear to function as a circulating hormone; it is biosynthesized from androgens by the cytochrome P450 enzyme complex called aromatase [3] which is a product of the CYP19 gene, with the highest levels of this enzyme present in the peripheral adipose tissues of postmenopausal women [3]. Estrogen acts mainly at a local level as a paracrine or intracrine factor.
Aromatase has been found and measured in the stromal cell component of the normal breast as well as the breast tumour. Also, the enzyme has been detected in the breast epithelial cells in vitro [4-8]. Furthermore, expression of aromatase is highest in or near breast tumour sites [5,6,9,10]. It has been observed that the aromatase activity and expression is highest in the breast quadrant containing the tumour, such expression in the tumour containing quadrant is equal to that in the tumour itself, but double that in a quadrant of the same breast which does not contain a tumour which in turn is double the expression in the cancer free breast [10].
Evidence that postmenopausal obesity and weight gain are positively associated with postmenopausal breast cancer risk has been substantiated especially in women who never used hormone replacement therapy [11].
The aim of this study was to examine the hypothesis questioning the relationship between oestrogen and progesterone receptor status and breast tissue weight in postmenopausal women who had mastectomy for operable breast cancer.
Patients and methods
The mastectomy specimen weight, estrogen receptor (ER) and progesterone receptor (PgR) status for 62 consecutive patients who had a total mastectomy for operable breast cancer were retrospectively reviewed. Breast specimen weight was obtained from the pathology laboratory reports in grams. ER and PgR status was determined by immunohistochemistry (IHC) using the Quickscore system by a breast pathologist. ER/PgR status was considered positive if the score was 4 – 8. We also examined other parameters including tumour size, grade, nodal status, c-erb B2 expression and patient's age.
Correlation between breast weight and receptor status was examined in two groups of patients according to their age, 50 years and older and younger than 50 years.
Results
59 patients had invasive breast cancer and 3 patients had ductal carcinoma in situ (DCIS). Estrogen receptors were positive (ER+) in 50 patients (Quickscore: 4–8) and negative (ER-) in 12 patients (Quickscore: 0)
In general, breast weight was higher in patients with ER+ disease; median weight was 570 grams in ER+ patients compared to 413 grams in ER- patients (p = 0.04)
Patients were divided into two groups according to age. The first group consisted of patient aged 50 years and over (n = 43) with a median age of 60 years (range 51–83) and the second group contained those patients aged less than 50 years (n = 19) with a median age of 42 years (range 29–48).
Within the first group, breast weight was found to be significantly higher in women with ER+ tumours (669 vs. 220 grams, p = 0.015). Figure (1) demonstrates the relationship between the mastectomy specimen weight and ER status in the first group. Furthermore, when ER- and PgR – tumours were considered together the association was stronger (p = 0.003). On the other hand, there was no significant difference in breast weight between ER+ and ER- tumours in women in the second group (median weight: 440 vs.408 grams, p = 0.379).
Figure 1 ER status and mastectomy weight. (Age = or > 50 years, p = 0.015)
There was no significant difference in tumour's size between ER+ and ER- patients. Furthermore, the tumour's grade was higher in ER- patients (p = 0.009) and patient's age had no significant impact on the receptor status.
We observed a non-significant association between higher breast weight and PgR positivity (809 vs. 510 grams, p = 0.084) and absence of c-erb B2 (p = 0.088).
Finally, there was no association between breast weight and nodal status or tumour's grade.
Discussion
It has been established that obesity and weight gain are risk factors of breast cancer in postmenopausal women [12]. This relationship is explained by the fact that increased body weight is due to increased amount of adipose tissue in the body including breasts and subsequently increased of aromatase activity, thus increasing the local production of estrogen by aromatization of circulating androgens [13-16]. This process is a very important growth stimulating system in hormone dependent breast cancer [17].
Breast tissue and particularly the breast adipose tissue in postmenopausal women with breast cancer seem to have an increased aromatase expression. Agrawel et al studied 9 women undergoing breast reduction mammoplasty and 18 breast cancer patients undergoing mastectomies. Non-tumour bearing adipose samples from mastectomies expressed significantly more aromatase than adipose tissue taken from mammoplasty patients, a difference that is unlikely to be due the tumour's influence on aromatase expression [10].
The relationship between a higher body mass index (BMI) and the risk of ER+/PgR+ breast cancer has been studied previously [18-23]. In a case control study by Shelley et al a significantly increased risk of ER+/PgR+ breast cancer with increasing body size was observed. On contrast, other studies showed a greater frequency of ER- breast cancer among obese women [24-27].
The relationship between increased breast weight in postmenopausal women and receptor status has not been previously examined. In our study we hypothesised that in breast cancer patients; ER+ breast cancer was associated with a higher breast weight than ER- disease. This hypothesis was proved to be correct in postmenopausal women. Furthermore, such an association was not observed in women aged less than 50 years.
Our study has several limitations; firstly, we did not include other risk factors such as body mass index. Although a higher BMI is a recognised risk factor for ER+ disease, larger breasts are not always associated with a high BMI; higher breast weight could be found in a normal or low BMI postmenopausal women.
Secondly the family and HRT histories were not included in the analysis. Thirdly, the weight of the formatin-fixed mastectomy specimen was measured rather than the weight of the fresh specimen. However this applies to all specimens and is therefore unlikely to alter our findings. Finally we used the age of 50 years rather than the date of the last menstrual period to define the menopausal status. These limitations could be tackled in a larger prospective study.
In summary, in women aged 50 years or older with breast cancer, a higher breast weight seems to be significantly associated with ER+ disease. If this is confirmed in larger prospective studies, our findings may have implications regarding breast cancer prevention with anti-estrogens.
==== Refs
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Bulun SE Price TM Aitken J Mahendroo MS Simpson ER A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription J Clin Endocrinol Metab 1993 77 1622 1628 8117355 10.1210/jc.77.6.1622
Miller WR Mullen P Sourdaine P Watson C Dixon JM Telford J Regulation of aromatase activity within the breast J Steroid Biochem Mol Biol 1997 61 193 202 9365190 10.1016/S0960-0760(96)00202-6
Reed MJ Topping L Coldham NG Purohit A Ghilchik MW James VH Control of aromatase activity in breast cancer cells: the role of cytokines and growth factors J Steroid Biochem Mol Biol 1993 44 589 596 8476771 10.1016/0960-0760(93)90264-W
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Agarwal VR Bulun SE Leitch M Rohrich R Simpson ER Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients J Clin Endocrinol Metab 1996 81 3843 3849 8923826 10.1210/jc.81.11.3843
Huang Z Hankinson SE Colditz GA Stamper MJ Hunter DJ Manson JAE Hennekens CH Rosner B Speizer FE Willett WC Dual effects of weight and weight gain on breast cancer risk Journal of the American Medical Association 1997 278 1407 1411 9355998 10.1001/jama.278.17.1407
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| 15904529 | PMC1164427 | CC BY | 2021-01-04 16:38:37 | no | Int Semin Surg Oncol. 2005 May 16; 2:11 | utf-8 | Int Semin Surg Oncol | 2,005 | 10.1186/1477-7800-2-11 | oa_comm |
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Int Semin Surg OncolInternational seminars in surgical oncology : ISSO1477-7800BioMed Central London 1477-7800-2-121592706710.1186/1477-7800-2-12ResearchDurable remissions are rare following high dose therapy with autologous stem cell transplantation for adults with "paediatric" bone and soft tissue sarcomas Nath Shriram V [email protected] H Miles [email protected] Peter FM [email protected] Guy C [email protected] Haematology Service, Peter MacCallum Cancer Centre, St. Andrew's Place, East Melbourne, Australia2 Sarcoma Service, Peter MacCallum Cancer Centre, St. Andrew's Place, East Melbourne, Australia3 University of Melbourne, Australia2005 31 5 2005 2 12 12 6 3 2005 31 5 2005 Copyright © 2005 Nath et al; licensee BioMed Central Ltd.2005Nath et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The role of high dose therapy (HDT) with autologous stem cell transplantation (AuSCT) for the treatment of bone and soft tissue sarcomas remains investigational. There are few reports examining this strategy focusing on the adult population.
Methods
We retrospectively reviewed our experience of adult patients undergoing HDT and AuSCT for 'paediatric' sarcomas.
Results
A total of 17 patients (14 male, 3 female) with median age at transplant of 24 years (range 20 – 41) were identified. The diagnosis was Ewings sarcoma/PNET (10), osteosarcoma (5) and rhabdomyosarcoma (2). Status prior to HDT, following conventional-dose chemotherapy +/- surgery +/- radiotherapy, was complete remission (CR) (6), partial remission (PR) (6), stable disease (1) and progressive disease (4). There was no transplant-related mortality. Two patients remain disease free beyond four years and both received HDT as part of their primary therapy (CR1 and PR1) however, the median progression free survival and overall survival following AuSCT for the entire cohort was only 7 months (range: 2–92 months) and 13 months (range: 2 – 92 months), respectively.
Conclusion
HDT and AuSCT infrequently achieves prolonged remissions in adult patients and should only be considered in patients who are in a PR or CR following conventional-dose therapy. Further studies are required to define the role of HDT with AuSCT for adult patients with sarcoma.
Ewings sarcomaOsteosarcomaRhabdomyosarcomaHigh dose chemotherapytransplantation
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Introduction
The role of high dose chemotherapy (HDT) and autologous stem cell transplantation (AuSCT) for the treatment of patients with sarcoma remains controversial. [1-3]
The most common sarcoma for which HDT and AuSCT has been investigated is the Primitive Neuroectodermal Ectodermal Tumor (PNET) family of tumours which includes Ewing's sarcoma (ES). Indeed, the median age at diagnosis of PNET is14 years with 90% of patients being under the age of 20 years.[4] In adults with PNET, the 5-year overall survival (OS) following conventional-dose therapy is between 38–60% while the progression-free survival (PFS) is 27–59%. [5-7] Conversely, adult and paediatric patients who fail to achieve a complete response following surgery and conventional-dose chemotherapy are considered incurable and those with distant metastases have only 9% – 33% long term survival.[8] With respect to the role of HDT with AuSCT, no randomized studies have been performed and most published studies have included predominantly paediatric patients. [9-16]
Similarly, there is limited data on the role of HDT and AuSCT for osteosarcoma.[17] Again, studies to date have predominantly involved paediatric patients.[1,18,19]
The most common soft tissue sarcoma (STS) in children is rhabdomyosarcoma (RMS). Rare in adults, the disease free survival at 5 years of adult STS (different histologies) is approximately 10%. [20-25] In general, aggressive local therapy consisting of maximum tumor bulk reduction with surgery, with or without radiotherapy, is the cornerstone of initial management and patients with STS who are rendered disease free after surgery have a superior DFS than those who are inoperable.[26] However, many patients with STS are diagnosed with advanced-stage of the disease and/or complete surgical resection of the tumor is not feasible. Moreover, 40 to 60% of patients, even after a primarily curative local therapy, will develop metastases most frequently occurring within 2 to 3 years, and they will ultimately die of the disease.[27] Adult patients with RMS treated with conventional-dose therapy have an even worse prognosis than children.[28] The role of HDT and AuSCT for RMS remains unclear.[11,29]
Given the results with conventional-dose therapy, the role of HDT with AuSCT for sarcomas has largely been explored for patients with relapsed disease or those at high-risk of relapse. Patients with adult STS generally are poorly responsive to conventional-dose chemotherapy, with doxorubicin and ifosfamide being the only available agents showing response rates of greater than 20%. Combination regimens generally do not add efficacy, but do add toxicity.[21,24,27] For both agents, a dose-response relationship has been shown. Despite this theoretical rationale, the benefit of HDT is far from established in paediatric population, let alone for adult patients with sarcomas, with only a few studies reported and with small patient numbers.[9-11,17,18,27,30]
Of the studies reported to date, improved survival rates appear to be achieved for patients who achieve a complete remission (CR) prior to HDT or for those where total resection of tumor is feasible either before or after HDT. [10-12,26,30] Conversely, HDT with AuSCT appears not to improve outcome for PNET patients with metastatic disease at presentation.[13-16,31] Here we report our experience in treating adult patients with PNET, osteosarcoma and RMS.
Materials and methods
A retrospective review of adult patients who underwent HDT with AuSCT for sarcoma since 1992 at our institute was performed. Toxicity was assessed according to the World Health Organisation scale. Survival curves were generated according to the Kaplan-Meier method with OS and PFS calculated from the date of first stem cell infusion.
Results
A total of 17 patients underwent HDT and AuSCT and their characteristics are presented in Table 1 and included ES (9), PNET (1), osteosarcoma (5) and RMS (2). There were 14 males and 3 females with the median age at time of AuSCT of 24 years (range 20–41 years). HDT was administered as part of initial treatment (8), of which only one was in CR, five in PR, one with progressive disease (ProD) and one with stable disease (SD); in first relapse (7), of which four were in CR, one in PR and two with ProD; fourth relapse (1) who was in CR. Disease status immediately prior to HDT (following conventional-dose chemotherapy and/or surgery and/or radiotherapy) was CR (6), partial remission (PR) (6), stable disease (SD) (1) and ProD (4). The source of autologous stem cells was peripheral blood for 15 patients and bone marrow for two patients. Eleven patients underwent a planned tandem transplant.
Table 1 Summary of Patient Characteristics and Outcome
No age at AuSCT gender diagnosis surgery pre AuSCT disease site at HDT status at AuSCT time between diagnosis and AuSCT (months) Regimen* status post AuSCT surgery post AuSCT TTP (months) status time to last follow up/death (months)
1 39 M ES Y Rib CR1 11 e CR1 N NA A 57
2 20 F ES Y Lung CR2 38 c CR2 N 9 D 13
3 24 F ES Y Brain CR2 37 f CR2 N NA A 3
4 32 M ES N Pelvis PR1 4 g, b PR1 N 32 D 64
5 20 M ES N Scapula Clavicle PR1 6 g, b PR1 N 4 D 5
6 24 M PNET Y Brain PR2 52 f, b SD2 N NA A 4
7 41 M ES Y Tibia PR1 8 a, a PROG1 N 3 D 4
8 24 M ES N Lung Bones ProD2 40 c PR2 Y 5** D 5
9 27 M ES N Spine ProD1 7 d PROG1 N 5 D 2
10 26 M ES Y Spine SD1 23 g, b SD1 N 5 D 6
11 25 M OS Y Lung CR2 17 a, a CR2 N 7 D 15
12 21 M OS Y Lung CR4 84 a, b CR4 N NA A 12
13 24 M OS Y Node, Rib, Scapula CR2 24 a, a CR2 N 6 D 25
14 22 M OS Y Lung ProD2 34 a PR2 N 3 D 5
15 28 M OS Y Lung ProD3 32 a, b PR3 N 10 D 13
16 21 M RMS Y bladder PR1 13 g, h CR1 N NA A 92
17 34 F RMS Y face PR1 5 g, i CR1 N NA D 7
AuSCT = autologous stem cells transplant, TTP = time to progression,*See text for details of conditioning regimen, ES = Ewing's Sarcoma, PNET = Peripheral Neuroectodermal Tumor, OS = osteosarcoma, RMS = rhabdomyosarcoma, CR = Complete Remission, PR = Partial Remission, ProD = Progressive disease, SD = stable disease, NA = Not Applicable, A = alive, D = dead, **Travelled back to his native country and died (cause unknown but assumed to be disease-related).
HDT regimens were selected depending on prior therapy or involvement in specific clinical trials. Patients with osteosarcoma received carboplatin (700 mg/m2) and etoposide (750 mg/m2)a. Four patients subsequently received a second HDT and AuSCT with the same regimen (2) or melphalan (180 mg/m2) and etoposide (60 mg/kg)b (2). Patients with PNET/ES received a single course of melphalan (180 mg/m2)c(2) or radionuclide Samarium153 EDTMPd(1) or carboplatin (700 mg/m2) and etoposide (750 mg/m2)e(1) or carboplatin (AUC = 21), thiotepa (900 mg/m2) and etoposide (750 mg/m2)f (1) or tandem cycles with ifosfamide (12 g/m2), carboplatin (AUC = 20) and etoposide (60 mg/kg)g followed by and melphalan (180 mg/m2) and etoposide (60 mg/kg)b (3);or tandem cycles with two cycles of carboplatin (700 mg/m2) and etoposide (750 mg/m2)a (1); or tandem cycles with carboplatin (AUC = 21), thiotepa (900 mg/m2) and etoposide (750 mg/m2)f followed by melphalan (180 mg/m2) and etoposide (60 mg/kg)b (1). The two patients with RMS were treated with tandem cycles of HDT with AuSCT consisting of ifosfamide (12 g/m2), carboplatin (AUC = 20) and etoposide (60 mg/kg)g followed by mitoxantrone (48 mg/m2), carboplatin (AUC = 20) and etoposide (60 mg/kg)h (1) or ifosfamide (12 g/m2), carboplatin (AUC = 16) mitoxantrone (64 mg/m2)i (1). (Superscripted letters- indicating conditioning regimens used in Table 1)
All patients experienced WHO grade 4 leukopenia, neutropenia and thrombocytopenia after the HDT. The other WHO grade 3–4 toxicities were mucositis (3), pulmonary haemorrhage (1), ileitis (2) and haemorrhagic cystitis (1). There were no transplant-related deaths.
Median follow up for all surviving patients was 12 months. Of patients in CR at the time of HDT (6), none progressed during HDT and three of these patients have subsequently relapsed and died with a median PFS of nine months (range: 3–57 months). In patients entering HDT in PR (6), two achieved CR after AuSCT – one (pt 17) subsequently died of bilateral frontal haemorrhage at 7 months (not disease related), two achieved further reduction in tumour without achieving CR, one had SD and one had ProD. All of the patients who had either ProD (4) or SD (1) at the time of HDT subsequently progressed and died.
Of the eight patients who were in CR following HDT, three relapsed and died, four remain alive and disease free (3 – 92 months). Two patients remain disease free beyond four years (Pt 1 and Pt 16) and both received HDT as part of their primary therapy (CR1 and PR1) however, the median PFS and OS following AuSCT for the entire cohort was only 7 months (range: 2–92 months) and 13 months (range: 2 – 92 months), respectively. (Figures 1 &2).
Figure 1 Kaplan-Meier Curve of Progression free Survival for the entire cohort.
Figure 2 Kaplan-Meier Curve of Progression free Survival for the entire cohort.
Discussion
Given the relative chemotherapy-sensitivity of paediatric sarcomas and the poor prognosis with conventional-dose therapy for high-risk or relapsed disease, HDT with AuSCT has been investigated. Due to the rarity of these tumours, most studies have been retrospective, relatively small and have examined heterogeneous patient populations. Thus, despite some promising results demonstrating longer than expected survival, the benefits of this strategy remains uncertain.[27]
There is currently little data on the role of HDT for "paediatric" sarcomas in the adult sarcoma population. Our results in this group are disappointing. The median OS for the entire population was only thirteen months (range: 2–92 months) with a median PFS of seven months (range: 2–92 months) with only two patients remaining disease free beyond four years. Meyer et al. demonstrated that consolidation with high dose chemo-radiotherapy followed by stem cell support failed to improve the probability of EFS in a cohort of patients with newly diagnosed metastatic Ewings Sarcoma.[16]
With respect to osteosarcoma, we utilized a tandem transplant approach similar to that of the Italian sarcoma group (two courses of high dose carboplatin and etoposide) and they have recently demonstrated in 32 patients, with a median age of 15 years, a transplant-related mortality of 3.1% and a progression rate of 84% with a three-year OS and DFS of 20% and 12%, respectively.[24]
Comparative results of other published trials in STS, most of which include a paediatric population are presented in Table 2. Of note, Blay et al. reported an eight percent five year survival in adult patients with metastatic or locally advanced irresectable STS [12] and Bokenmeyer et al[32] reported results similar to ours in a cohort of 18 adult patients with STS.
Table 2 Summary of High Dose Chemotherapy Studies in Soft Tissue Sarcomas
Author Number of Pts Median Age (Range) R.F.S (months) O.S. (months)
Kasper 29 27 30.6 (13–59) 12 16.5
Blay 12 30 34 (17–57) 7 19
Samuel 34 23 not stated - 5.1
Dumontet35 22 16 (3–45) 15 19
Bokemeyer32 18 45 (25–37) 8 13
Kang36 24 24 (11–53) 6 10
Schlemmer37 55 not stated - 23
The European Organization for Research and Treatment of Cancer demonstrated that predictors for improved survival for STS and bone sarcomas following HDT were performance status, female gender, grade I tumors and the achievement of a CR after first line treatment.[33] In our study there was no clear predictors of durable remissions; three of the six patients who underwent HDT in CR relapsed, with a median PFS of nine months. (Range 3–57 months). Similarly, four of the eight who were in CR following HDT relapsed and died. It is of interest that one patient remains disease free at 12 months undergoing HDT and AuSCT in fourth CR.
Conclusion
Our results demonstrate that HDT and AuSCT infrequently achieves prolonged remissions in adult patients and only prospective studies will definitely determine the place of HDT in this group. Our data support that contention that HDT should only be considered in patients who are in a PR or CR following conventional-dose therapy.
Abbreviations
AuSCT Autologous stem cell transplantation
CR Complete remission
ES Ewing's sarcoma
HDT High dose therapy
OS Overall survival
PR Partial remission
PNET Primitive Neuroectodermal Ectodermal Tumor
PFS Progression free survival
ProD Progressive Disease
RMS Rhabdomyosarcoma
STS Soft tissue sarcoma
SD Stable disease
Authors' contributions
SVN, HMP, PFMC, GCT conceived the study and participated in its design and helped draft the manuscript. Authors read and approved the final manuscript.
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| 15927067 | PMC1164428 | CC BY | 2021-01-04 16:38:37 | no | Int Semin Surg Oncol. 2005 May 31; 2:12 | utf-8 | Int Semin Surg Oncol | 2,005 | 10.1186/1477-7800-2-12 | oa_comm |
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J Immune Based Ther VaccinesJournal of Immune Based Therapies and Vaccines1476-8518BioMed Central London 1476-8518-3-31590452510.1186/1476-8518-3-3ReviewGranulocyte-macrophage colony-stimulating factor as an immune-based therapy in HIV infection Brown Pierre Antoine [email protected] Jonathan B [email protected] Department of Medicine, University of Ottawa, 501 Smyth, Box 210, Ottawa, Canada, K1H 8L62 Division of Infectious Diseases, Ottawa Hospital – General Campus, 501 Smyth, Room G-12, Ottawa, Canada, K1H 8L62005 18 5 2005 3 3 3 4 2 2005 18 5 2005 Copyright © 2005 Brown and Angel; licensee BioMed Central Ltd.2005Brown and Angel; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The HIV/AIDS epidemic continues to spread despite more than 20 years of significant research and major advances in its treatment. The introduction of highly active antiretroviral therapy in recent years has significantly improved disease treatment with a dramatic impact in HIV/AIDS associated morbidity and mortality in countries which have access to this therapy. Despite these advances, such therapies are imperfect and other therapeutic modalities, including immune-based therapies, are being actively sought. Potential benefits of immune-based therapies include: 1) the improvement of HIV-specific immunity to enhance control of viral replication, 2) the improvement of other aspects of host immunity in order to prevent or delay the development of opportunistic infections and 3) the potential to purge virus from cellular reservoirs which are sustained despite the effects of potent antiretroviral therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been studied as one of these immune-based therapies. Several randomized, controlled trials have demonstrated benefits of using GM-CSF as an adjunct to conventional anti-retroviral therapy, although such benefits have not been universally observed. Individual studies have shown that GM-CSF increases CD4+ T cells counts and may be associated with decreased plasma HIV RNA levels. There is limited evidence that GM-CSF may help prevent the emergence of antiretroviral drug resistant viruses and that it may decrease the risk of infection in advanced HIV disease. Despite its high costs and the need to be administered subcutaneously, encouraging results continue to emerge from further studies, suggesting that GM-CSF has the potential to become an effective agent in the treatment of HIV infection.
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Review
Introduction
More than 20 years after its discovery, and despite extensive research in the field, HIV-1 infection remains one of the most important public health problems in the world. The HIV/AIDS epidemic continues to spread and an increasing number of people continue to live with HIV/AIDS and die from it. The advent of highly active antiretroviral therapy (HAART) marked a cornerstone in HIV/AIDS treatment that drastically changed the prognosis of HIV infection, by its ability to induce sustained suppression of viral replication [1-4]. Yet HIV infection remains, to this day, incurable. Even with multiple available therapeutic options, failure of therapy, manifested by a rebound in plasma viral load accompanied by further decline in CD4+ T cell counts, remains frequent, leaving limited available options for the treatment of individuals experiencing such failures. The persistence of HIV infection in the face of HAART is due to its limited effect on the persistent cellular reservoir(s) of replication-competent virus. T cells and macrophages have been implicated as such reservoirs [5-7]. This discovery prompted research in the field of immune-based therapy, in the hopes of enhancing or restoring cell mediated immune responses to HIV, or even purging latent viral reservoirs. A number of different approaches have been and are being studied, including several cytokines and therapeutic vaccines that are at various stages of evaluation in human trials [8-10]. Only a limited numbers of these have however been evaluated in controlled clinical trials and only interleukin-2 (IL-2), Remune™ and GM-CSF have been the subject of phase III studies, with clinical events as the primary outcomes [11-14]. Initially used in the treatment of leukopenia in HIV-1 infection, GM-CSF has also been used in clinical trials as an adjunct to HAART in which some of the results appear promising [12,15-18]. In this review, results from published randomized controlled trials that have evaluated the potential role for GM-CSF in the management of patients with HIV infection will be summarized (see Table in Additional file: 1).
Pre clinical and early clinical studies and the rationale for GM-CSF as an adjunctive treatment in HIV infection
GM-CSF is a pleiotropic growth factor that enhances the number and function of various cells from both the myeloid and lymphoid lineages, including neutrophils, monocytes and lymphocytes [19]. It is one of the many cytokines profoundly affected by HIV infection with its production being significantly reduced [20,21]. This has been one of several rationales for its use in HIV-infection. First, replacement therapy is seen as a way of enhancing the bone marrow's production of cells important in cell-mediated immunity, including CD4+ lymphocytes. Second, GM-CSF has also been shown in vitro to enhance the activity of the antiretroviral agent zidovudine (AZT) in macrophages [22,23] and thus may be an approach to enhance clearance of viral reservoir when used in combination with HAART. Third, GM-CSF also has an effect on monocyte-derived macrophages. Maturation of monocytes into macrophages is usually accompanied by an increase in the expression of CCR5, the co-receptor for the M-tropic HIV strains, a finding that seems to explain the observation that HIV entry is more efficient in macrophages than in monocytes [24]. In vitro, the presence of GM-CSF suppresses the expression of CXCR4 mRNA and CCR5 mRNA by monocytes differentiating in macrophages, resulting in macrophages that are relatively resistant to M-tropic HIV infection [25].
In addition to in vitro studies that have suggested that GM-CSF enhances the action of anti-retroviral drugs (ARVD) in macrophages [22,23], data supports the idea that GM-CSF can also lower the frequency of ARVD-resistant HIV-1 mutants in vivo. There appears to be lower frequency of resistant-mutations among subjects on zidovudine and GM-CSF, as part of their anti-retroviral regimens, versus those on AZT alone [16]. This finding is of potential significance, as the management of drug resistant strains of HIV remains a major issue. However, which specific mutations were observed at what frequency was not reported. This has an impact on the importance of this finding, as not all mutations have the same clinical significance. As well, whether these observations with AZT occur with other ARVD and how relevant this is given the current management of HIV-infected individuals remains to be established. Although it is used effectively in patients with neutropenia, typically caused by medication or bone marrow dysfunction [26-28], the positive effect of GM-CSF on CD4+ lymphocyte count in HIV had not been studied or well documented in early observational studies [29-31]. Following these in vitro findings and early in vivo studies, several randomized controlled trials were developed that studied the effect of GM-CSF as a treatment for HIV-1 infected individuals.
Impact of GM-CSF use in HIV infected individuals
Effect of GM-CSF on plasma HIV RNA levels
Few randomized controlled trials of GM-CSF have shown a clear, significant reduction in HIV replication. The first randomized controlled trial on the use of GM-CSF in non-neutropenic HIV-1 infected subjects, published in 1999, did not show any significant effect of GM-CSF on plasma HIV RNA levels [15]. This trial enrolled 20 patients, ten in the placebo group and ten in the treatment group. Subjects had similar baseline characteristics; the mean HIV RNA load was 3.95 log10 copies/ml in the placebo group compared with 4.21 log10 in the treatment group (p = 0.29) and the mean CD4+ T cells count in the placebo group was 243 cells/mm3 compared with 178 cells/mm3 in the GM-CSF group. All subjects were on stable antiretroviral therapy, including either indinavir or ritonavir, for a mean period of 5.0 months in the placebo group and 4.8 months in the treatment group. They received either 250 μg of GM-CSF or placebo subcutaneously 3 times per week for a total of eight weeks. All subjects were followed closely every two weeks during the study and twice at week 3 and week 5 after the study ended. During the study and at both follow up time points, the viral load remained within 0.5 log10 copies/ml of the baseline values for both groups.
Despite no overall changes in the mean HIV RNA load between groups, more subjects in the GM-CSF group than in the control group had HIV RNA values decreased by >0.5 log10 from baseline (50% vs. 10%). Since this size of a viral load decrease has been associated with clinical benefits, this study suggests that GM-CSF may have a beneficial effect in a subset of individuals.
In the largest double blind, randomized controlled trial on GM-CSF use in HIV infected individuals published to date, 309 subjects stratified according to viral load (≤ 30000 copies/ml vs. > 30000 copies/ml) received either 250 μg of GM-CSF or placebo three times per week for 24 weeks [12]. In total, 70% of subjects completed the full 24-week period. In the treatment and control arm respectively, 89 and 90% were males, 80% and 79% of subjects were on at least 3 antiretroviral agents, and 82% in both groups previously had one or more opportunistic infection. The mean CD4+ T cell count was 49.8 cells/mm3 in the control group and 50.8 cells/mm3 in the GM-CSF group. The majority of subjects entered the study with a viral load over 30000 copies/ml (62% for the placebo arm and 63% for the GM-CSF arm). There was no significant decrease in HIV-RNA in the combined strata in either the placebo or treatment group. However, GM-CSF had a positive influence on other viral parameters. GM-CSF use delayed virologic failure in those patients with plasma HIV RNA levels less than 400 copies/ml before initiation of GM-CSF therapy. At 6 months, 24 out of 29 (83%) of subjects on GM-CSF maintained viral loads below the limit of detection compared to 15 out of 28 (54%) of those on placebo (p = 0.02). This, in turn, reduced the need for antiretroviral regimen change. In this trial, ARVD regimen changes were allowed, which could have obscured a preferential decrease in viral load by GM-CSF. As such, there were fewer changes in ARVD regimens in the GM-CSF group (19%) than in the placebo group (38%) for the lower viral load stratum (p = 0.03). In the higher stratum, no significant difference in treatment change was observed (62% placebo versus 62% GM-CSF; p = 0.68). Again, this supports the idea that low-dose GM-CSF may have the potential to limit HIV replication and prevent or delay the development of drug resistant viruses, as described earlier in in vitro and in vivo studies.
In a Brazilian study, 105 individuals with AIDS were enrolled in a placebo-controlled, double-blind randomized control trial to receive AZT along with GM-CSF (125 μg) or placebo twice weekly for 6 months [16]. Subjects were required to have an AIDS defining diagnosis based on 1993 Center for Disease Control and Prevention criteria within the last three months or a CD4+ cell count <300 cells/mm3. Patients were excluded if they had an active AIDS defining diagnosis at the time of randomization or if they had been exposed to zidovudine for >6 months prior to study entry. The mean HIV RNA plasma levels at baseline were 93000 copies/ml in the placebo group and 155000 copies/ml in the GM-CSF group (p = 0.21). All the subjects received AZT, and 65% and 68% of subjects in the placebo and GM-CSF group respectively were also on a second agent, either ddI, ddC, 3TC or Saquinavir. Prior opportunistic infection rates were 58% in the placebo group and 70% in the treatment group (p = 0.14). This study did show a statistically significant effect of GM-CSF on viral loads. Mean HIV RNA levels declined in the GM-CSF group throughout the 6 months of the study. Over this period, the change was -0.07 log10 copies/ml in the control group as opposed to -0.60 log10copies/ml in the treatment group (95% CI -0.94-0.12; p = 0.02). As well, there was a greater number of subjects in the GM-CSF group with a decrease of 1 log10 or greater in viral load (20/52; 38%) compared with the placebo group (9/53; 17%) (p = 0.02). The reason why a decrease in viral load was observed in this study and not in other trials is unclear. It was the only trial with a smaller dose of GM-CSF (125 μg twice weekly vs. 250 μg thrice weekly for most other trials) and all patients were receiving AZT, both of which might have played a role in this difference.
More recent clinical data on the use of GM-CSF in combination with HAART continues to show some effect of GM-CSF on viral load [17]. These data stem from a randomized controlled trial in which 116 subjects were required to remained virologically stable (within a difference of 0.7 log10 copies/ml) for at least 7 days prior to entry and where no HAART regimen change was allowed during the 16 weeks period of the trial. Subjects were divided in 2 groups, depending if their CD4+ T count was below or above 200 cells/mm3 at baseline and then randomized to either 250 ug of GM-CSF or placebo three times per week for 16 weeks. All patients subsequently received a 32-week course of open label GM-CSF. Baseline characteristics were similar in both groups. At baseline, in the ≥ 200 and <200 CD4+ cells/mm3 strata, median plasma RNA levels were 3.81 log10 and 4.46 log10 copies/ml, with no difference between control and treatment groups. After the 16 weeks of double-blinded treatment, the change in HIV RNA levels was +0.048 log10 copies/ml in the GM-CSF group compared with -0.103 log10 copies/ml (p = 0.036) in the placebo group, both strata combined. However, when the two strata (≥ 200 and <200 CD4+ cells/mm3) were studied individually, the changes in mean viral loads were not significant. Thus, in this trial, subjects in the GM-CSF group, irrespective of their initial CD4+ count, tended to have a modest increase in HIV RNA levels at the end of the 16-week randomized period. Although the modest increase in viral load was significant, it was not associated with a decrease in CD4 counts or an increase in clinical events, as is discussed later.
Finally, a Swiss study evaluated the use of GM-CSF 300 μg three times a week for the first four weeks of a 12-week HAART interruption period [18]. This small study randomized 33 subjects who had previously been stable on HAART for at least six months, with viral load below 50 copies/ml and CD4+ T cell counts >400 cells/mm3. In both groups the viral load peaked at 6 weeks and trended down afterwards. In the GM-CSF group, the maximum viral load reached a mean of 4.97 log10 compared with 5.54 log10 in the scheduled treatment interruption-only (STI-only) group (p = 0.03). Over a period of twelve weeks, the mean area under the curve for viral loads were 47.77 log10 in the GM-CSF group and 51.88 log10 in the STI-only group (p = 0.07) This suggests not only that there is no deleterious effect of GM-CSF on plasma HIV RNA levels but that GM-CSF may help control the viral load in patients who need to stop HAART for a short period.
Overall, the evidence regarding the effect of GM-CSF on plasma HIV-1 RNA levels is somewhat conflicting. Four of the five trials reviewed show either a significant decline or no statistical changes in the viral load. The explanation for the observed increase in viral load in the GM-CSF group in the trial by Jacobson et al. is not clear. This study was somewhat unique in that it included only patients with uncontrolled viral replication. It appears likely that the impact of GM-CSF on viral load is dependant upon the setting in which GM-CSF is used. Furthermore, GM-CSF may selectively enhance the antiviral activity of specific antiretroviral agents (e.g. AZT). Also, as may be becoming apparent with other immune-based therapies, the greatest effect of GM-CSF may be observed in situations where there is the greatest degree of virologic suppression and associated immunologic recovery.
Effect of GM-CSF on CD4+ T cells
The initial randomized control trial of GM-CSF use in non-leukopenic HIV infected individuals, referred to previously, reported other important findings. CD4+ T cell counts reached higher levels in the treatment group, but these results did not reach statistical significance [15]. The mean maximal increase in the treatment group was 129.6 ± 149.9 cells/mm3 and 57 ± 58.9 cells/mm3 in the control group (p = 0.02). A significant majority (70%) of subjects treated with GM-CSF demonstrated an increase of >30% of their CD4+ T cell counts over baseline at any given time versus a minority (30%) in the placebo group (p = 0.07). When those patients with baseline CD4+ T cell counts of <50 cells/mm3 were excluded from the analysis, in order to ensure an increase of >30% was not due to daily variability, 6 of 7 patients in the GM-CSF group and 1 of 8 patients in the placebo group had a CD4+ T cell increase of >30% (p = 0.01). This may have a clinical impact as a >30% increase of the CD4+ T cell count in light of a stable viral load has been associated with a relative risk reduction of disease progression in a previous study [32].
An earlier randomized controlled study looking into the effect of GM-CSF on leukopenia in HIV-infected individuals also demonstrated an increase in CD4+ T cell counts. After 12 weeks of therapy (300 μg GM-CSF daily for 1 week then 150 μg twice-a-week for 11 weeks), absolute CD4+ T cell counts rose by 53% compared to baseline (p < 0.001) and was statistically different than that observed in the control group (p < 0.001) [26].
Other trials observed a trend towards modest, non-significant, increases in the absolute CD4+ counts. In the study by Brites et al, the authors reported a modest increase in the CD4+ T cell count in both groups at six months [16]. In the GM-CSF group, there was a small, non-significant increase in the CD4+ T cell count of 35 cells/mm3 compared with 12 cells/mm3 in placebo group (p = 0.42). As with the study by Skowron et al, they also observed a significant difference in the number of subjects who had a ≥ 30% increase of the CD4+ T cell count. Only 59% of subjects in the placebo group achieved this increase as opposed to 80% in the GM-CSF group (p = 0.03).
In the other, more recent trial, from Jacobson et al., the authors reported a change in the CD4+ T cell count of +29 cells/mm3 in the GM-CSF vs. -8 cells/mm3 in the placebo group for the stratum of subjects with >200 CD4+ T cells/mm3 at baseline (p = .20) [17]. They observed a similar trend in the <200 CD4+ T cells/mm3 stratum, with +5 cells/mm3 in the GM-CSF group at 16 weeks vs. -5 cells/mm3 in the placebo group but this did not reach statistical significance (p = 0.22).
Fairly convincing evidence of a significant increase in absolute CD4+ leukocyte count following treatment with GM-CSF comes from the phase III randomized control trial by Angel and colleagues, described earlier [12]. The baseline CD4+ T counts were 49.8 × 106 cells/L for the placebo group and 50.8 × 106 cells/L for the treatment group. At 12 months the mean CD4+ T cell count was 102 ± 15 × 106 cells/L in the placebo group vs. 152 ± 18 × 106 cells/L in the treatment group. This was also reflected by statistically significantly greater increases in the CD4+ T cell count at 1, 3 and 6 months in the GM-CSF group.
It might be speculated that, since this was the largest (n = 307) and one of the longest (24 weeks) randomized control trial of GM-CSF use in HIV infected individuals, it maybe the only study with enough power to demonstrate statistical significance. As other randomized control trials show trends towards higher CD4+ T cell counts in the GM-CSF group, and statistically significant difference in various sub-analysis, it is possible that an appropriately conducted meta-analysis would clarify the impact of GM-CSF on CD4+ T cell counts.
The recent study by the Swiss group also supports a positive effect of GM-CSF on CD4+ lymphocytes count [18]. In that trial, the CD4+ T cell counts fell from 720 × 106 cells/L at baseline to 537 × 106 cells/L at four weeks after stopping HAART in the STI-only group (p < 0.001). In the GM-CSF treated group, there was no significant change in the CD4+ T cell counts four weeks after stopping HAART. CD4+ T cell counts were 890 × 106 cells/L at baseline and 792 × 106 cells/L at week four (p = 0.6). This adds evidence that GM-CSF could have a beneficial effect on CD4+ T cell counts.
Impact of GM-CSF on clinical outcomes
GM-CSF has an excellent safety and tolerability profile when used in HIV-1 infected individuals [12,15-17]. In all the major randomized controlled trial, pain, local swelling and erythema were the most frequent side effects and reactions were almost all grade 1 or 2 with only rare grade 3 or 4 events. In the recent randomized controlled trial by Jacobson et al, a total of 4 patients had to discontinue GM-CSF use because of toxicity or acute allergic reactions [17]. That was not the case in other trials, where there were no discontinuations of therapy over many patient-months of therapy [12,15,16]. There were no hospitalizations or death attributable to GM-CSF in any study.
The large phase III trial by Angel et al. has been the only study to use clinical events as endpoints, using the Centers for Diseases Control and Prevention definition of opportunistic infections (OI), bacterial pneumonia or death as their primary endpoint [12]. An effect of GM-CSF on the rate of OI was not observed, with an event rate of 18% in the placebo group and 21% in the GM-CSF group (p = 0.61). Despite this, there were some important benefits to the use of GM-CSF on other clinical events. These same authors found that the incidence of overall infections (OI and non-OI) was significantly lower in the treatment group of their study; 78% in the placebo group versus 67% in the GM-CSF group (p = 0.03). They also found that time to occurrence of the first infection or death was also significantly longer when GM-CSF was used as an adjunctive treatment in HIV infection (97 days vs. 56 days for placebo; p = 0.04).
For individuals who do not have a history of OI, GM-CSF may decrease the risk of a first opportunistic infection[16]. Despite the fact that they did not observe differences in the rate of overall infections or OI, Brites et al. did noticed that all 17 subjects in the GM-CSF arm who developed an OI had a prior history of one or more of these infections. In the placebo group, only 50% of the 14 subjects who developed an OI during the study had a prior history of OI (p < 0.01). This might prove to be an important role for adjunct treatment with GM-CSF as OI are still an important cause of morbidity and mortality in HIV infected individuals.
The most recent randomized control trail by Jacobson et al. did show a non-significant reduction in clinical events in the GM-CSF group [17]. No HIV associated clinical events were seen in the treatment group versus 4 in the placebo group (p = 0.12), Again, all the subjects in this trial were on stable HAART prior to and during the study, which is likely responsible for a very low incidence of both overall and OI rates. This, combined with a smaller sample size, likely accounts for the lack of power of this trial to demonstrate an effect of GM-CSF on clinical events.
Finally, the study by Fagard et al. failed to show any impact of GM-CSF on clinical events during HAART interruption. However, they studied only 33 patients with high CD4 counts and off HAART for a limited period of time [18].
Despite all these results, questions still remain as to whether use of GM-CSF is associated with a reduction in the incidence of AIDS related morbidity and mortality, as even the authors of the largest phase III study published to date admit to a lack of power in their trial [12]. The introduction of HAART at the time of this trial, thereby likely lowering the incidence of OI in both the GM-CSF group and placebo group, could be expected to have had a significant impact on the outcome of that study.
Conclusion
In various studies GM-CSF has a positive effect on important parameters of HIV infection, namely plasma HIV RNA levels and CD4+ lymphocytes counts. Although the positive effects are modest and not universally observed, they are significant in many trials. Moreover, the positive effect on these measures may translate into significant clinical benefits. Clinical outcome results of current randomized controlled trials are, thus far, somewhat encouraging. Despite the frequent lack of statistical significance, there are positive trends towards clinical benefit of GM-CSF use in these studies. Moreover, the largest randomized control trial did show that GM-CSF produces a significant reduction in the time to first infection or death. The possibility of allowing longer disease free periods is a desirable outcome for HIV infected individuals, contributing to improved quality of life. However, the high cost of GM-CSF and its mode of administration may be difficult hurdles for patients to overcome.
Future trials designed to look at specific clinical outcomes, for example diseases free period, progression of HIV infection and quality of life, might bring to light additional beneficial effects of GM-CSF. This would require focusing on patients with advanced HIV disease and lower CD4 counts. Alternatively, future trials could focus on the use of GM-CSF as an adjuvant therapy, either to HAART or as an adjuvant with HIV or other vaccines. There is growing evidence that GM-CSF enhances the immune response to vaccines directed against both infectious agents and various cancers [33]. Clinical trials of GM-CSF as an adjuvant to hepatitis B vaccination have shown some positive results [34-37]. Moreover, GM-CSF when added as an adjuvant to HIV envelope vaccination in mice resulted in a greater HIV-specific cellular immune response [38]. Regardless of future studies, it would appear important that those trials focus on individuals with suppressed viral replication, as they seem more likely to realize the benefits of GM-CSF.
It remains to be seen if GM-CSF will ever loose its experimental status and become an accepted therapy for selected individuals HIV infection. The evidence for the role of immunotherapy in HIV/AIDS is ever increasing and GM-CSF might very well become a widely accepted treatment in the years to come.
Competing interests
The author(s) declare that they have no competing interests.
Supplementary Material
Additional File 1
Table 1 is a summary of clinical trials of GM-CSF in the treatment of HIV infection.
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| 15904525 | PMC1164429 | CC BY | 2021-01-04 16:37:45 | no | J Immune Based Ther Vaccines. 2005 May 18; 3:3 | utf-8 | J Immune Based Ther Vaccines | 2,005 | 10.1186/1476-8518-3-3 | oa_comm |
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J NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central London 1742-2094-2-121592979710.1186/1742-2094-2-12ResearchCu/Zn superoxide dismutase expression in the postnatal rat brain following an excitotoxic injury Peluffo Hugo [email protected] Laia [email protected] Maryam [email protected] Bernardo [email protected] Berta [email protected] Unit of Histology, Department Of Cell Biology, Physiology, and Immunology; Autonomous University of Barcelona, 08193, Spain2 Institute of Neuroscience, Autonomous University of Barcelona, 08193, Spain2005 1 6 2005 2 12 12 14 3 2005 1 6 2005 Copyright © 2005 Peluffo et al; licensee BioMed Central Ltd.2005Peluffo et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
In the nervous system, as in other organs, Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key antioxidant enzyme involved in superoxide detoxification in normal cellular metabolism and after cell injury. Although it has been suggested that immature brain has a different susceptibility to oxidative damage than adult brain, the distribution and cell-specific expression of this enzyme in immature brain and after postnatal brain damage has not been documented.
Methods
In this study, we used immunohistochemistry and western blot to analyze the expression of Cu/Zn SOD in intact immature rat brain and in immature rat brain after an NMDA-induced excitotoxic cortical injury performed at postnatal day 9. Double immunofluorescence labelling was used to identify Cu/Zn SOD-expressing cell populations.
Results
In intact immature brain, Cu/Zn SOD enzyme was widely expressed at high levels in neurons mainly located in cortical layers II, III and V, in the sub-plate, in the pyriform cortex, in the hippocampus, and in the hypothalamus. Glial fibrillary acidic protein-positive cells only showed Cu/Zn SOD expression in the glia limitans and in scattered cells of the ventricle walls. No expression was detected in interfascicular oligodendroglia, microglia or endothelial cells. Following excitotoxic damage, neuronal Cu/Zn SOD was rapidly downregulated (over 2–4 hours) at the injection site before neurodegeneration signals and TUNEL staining were observed. Later, from 1 day post-lesion onward, an upregulation of Cu/Zn SOD was found due to increased expression in astroglia. A further increase was observed at 3, 5 and 7 days that corresponded to extensive induction of Cu/Zn SOD in highly reactive astrocytes and in the astroglial scar.
Conclusion
We show here that, in the intact immature brain, the expression of Cu/Zn SOD was mainly found in neurons. When damage occurs, a strong and very rapid downregulation of this enzyme precedes neuronal degeneration, and is followed by an upregulation of Cu/Zn SOD in astroglial cells.
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Background
It has been shown that ~2–5% of the electron flow in isolated brain mitochondria produces superoxide anion radicals (O2-.) and hydrogen peroxide (H2O2) [1]. Cellular levels of O2-. are normally low due to the action of cytosolic copper zinc superoxide dismutase (Cu/Zn SOD) and mitochondrial manganese superoxide dismutase (Mn SOD). These key enzymes catalyze the dismutation of O2-. to oxygen and H2O2 [2]. Increased production of superoxide and its derivatives can induce injury by diverse mechanisms including initiation of lipid peroxidation, inactivation of enzymes, damage to DNA, and protein sulfhydryl oxidation. In particular, in the presence of nitric oxide (NO.), O2-. and NO. rapidly and spontaneously react to form the potent oxidant peroxynitrite (ONOO-), which is capable of nitrating tyrosine [3,4] contributing to the neuropathological process [5]. In this sense, superoxide radicals have been identified as important mediators of oxidative injury during ischemia-reperfusion and many other neurological injuries [6]. Cerebral ischemia [7-9] and traumatic brain injury [10] cause a rapid and sustained increase in the formation of O2-., which is accelerated during mitochondrial dysfunction, and may also result from increased activity of several cytosolic enzymes as phospholipase A2 [11] or cycloxygenase 2 (COX2) [7].
In the adult central nervous system (CNS), Cu/Zn SOD is widely expressed in different neuronal populations: hippocampal CA pyramidal neurons and granule neurons of the dentate gyrus, cortical neurons, especially pyramidal cells, neurons of the substantia nigra, and at very high levels in motor neurons of the spinal cord [12-17]. In regards to glial cells, it is generally assumed that microglial cells and oligodendrocytes do not show expression of Cu/Zn SOD [12,16-19], but the astroglial expression of this enzyme is controversial. Some studies have found Cu/Zn SOD expression primarily and most intensely in astrocytes [18,19], but a number of works have failed to detect this expression in astrocytes [13-15,17], or have reported expression only in some scattered astrocytes [12,16]. On the other hand, expression of Cu/Zn SOD has been well documented in reactive astrocytes several days after different types of adult brain injury, like transient cerebral ischemia [13], kainate treatment [14], quinolinic treatment [18], or Alzheimer's and Down's Syndrome [16].
According with its antioxidant role, in most adult CNS injury models the over-expression of Cu/Zn SOD is thought to be neuroprotective [20-22]. In agreement, synthetic O2-. dismuting metalloporphyrins protect against transient middle cerebral artery occlusion [23], and targeted deletion of the Cu/Zn SOD or extracellular SOD genes worsens outcome after focal ischemia [24,25]. However, contradictory results have been reported regarding the toxicity of O2-. after hypoxic/ischemic injury to the immature brain. Whereas several antioxidant molecules including SOD mimetics (as O2-. dismuting metalloporphyrins) have been shown to be neuroprotective [26], a slightly worsened neuropathological outcome is observed in transgenic mice over-expressing Cu/Zn SOD [27]. Several other differences in regards to oxidative stress and antioxidant defences have been reported in the immature versus the mature brain. For example, and in comparison to the adult brain, in the immature damaged brain glutathione peroxidase is not upregulated after trauma [28]; free iron accumulates more rapidly within 4 hours after transient cerebral ischemia stimulating Fenton reactions [29,30]; and metallothioneins, potent antioxidant enzymes that bind transition metals as Zn, are less concentrated [31,32]. Finally, the postnatal brain is more sensitive than the adult brain to the neurotoxic actions of N-methyl-D-aspartate (NMDA) [33], which will lead to increased O2-. generation [34,35]. Interestingly, it has been reported that Cu/Zn SOD is rapidly downregulated after several types of injury in the mature CNS [13,14,36,37], but no data is available regarding this phenomenon in immature brain, where overall findings suggest a differential scenario for oxygen and nitrogen reactive species in the evolution of a brain injury [38].
In this context, the aim of our study was to evaluate the temporal and spatial dynamics, and the identity of Cu/Zn SOD expressing-cells, in the intact immature rat brain, and following an excitotoxic injury.
Methods
Excitotoxic lesions
Nine-day-old Long-Evans black-hooded rat pups of both sexes were placed in a stereotaxic frame adapted for newborns (Kopf) under isofluorane anaesthesia. The skull was opened using a surgical blade, and 0.15 μl of saline solution (0.9% NaCl, pH 7.4) containing 18,5 nmol of N-methyl-D-aspartate (NMDA) (Sigma, M-3262, Germany) were injected into the right sensorimotor cortex at the level of the coronal suture (2 mm lateral from bregma and at 0.5 mm of depth). Control animals received an injection of 0.15 μl of vehicle saline solution. After suture, pups were placed in a thermal pad and maintained at normothermia for 2 hours before being returned to their mothers. Experimental animal work was conducted according to Spanish regulations, in agreement with European Union directives. This experimental procedure was approved by the ethical commission of the Autonomous University of Barcelona. All efforts were made to minimize animal suffering in every step.
Immunocytochemical study
Intact immature brains from P9, P10, P12, and P16 rats of both sexes where used for the analysis of Cu/Zn SOD expression under physiological conditions (2–4 animals per time). Moreover, at 2, 4, and 10 hours, and 1, 3, 5 and 7 days after NMDA (3–4 animals per time) or saline injection (2 animals per time), rats were anaesthetized and perfused intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were post-fixed in the same fixative for 2 hours and sunk in a 30% sucrose solution before being frozen with dry CO2. Coronal sections (30-μm-thick) were obtained using a Leitz cryostat. Free-floating parallel sections were treated with 10% foetal calf serum in Tris-buffered saline (TBS: 0.05 M Trizma base containing 150 mM of NaCl, pH 7.4) +1% triton X-100 for 1 hour, and incubated overnight at 4°C with sheep polyclonal anti-Cu/Zn SOD (574597, Calbiochem, Darmstad, Germany) (1:300) in the same solution. Afterwards, sections were rinsed and incubated for 1 hour at room temperature with Cy3-conjugated anti-sheep secondary antibody (AP147C, Chemicon, California, USA) (1:150). As negative controls, sections were incubated in media lacking the primary antibody.
Double labelling with specific cell markers
Double labelling was carried out in order to identify Cu/Zn SOD-expressing cells in sections previously immunoreacted for Cu/Zn SOD as reported above.
For neuronal identification, sections were further incubated with a monoclonal anti-NeuN antibody (MAB377, Chemicon, California, USA) (1:1000) and Cy2-conjugated anti-mouse secondary antibody (PA-42002, Amersham Pharmacia Biotech, England) (1:1000).
For astroglial labelling, sections were incubated with a polyclonal anti-GFAP antibody (Z-0334, Dakopatts, Denmark) (1:1800), and immunostaining was visualized with Cy2-conjugated anti-rabbit secondary antibody (PA-42004, Amersham Pharmacia Biotech, England) (1:1000).
As a microglial marker, sections were processed for double staining with tomato lectin histochemistry by incubating with the biotinylated lectin from Lycopersicon esculentum (tomato) (Sigma, L-9389, Germany) diluted to 6 μg/ml, followed by Cy2-conjugated streptavidin (PA-42000, Amersham Pharmacia Biotech, England) (1:1000).
Selected sections of all double labelling techniques were incubated for 5 min with a 0.00125 μg/ml solution of 4, 6-diamino-2-phenylindole (DAPI) in TBS. Double-stained sections were analyzed using a Nikon Eclipse E600 epifluorescence microscope and a LEICA TCS SP2 AOBS confocal microscope.
TUNEL labelling
Terminal dUTP Nick End Labelling (TUNEL) staining for detection of DNA fragmentation was performed on parallel sections mounted on slides. Tissue sections were rinsed in Tris buffer (10 mM, pH 8) and EDTA (5 mM) and then incubated in the same buffer plus Proteinase K (20 μg/ml) for 15 min. at room temperature. After several washes with EDTA (5 mM), sections were incubated for 10 min. in TdT buffer (Tris 30 mM, 140 mM Sodium Cacodilate, 1 mM Cobalt chloride, pH 7.7). Sections were then incubated in TdT buffer plus 0.161 Units/ μl TdT enzyme (Terminal Transferase, 3333566 Roche, Manheim, Germany) and 0.0161 nmol/ μl of biotin-16-dUTP (1093070, Roche, Manheim, Germany) for 30 min. at 37°C. The reaction was stopped by washing the sections in citrate buffer (300 mM sodium chloride, 30 mM sodium citrate, 5 mM EDTA). After several washes with TBS, sections were incubated with avidin-peroxidase (P-0364, Dakopatts, Denmark) (1:400) for another hour at room temperature. Finally, the peroxidase reaction product was visualised in 100 ml of Tris buffer containing 50 mg of 3'-diaminobenzidine and 0.01% of hydrogen peroxide.
Western blotting and densitometry
Three or four NMDA-injected and two intact and saline-injected animals for each survival time were decapitated, and the complete injected cortex quickly extracted, chopped and frozen in liquid nitrogen. Samples were resuspended in Tris/HCl (50 mM, pH7.8), EDTA (1 mM), DTT (1 mM), PMSF (100 μg/ml), pepstatin A (2 μg/ml), leupeptin (2 μg/ml), trypsin inhibitor (10 μg/ml), benzamidine (0.2 mM) and submitted to mechanical dissociation. Total protein concentration was measured by the bicinchoninic acid method and equal quantities of protein were run on a 15% SDS-polyacrilamide gel electrophoresis (SDS-PAGE). Electrotransfered protein samples to polyvinylidene fluoride (PVDF) membranes were incubated overnight at 4°C with TBS+0.3% Tween 20 and 5% non-fat milk, and for 2 hours at room temperature with sheep polyclonal anti-Cu/Zn SOD (574597, Calbiochem, Darmstad, Germany)(1:1000). Membranes were rinsed and incubated in biotinylated anti-sheep antibody (1:1000) (RPN-1025, Amersham Pharmacia Biotech, England), avidin-peroxidase (1:2000) (P0364, Dakopatts, Denmark) and finally in the chemiluminiscent substrate SuperSignal West Pico (PIERCE) combined with exposure on Hyperfilm ECL (Amersham).
Semi-quantitative estimation of western blot protein signals were performed by measuring band integral intensity with analySIS® software after high resolution scanning.
Statistical analysis
All results are expressed as mean ± standard error mean (SEM). ANOVA followed by Fisher's PLSD post-hoc test was used to determine significant differences (p<0.05) after western blot densitometry.
Results
Cu/Zn superoxide dismutase expression in the immature rat brain
As there is no available description of Cu/Zn SOD cell-specific expression in the immature brain, we began our study with this general description. No changes in the distribution pattern and cellular identity of Cu/Zn-expressing cells were found between P9 to P16 and thus will be described in general. The immature rat brain showed widespread immunoreactivity for Cu/Zn SOD, mainly located in neurons. The most intense staining was observed in pyramidal neurons located in cortical layers V, III and II (Fig. 1A, B), in the pyriform cortex, and in the pyramidal neurons of the olfactory tubercle. Sub-plate neurons, a transient cell population located beneath the cortical plate [39] were also among the most intense cells (Fig 4A). In addition, hippocampal CA pyramidal neurons and some inter neurons (Fig. 1C), as well as dentate gyrus granular and sub-granular neurons, medial septum neurons, and hypothalamic neurons also displayed intense staining. There was also more diffuse staining in many other neuronal populations as for example in various thalamic nuclei, substantia nigra (Fig. 1D), and striatum. Most of the immunoreactivity was mainly observed in neuronal soma although less intense staining was also present in the nuclei, sparing the nucleolus.
Figure 1 Neuronal Cu/Zn SOD immunoreactivity in intact immature brain. Widespread immunoreactivity for Cu/Zn SOD was observed mainly in neurons, both in the cytoplasm and nucleus, sparing the nucleolus. In the cortex, the highest expression was observed in cortical pyramidal neurons of layers V and III and II (A, B: Parietal cortex). In addition, the hippocampal pyramidal layer (CA1; C), and of substantia nigra (D) also displayed immunoreactivity. Scale bars: 50 μm in A and B; 20 μm in C and D.
Figure 4 Cell-specific Cu/Zn SOD expression in the early excitotoxicaly lesioned immature brain. In the injection site, Cu/Zn SOD immunoreactivity decreased from 2–4 hours after the NMDA injection, and continued to be downregulated until 24 hours after injection (A-B,SPN: sub-plate neurons, V and II-III: cortical layers). Neurons from the injected cortex after 4 hours showed slightly condensed chromatin (compare C and D: arrows), and a shift of NeuN staining from the soma and nucleus towards the nucleus (compare F and G: arrows) in the absence of TUNEL staining (H). From 10 hours after lesion onward, neuronal nuclei showed clear apoptotic signals like condensation and blebbing (E: arrows) and TUNEL staining (I). Scale bars in A-B: 200 μm; in C-G 5 μm; and in I-H: 40 μm.
No specific Cu/Zn SOD immunoreactivity was detected in parenchymal grey matter GFAP-positive astrocytes (Fig 2A). However, co-expression of Cu/Zn SOD and GFAP was observed in scattered tanycytes of the third ventricle wall (Fig 2B), in corpus callosum, and in astrocytes forming the glia limitans (Fig 2C). Most ciliated ependymal cells expressed Cu/Zn SOD (Fig. 2B). Moreover, besides the ependymal cells described above, co-localization confocal studies with tomato lectin, which specifically detects microglial cells as well as endothelium [40], did not show overlap with Cu/Zn SOD staining (Fig 2D). No consistently Cu/Zn SOD-labelled cells were observed in white matter tracts, which indicated that oligodendrocytes at this location do not seem to express this enzyme.
Figure 2 Glial distribution of Cu/Zn SOD immunoreactivity in intact immature brain. Cu/Zn SOD was not observed in the GFAP-expressing astrocyte population of the brain parenchyma (A: cortex, confocal image). However, GFAP-expressing astrocytes showed co-localization with Cu/Zn SOD in the glia limitans (C, confocal image) and in the ventricle walls (B: third ventricle; arrow: tanycyte). Microglial cells and endothelium (arrow), identified with tomato lectin histochemistry, were negative for Cu/Zn SOD (D: cortex; confocal image). Scale bars in A: 40 μm; in B, C and D: 20 μm.
Finally, western blots showed a significant developmental increase in the total amount of brain Cu/Zn SOD between P9 and P16 (Fig. 3B), evidenced as a single band of 16 KDa corresponding to the expected molecular mass of the enzyme monomer.
Figure 3 Total Cu/Zn SOD expression in control, and saline or NMDA injected postnatal cortex. Western blot (A) and semi-quantification of total levels of Cu/Zn SOD protein from intact, saline-injected, or NMDA-injected brain cortical extracts are represented as percentage of Cu/Zn SOD level in P9 (B). A single band of the expected molecular weight of 16 KDa of the Cu/Zn SOD monomer was observed in all samples (A). The intracortical injection of saline solution induced a trend towards the transient upregulation of Cu/Zn SOD which showed a peak at 10 hours post-injection (B: solid circles). In contrast, after NMDA injection, total cortical Cu/Zn SOD expression showed a trend towards rapidly diminishing (A, B: triangles). This trend of Cu/Zn SOD reduction at 4 hours after NMDA injection in total cortical extracts is comparable to the % of lesioned cortex which was around 20% (not shown). However, at later time points, Cu/Zn SOD was significantly upregulated peaking at 7 days (168 hours) post-lesion. In intact control brain (B: opened circles), total enzyme level significantly increased from postnatal day 9 to postnatal day 16. (# p < 0.05 in relation to P9 control animals and * p < 0.05 in relation to NMDA 4 hours).
Control intracortical saline injection caused a local and transient increase in Cu/Zn SOD immunoreactivity mainly in cortical neurons (data not shown) 10 hours later surrounding the injection site. A trend towards a rapid and transient increase could also be observed by western blots (Fig 3B).
Cu/Zn superoxide dismutase expression after excitotoxic injury to the immature brain
As shown before, intracortical NMDA administration is a model of excitotoxic damage that triggers rapid neuronal death and tissue injury, which expands rostro-caudally and includes part of the cortex, corpus callosum, dorsal striatum, septum and rostral hippocampus [33,41].
In contrast to saline injected animals, as observed both by western blot tendencies (Fig. 3) and immunohistochemistry (Fig. 4, 5) at 2–4 hours after NMDA injection, the lesioned neural parenchyma showed a drastic downregulation of total Cu/Zn SOD immunoreactivity in neuronal cells, which returned to basal levels 24 hours after the lesion and increased significantly thereafter due to expression in astroglial cells. Interestingly, the early decrease in neuronal Cu/Zn SOD immunoreactivity at 2–4 hours (Fig. 4A–B) was accompanied by a slight condensation of nuclear chromatin (Fig. 4C–D) and changes in NeuN neuronal marker (Fig. 4F–G), in the absence of apparent signs of neuronal degeneration or TUNEL staining (Fig. 4H). At later time points, from 10 hours, degenerating neurons still showed downregulated Cu/Zn SOD immunoreactivity, but displayed condensed nuclei, nuclear blebbing (Fig. 4E) and also DNA fragmentation observed by TUNEL staining (Fig. 4I). Downregulation of Cu/Zn SOD and signs of neuronal degeneration were observed from 10 hours in primary cortical degenerating areas and from 1 day post-lesion in secondary degenerating regions such as the cortical, striatal and hippocampal penumbra (Fig. 5A). However, the total Cu/Zn SOD enzyme level increased from 1 day post-lesion, reaching a maximum induction after 3–7 days (Fig 3B and 5B), due to astroglial upregulation of the enzyme. Cu/Zn SOD was induced in the soma and proximal projections of the most hypertrophied and most intensely GFAP-immunopositive astroglial cells of the whole degenerative area including the white matter (Fig. 5C), but not in slightly activated astrocytes with lower GFAP immunoreactivity. This elevated expression continued at 5 and 7 days post-lesion when most astrocytes of the degenerative area were strongly hypertrophic and formed the glial scar (Fig. 5D). Only scattered reactive microglial cells expressed Cu/Zn SOD at 3 days after lesion, but most reactive microglial cells of the lesioned parenchyma remained negative.
Figure 5 Cell-specific Cu/Zn SOD expression in the late excitotoxicaly lesioned immature brain. One day after the lesion (A), neuronal bodies were condensed in the lesioned zone (*) when compared to the penumbra zone (Pen). Moreover, the lesion core (*) showed very low or no expression of Cu/Zn SOD. In contrast, 3 days after injection, cortical expression of Cu/Zn SOD increased in the whole lesion (B, *). Neurons in the penumbra (B, Pen) and reactive hypertrophic astrocytes (C) within the degenerative core (*) expressed Cu/Zn SOD after 3 days. These GFAP-positive hypertrophic astrocytes were the main cell type expressing Cu/Zn SOD in the lesion core (D). This pattern of expression was maintained until 7 days post-lesion (the last time analyzed), when the glial scar was evident. Scale bars in A-D: 20 μm.
Discussion
In the last decade, progress has been made regarding the complex evolution of damage in the developing brain [38] and how it differs with adult brain injury. In this sense, one of the divergences proposed is that immature brain deals poorly with oxidative stress. In this study, we have shown that the expression of the key antioxidant enzyme Cu/Zn SOD is mainly found in neuronal cells. When damage occurs, a strong neuronal downregulation of this enzyme precedes both neuronal cell death and the subsequent Cu/Zn SOD upregulation in astroglial cells.
Cu/Zn SOD in the intact immature brain
It is well known that total brain levels of antioxidant enzymes vary throughout life [32,41,42]. Accordingly, brain glutathione peroxidase and Mn SOD were reported to increase during the first month of postnatal rat life, and to continue increasing slightly during adulthood and aging [28,43-46]. Catalase has been reported to peak around the first week of life and then decline to reach a plateau by the first month [43,44]. Regarding Cu/Zn SOD, it is known that its levels increase rapidly after birth, peaking around the second postnatal week, in agreement with our western blot studies. Later on, Cu/Zn SOD decreases slightly to reach adult levels, but increases slightly again in aging [42,44,45]. Thus, it seems clear that immature rat brain has a different balance of antioxidant enzymes, which reach the adult overall pattern only after the first month of life.
We have shown that in the postnatal brain the majority of Cu/Zn SOD immunoreactivity is observed in neuronal somas with less intense staining present in the nuclei, sparing the nucleoli, as has been reported earlier for adult animals [12-17,47]. Accordingly, even though changes in Cu/Zn SOD levels occur in postnatal development, the cell type distribution of this enzyme does not change. Neurons are the brain cells with the highest oxygen consumption, constantly submitted to oxidative stress as it is suggested by O2-. mediated oxidation of hydroethidine [9], or by the presence of nitrotyrosine [48,49]. Accordingly, it is not surprising that high levels of Cu/Zn SOD are observed in a wide number of neuronal populations, specifically in the large neurons with high energetic requirements, like pyramidal neurons, or catecholaminergic neurons which are submitted to elevated oxidative stress due to catecholamine metabolism [50]. Very recently, a site for the pro-inflammatory transcription factor NFκB has been reported at the human Cu/Zn SOD promoter, which can induce its expression [51]. Accordingly, in the CNS, neurons are the main cells that display constitutively activated NFκB [52,53], and its activity is required for their survival [54]. In addition, the other important cellular superoxide dismutase, the Mn SOD, is also enriched in subsets of neurons [55], although different from those enriched in Cu/Zn SOD.
No immunoreactivity for Cu/Zn SOD was observed in the GFAP-expressing astrocyte population of the neural parenchyma in the immature brain, as has been previously reported for adult brain [13-15,17,47], and despite the fact that immature astrocytes differ from adult astrocytes. This lack of Cu/Zn SOD expression contrasts with the higher tolerance of astrocytes to oxidative stress [48,56]. Accordingly, and in comparison with neuronal cells, astrocytes in culture have been shown to have higher levels of glutathione and the lipophilic antioxidant vitamin E [57], and they are capable of synthesizing their own glutathione from cysteine [58], mechanisms that probably confer upon astrocytes an elevated antioxidant status and increased resistance towards oxidative stress, despite the absence of detectable levels of Cu/Zn SOD. Noteworthy, Cu/Zn SOD immunoreactivity is only found in glia limitans astrocytes and in GFAP-positive tanycytes, which could be attributed to the contact of these astrocytes with non-CNS molecules, which are also known to induce the expression of several other activation markers observed previously in these regions, like GFAP overexpression, vimentin or metallothioneins [41]. Regarding microglial cells, neither resting ramified microglia nor amoeboid microglial cells found in the immature brain showed Cu/Zn SOD immunoreactivity, as has been shown for adult resting microglial cells [12,17,47]. In addition, we were unable to find consistent immunoreactivity for Cu/Zn SOD in white matter oligodendrocytes of immature brain, as has been described in the adult [12,17,47]. The lack of this enzyme could help explain the well known high sensitivity of oligodendrocytes to excitotoxicity and oxidative stress [59], especially in the immature brain where white matter injury is thought to be the underlying mechanism of brain damage [38].
Cu/Zn SOD in the injured immature brain
After an excitotoxic injury to the postnatal brain, we have observed a dramatic and rapid neuronal downregulation of Cu/Zn SOD in the NMDA injection site, which is early evident 2–4 hours after injection in neurons that only show slight and very early signs of degeneration and are negative for TUNEL staining. In fact we have previously shown that these neurons display, 10 hours after NMDA injection, NFκB activation and COX2 upregulation, suggesting that they are still active and functional [53,60]. The Cu/Zn SOD downregulation also coincides with neuronal nitration [48] suggesting endogenous O2-./peroxynitrite formation at these very early time points in compromised neurons. Although to our knowledge this is the first study describing the expression of Cu/Zn SOD after immature brain damage, studies on adult brain injury have also shown, as early as 4 hours after transient cerebral ischemia, Cu/Zn SOD immunoreactivity downregulation in striatum and cortex [37] and in neurons of the hippocampal CA region [13]. Moreover, 24 hours after injection of kainate to adult rat hippocampus, Cu/Zn SOD immunoreactivity is also downregulated in neurons of CA, despite an absence of apparent neuronal degeneration at that time-point [14]. Accordingly, it is known that superoxide scavengers protect from injury at these early time points [23,26,61]. Although the mechanism whereby this rapid downregulation occurs is not clear, it appears that it could be mediated by oxidative stress, as PC12 cells treated with H2O2 rapidly (after 4 hours) downregulate Cu/Zn SOD [51]. Interestingly, hypothermia, the most powerful neuroprotective strategy known, not only inhibits the rapid downregulation of Cu/Zn SOD after a traumatic brain injury but also induces its over-expression [36]. Most surprisingly, this effect is specific for Cu/Zn SOD, and in fact hypothermia induces a less significant upregulation of other antioxidant enzymes such as catalase and glutathione peroxidase in comparison with non-hypothermic brain.
Contradictory results have been reported regarding the toxicity of O2-. after hypoxic/ischemic injury to the immature brain. Whereas slightly worsened neuropathological outcome was observed in transgenic mice overexpressing Cu/Zn SOD and submitted to severe hypoxia/ischemia [27], several antioxidant molecules including SOD mimetics as O2-. dismuting metalloporphyrins were shown to be neuroprotective [26]. One hypothesis is that Cu/Zn SOD transgenic mice produce excess H2O2 that in the immature brain is not cleared by the upregulation of the glutathione peroxidase as has been reported for adult animals [28,62]. However, an additional explanation would be that as the postnatal brain express increased amounts of the NMDA receptor [63], whose activation leads to increased O2-. generation [34,35], an initial enhanced oxidative stress would occur. Taken together, this data suggest that in the immature brain subjected to ischemia/excitotoxicity, neurons at the lesion zone are very early submitted to an elevated oxidative stress, and therefore Cu/Zn SOD downregulation contributes to the further amplification of cell damage and neuronal cell death.
Although Cu/Zn SOD expression remains very low in compromised neurons, a return to normal expression levels is seen by 24 hours after lesion, and an increase in total enzyme level is observed later on. This secondary Cu/Zn SOD induction is due to upregulation in reactive hypertrophic astrocytes within the lesion site. We have shown in previous studies that these reactive astrocytes display activated NFκB from 10 hours after the excitotoxic lesion [53], which could contribute to the induction of the Cu/Zn SOD observed here. We have also shown that the hypertrophic Cu/Zn SOD-overexpressing astrocytes are heavily nitrated, and also display metallothionein I-II expression, suggesting an elevated rate of oxidative stress in this particular group of cells [48]. Our findings in immature brain are in accordance with studies of adult brain damage that have shown induction of Cu/Zn SOD and Mn SOD in astrocytes several days after focal ischemia [37] excitotoxicity [14,18], or in Alzheimer's disease and Down's Syndrome [16]. In addition to the upregulated enzymatic antioxidant defences, astrocytes have been shown to produce their own glutathione and also provide neurons with cysteine, a rate-limiting precursor in neuronal glutathione synthesis [58]. Thus, astrocytes seem to be the main cell type increasing the total antioxidant capabilities in the nervous tissue after a lesion, which can in addition explain their elevated resistance to cell death after an injury. In this sense, previous studies showed that Cu/Zn SOD-overexpressing astrocytes have increased resistance to oxidative damage [64] and attenuated oxidative inhibition of glutamate uptake [65], allowing for a maintenance of their physiological functions after a lesion.
Regarding microglial cells, it is somehow surprising that only a reduced number of reactive ameboid microglial cells express Cu/Zn SOD, and that this occurs very transiently, as in some circumstances activated microglia produce large amounts of oxygen radicals after a lesion including O2-. [66].
We believe that the results presented here highlight the importance of in vivo cell-localization studies for antioxidants, as some of the reactive species like O2-. do not diffuse across cell membranes and thus will most probably react within the cell where it is formed.
Conclusion
In conclusion, we show that in the intact immature rat brain during the plasticity window, Cu/Zn SOD is mainly expressed in neurons, though it is also expressed at the central nervous system boundaries like the glia limitans or the ependymal cells. Moreover, we show that brain Cu/Zn SOD expression levels vary after an excitotoxic injury: it is rapidly downregulated in neurons, rendering the affected neurons even more susceptible to oxidative damage, and later on it is upregulated in highly hypertrophic astrocytes. Therefore, as no changes in cell specificity are found in the immature versus the adult brain, further studies would be needed to elucidate the mechanisms underlying the different susceptibility to oxidative damage in these two lesion paradigms.
List of abbreviations
SOD: superoxide dismutase; O2-.: superoxide; H2O2: hydrogen peroxide; NO.: nitric oxide; COX: cycloxygenase; CNS: central nervous system; NFκB: nuclear factor kappa B; NMDA: N-methyl-D-aspartate; TUNEL: terminal dUTP nick end labelling; DAPI: 4, 6-diamino-2-phenylindole; GFAP: glial fibrilary acidic protein.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
HP carried out part of the brain lesions and animal work, performed most of the immunohistochemical and western blot studies, conceived the study and drafted the manuscript. LA carried out part of the brain lesions and animal work, participated in the design of the study and helped to draft the manuscript. MF carried out part of the immunohistochemistry and helped to draft the manuscript. BC and BG coordinated and supervised the development of the study, were responsible for the project giving economical support and helped in the last version of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We would like to thank M. A. Martil for his excellent technical help. This work was supported by Spanish Ministry of Education (DGES BFI2002-02079) and "la Caixa" 00/074-00. H.P. is currently a F.I. fellow of the Generalitat de Catalunya (AGAUR).
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| 15929797 | PMC1164430 | CC BY | 2021-01-04 16:38:22 | no | J Neuroinflammation. 2005 Jun 1; 2:12 | utf-8 | J Neuroinflammation | 2,005 | 10.1186/1742-2094-2-12 | oa_comm |
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Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-4-231589288610.1186/1475-2875-4-23ResearchThe unexpected importance of mosquito oviposition behaviour for malaria: non-productive larval habitats can be sources for malaria transmission Menach Arnaud Le [email protected] F Ellis [email protected] Antoine [email protected] David L [email protected] Fogarty International Center, National Institutes of Health, Bethesda, MD 20892, USA2 Université Pierre et Marie Curie, Inserm U707, 27 rue Chaligny, 75012 Paris, France2005 13 5 2005 4 23 23 14 1 2005 13 5 2005 Copyright © 2005 Menach et al; licensee BioMed Central Ltd.2005Menach et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Mosquitoes commute between blood-meal hosts and water. Thus, heterogeneity in human biting reflects underlying spatial heterogeneity in the distribution and suitability of larval habitat as well as inherent differences in the attractiveness, suitability and distribution of blood-meal hosts. One of the possible strategies of malaria control is to identify local vector species and then attack water bodies that contain their larvae.
Methods
Biting and host seeking, not oviposition, have been the focus of most previous studies of mosquitoes and malaria transmission. This study presents a mathematical model that incorporates mosquito oviposition behaviour.
Results
The model demonstrates that oviposition is one potential factor explaining heterogeneous biting and vector distribution in a landscape with a heterogeneous distribution of larval habitat. Adult female mosquitoes tend to aggregate around places where they oviposit, thereby increasing the risk of malaria, regardless of the suitability of the habitat for larval development. Thus, a water body may be unsuitable for adult mosquito emergence, but simultaneously, be a source for human malaria.
Conclusion
Larval density may be a misleading indicator of a habitat's importance for malaria control. Even if mosquitoes could be lured to oviposit in sprayed larval habitats, this would not necessarily mitigate – and might aggravate – the risk of malaria transmission. Forcing mosquitoes to fly away from humans in search of larval habitat may be a more efficient way to reduce the risk of malaria than killing larvae. Thus, draining, fouling, or filling standing water where mosquitoes oviposit can be more effective than applying larvicide.
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Background
Malaria is responsible for 700,000 to 2.3 million deaths each year, mainly among children [1]. It is caused by four species of Plasmodium, protozoan parasites that are most common in the tropics, especially Africa, and are transmitted between humans by the bites of female Anopheles mosquitoes. Thus, the distribution of Anopheles mosquitoes is an important factor in determining the prevalence of Plasmodium infections in humans. At large spatial scales (i.e. 100–1,000 kilometers), the distribution of malaria is best described by climate: warm, humid places with standing water support large mosquito populations and high malaria prevalence. At local scales (i.e. 100 metres to one kilometre), the risk of malaria is determined by mosquito behaviour and ecology, especially the distribution of blood-meal hosts and water. Mosquitoes alternate between blood feeding and oviposition, and suitable hosts and water are heterogeneously distributed [2]. Thus, human biting reflects the mosquitoes' commute to complete its gonotrophic cycle, as well as inherent differences in the attractiveness, suitability and distribution of blood-meal hosts [3]. Here, mathematical models are used as conceptual tools to explore mosquito oviposition behaviour and the availability of water as an explanation for variability in the risk of malaria.
Mathematical models have played an important role in malaria epidemiology. The mathematical models of Ross illustrated the role of mosquitoes in the dynamics of malaria, placing mosquito control at the centre of anti-malaria intervention strategies [4]. The basic concepts of the entomological inoculation rate (EIR), vectorial capacity and the basic reproductive number for malaria (R0) were all based on mathematical models of malaria transmission, linking entomology and malaria epidemiology [5]. Several studies and mathematical models have emphasized the role of heterogeneous biting in the dynamics and control of malaria [6,7]. Approximately 20% of the human population contributes 80% of the net transmission of malaria, because mosquitoes bite some people more than others [8].
Heterogeneous biting is due, in part, to the ecology and behaviour of Anopheles mosquitoes. A number of surveys, field and lab experiments have been carried out to better understand which cues are responsible for mosquitoes' differential attraction [9]. Mosquitoes emerge from water sources and then fly to a blood-meal host, locating a host using a set of cues, including host movement, odour, CO2 and body temperature. Thus, the proximity of households to larval habitat [10], domestic animals [11,12], human avoidance and defensive behaviour [13,14] and individual attractiveness, depending mainly on odour [15,16] or infection status [7], help to explain why some humans are bitten more often than others. Studies have most consistently reported gradients in vector density away from the breeding sites: in the vicinity of the aquatic habitat, the number of adult mosquitoes is higher [17,18] as is malaria prevalence [19,20] without being always correlated with clinical illness [21]. Thus, vector dispersal is driven by the search for oviposition sites as well as the search for hosts: the distance vectors have to fly to lay their eggs influences the radius of control measures [22].
One factor that has been neglected in these studies is the oviposition behaviour of mosquitoes. The cues used by any Anopheles species to select the sites at which they oviposit between blood-meals remain poorly understood, except in very general terms. For example, Anopheles arabiensis and Anopheles gambiae s.s. typically breed in very transient habitats like shallow sunlit fresh water pools or human-made habitats [23], though they may also be common in rice fields [24,25]. In contrast, Anopheles funestus breeds mainly in marshes and other types of sheltered habitats that contain vegetation [26,27]. What is suitable one week may become unsuitable the next, due to abiotic (e.g. drying or flooding) or biotic factors (e.g. increased predation or competition). Furthermore, eggs, larval instars and pupae may have different ecological requirements. The standard way to locate the "breeding sites" of malaria vectors is to look for larvae, sample them and identify their species. Often, most of the sites that seem most suitable may be unoccupied by immature Anopheles of any stage, at least temporarily. Breeding sites are prone to change, e.g. in accord with agricultural development, deforestation or irrigation [28]. Environmental management [29] allows for vector control focusing on long-term change in vector habitat (draining breeding sites) or on using means that reduce vector reproduction, survival or abundance (i.e. spraying breeding sites with larvicide) [30]. Water source reductions may have played a role in eliminating malaria from Israel, the United States and Italy [31].
It seems clear that larval habitat should be a focal point for malaria transmission, but what are the effects of non-productive water sources where mosquitoes oviposit but eggs fail to develop to adults (e.g. if sprayed with larvicide or desiccated), compared to truly productive water sources (breeding sites)? Does the presence of these unsuitable water sources increase or decrease malaria prevalence? Here, the heterogeneous distribution of water and mosquito oviposition behaviour are explored as factors in heterogeneous biting.
Methods
1. Model
Here, a recent spatial model for malaria epidemiology on heterogeneous landscapes was modified by incorporating a more detailed description of the gonotrophic cycle into models for mosquito infection dynamics [32] (Figure 1). Let S denote the density of susceptible (i.e. uninfected) mosquitoes, L denote the density of latent (i.e. in the incubation period: from the onset of infection to the beginning of the infective period) and I the density of infective mosquitoes. Whatever her state of infection, the mosquito alternates between the activities of blood-meal feeding and ovipositing. Fed, gravid mosquitoes, denoted with subscript f, have taken a blood-meal and seek a place to oviposit, while unfed mosquitoes, denoted with subscript u, have recently oviposited and seek a blood-meal host.
Figure 1 Malaria transmission dynamics within a patch between mosquito and human population. Mosquito population (in blue): Mosquitoes emerge from a water source uninfected and unfed (Su). Susceptible, unfed mosquitoes feed at rate Au and they are then considered fed and susceptible (Sf), unless the blood-meal infects the mosquito with malaria (with probability cX) in which case they become latent and fed (Lf). Latent mosquitoes become infectious at the rate θ, regardless of whether they are fed or unfed (Lf to If, or Lu to Iu). Fed mosquitoes, regardless of their infection status, return to being unfed after ovipositing, at rate Af. (Su to Sf, Lu to Lf, or Iu to If). All mosquitoes die at a rateμ. Human population (in green): Susceptible Human (H) may become infective (X) after an infective bite at a rate b.Au. They would return to the susceptible state at a rate r.
Spatial heterogeneity was incorporated by subdividing the landscape into i patches in an array or grid (see below), with subscripts denoting the mosquito location and state. Thus, Ii, f describes the density of fed, infective mosquitoes in patch i (Eq. 1). Let Ai,u, denote the rate (speed of an event over time) at which unfed mosquitoes feed on a human host in patch i, if one is available; upon biting, the mosquito changes state from "unfed" (u) to "fed" (f). Similarly, let Ai,f denote the rate at which fed mosquitoes oviposit in patch i, if water is available; upon ovipositing, mosquitoes change state from fed to unfed. Thus, if hosts and water are available in patch i, the expected time to find a host is Ai,u-1, and the expected time to oviposit is Ai,f-1, so the duration of one gonotrophic cycle in patch i is Ai,f-1 + Ai,u-1. Note that the human biting rate (HBR) in each patch includes biting only by unfed mosquitoes: HBRi = Ai,u (Si,u + Li,u + Ii,u) / Hi, where Hi denotes human density in the ith patch. In this model, the entomological inoculation rate (EIR) in each patch includes biting only by infective, unfed mosquitoes, . Similarly, the sporozoite rate in unfed mosquitoes is defined as ratio of unfed infective mosquitoes to total number of unfed mosquitoes .
Let εi denote the local emergence rate of adults. If εi > 0, a patch was considered to be a productive source for mosquitoes. Some patches might have water where mosquitoes can oviposit, but no adults emerge. Thus, if Ai,f > 0, but εi = 0, the patch was called a non-productive water source. Obviously εi = 0 if no water was available. Thus, it is implicitly assumed that the emergence rate of adult mosquitoes is regulated at the pre-adult stages, not limited by the availability of eggs.
It is assumed that mosquitoes move among patches [33], depending on their gonotrophic state and the availability of hosts and water. Since water or hosts might not be available in some patches, Ai,f and Ai,u denote the local biting rates, subject to host availability [34] and local oviposition rates subject to the availability of oviposition habitat [35]. If humans were not available, Ai,u = 0, and if water was not available, Ai,f = 0: a mosquito in an unfed or fed state, respectively, would migrate to another patch in search of a blood-meal host or larval habitat (see below). Thus, the residence time in each disease state is longer if no host or no water is available. Otherwise, the parameters assume a positive value (Table 1). It is assumed that emigration of mosquitoes depends on the presence of water for fed mosquitoes and on human density for unfed mosquitoes. Let w denote the per-capita emigration rate of a fed mosquito, which depends on water availability: it describes the expected number of patches a mosquito would cross in one day if no water were available w = 0 if water is available). Similarly, it is assumed that the migration of unfed mosquitoes is a function of local human density, Hi. Let γ denote the per capita emigration rate of an unfed mosquito; thus γ = ςe-ψHi, where ς corresponds to the maximum daily number of patches a mosquito would visit in a day if no humans were available and ψ describes her responsiveness to human density. Another parameterωi, j describes the proportion of mosquitoes leaving patch j that fly into patch i; thus, . It is assumed that ωi,j = 0 unless two patches are adjacent and that mosquitoes move in a random direction. The same migration rates were applied for mosquitoes without regard for infection status. Let Ω(Cij) denote the total migration rate for mosquitoes in state C and location i; for example, represents the net migration of fed susceptible mosquitoes moving from patch i, and the net migration of unfed susceptible mosquitoes from patch i.
Table 1 Values, definition and bounds of the parameters used in the model and for the sensitivity analysis (* indicates the parameters used in the multivariate sensitivity analysis). The parameters' values were chosen to mimic an infection by Plasmodium falciparum carried by An. gambiae s.s. in an adult.
Symbol Definition Values (bounds) References
r-1 Human recovery period 100 days [47, 48]
Au Human biting rate 0.5 bites.mosquitoes-1.day-1 [1, 7, 47]
b Probability that a bite leads to infection among humans 0.5 [49]
c Probability that a bite leads to infection among mosquitoes 0.15 [49]
ς ψ Migration rate during host seeking 10 patches, 2 (This paper)
w* Migration rate during oviposition 10 (1–17) patches (This paper)
Af-1* Resting period before oviposition 2 (1–3) days [1, 45, 46]
μ-1* Mosquitoes lifespan 10 (5–20) days [47, 48]
θ-1 Incubation period 10 days [7, 47-49]
Finally, it is assumed that a susceptible mosquito biting an infective human becomes infected with probability c, that latent mosquitoes become infective at the rate θ and that mosquitoes die at rate μ. Let Xi denote the proportion of humans in patch i who are infective, assuming no latency (i.e. no pre-patent period) and no delay between the appearance of merozoites and gametocytes. Let b denote the probability that a bite by an infective mosquito produces a human infection, and r denote the rate at which a human infection is cleared; in other words, it is assumed that the recovery period is exponentially distributed with average duration r-1. The infection dynamics of malaria in mosquitoes and humans over time and space, including the mosquito gonotrophic cycle, are described by the following equations:
Parameter values are listed in Table 1. The emergence rate was manipulated such that the overall ratio of mosquitoes to humans was 2. The equations were numerically solved over a period of four years; by that date, the system of equations was at the equilibrium . The resulting static spatial distributions of the variables were plotted on two kinds of hypothetical landscapes.
First, simulations of malaria dynamics were performed on a linear array of 17 patches. Humans were uniformly distributed in patches 2 to 16 and absent from the edges. For mosquitoes, three scenarios were considered:
(a) Patch 1 was a productive water source. No water was available elsewhere.
(b) Patch 1 and Patch 9 were productive water sources. No water was available elsewhere.
(c) Patch 1 was a productive water source. Patch 9 was a non-productive water source. No water was available elsewhere.
This landscape is similar to that in a recent study in Tanzania that evaluated mosquito dispersion within three hamlets and showed that marked vectors dispersed differently in relation to the distribution of breeding sites [36].
Second, simulations of malaria dynamics were performed on a square grid of 100 patches (10 × 10). All 10 patches on the left side of the grid were productive water sources (e.g. a stream or pond edge). A non-productive water source was located near the centre of the grid and the human population was uniformly distributed on all the patches except on the left side of the grid. Among the total number of mosquitoes leaving a patch, it was assumed that 80% were randomly flying to patches that shared a side (up, down, left and right patches) and 20% to patches that shared a corner (on the diagonal). This scenario is based on a real-world example: we simulate a village away from a river where the breeding sites are mainly located [37] and with a water source in its centre that is non-productive because it was sprayed with larvicide or prone to desiccation.
2. Sensitivity analysis
A sensitivity analysis on the linear array from the scenario (c) above was performed. The evaluation of the influence of water on mosquito migration and distribution focused on the segment of the mosquito population that was fed and seeking a site to oviposit. A multivariate sensitivity analysis [38] to assess the impact of the parameters that describe the behaviour of fed mosquitoes on the proportion of infected humans was performed; parameters examined were the migration parameter w (the expected number of patches a mosquito would fly without water), the oviposition parameter Af-1 (the time between taking a blood-meal and oviposition) and mortality μ-1 (mosquito lifespan). These three parameters were sampled in accord with a Latin Hypercube Sampling (LHS) scheme [39]. The distribution of malarial infections in humans was computed using 10,000 sets of parameters drawn from a uniform distribution, with bounds described in Table 1. To evaluate the impact of uncertainty in these parameters we calculated the 5th and 95th percentile of the resulting distribution of the human malaria prevalence for each patch. The Partial Rank Correlation Coefficients (PRCCs) were also calculated using the 10,000 values for each parameter and the 10,000 predicted values in malaria prevalence over each patch. A univariate sensitivity analysis on the three parameters described above was performed using the extreme values.
Results
The distribution of risk along the linear array of patches, as measured by EIR, is related to the distribution of productive water sources and non-productive water sources. EIR is proportional to the density of unfed, infective mosquitoes (see Methods) and thus followed the same distribution (Figure 2). When mosquitoes emerged from a single productive point source (scenario a), EIR peaked in the vicinity of the source (daily EIR = 0.22 in patch 2, Figure 2a). The presence of a second productive water source at an intermediate distance (scenario b) produced a bi-modal distribution of infective mosquitoes (EIR = 0.13 in patch 2 and EIR = 0. 17 in patch 9, Figure 2b). A similar bi-modal distribution was observed when a non-productive water source was located at the same intermediate distance (scenario c, EIR = 0.18 in patch 2 and EIR = 0.14 in patch 9, Figure 2c). Thus, a non-productive water source near a mosquito productive water source acts as a focal point for malaria transmission, even if no adults emerge.
Figure 2 Distribution of the ratio of infectious mosquitoes (I) to Human (H), of malaria prevalence (PIH: Proportion of infected humans) and of the sporozoite rate for unfed mosquitoes among various aquatic habitats. a) One productive water source in patch 1. b) Two productive water sources in patch 1 and 9. c) One productive water source in patch 1 and one non-productive water source in patch 9. Human population was uniformly distributed from patch 2 to 16. The dotted black lines correspond to the ratio of fed infectious mosquitoes (If) and the dashed black lines to the ratio of unfed infectious mosquitoes Iu. The solid black lines correspond to the malaria prevalence (PIH) and solid red lines to the sporozoite rate for unfed mosquitoes. Dark gray bars represent the presence of a productive water source and light gray bars the presence of a non-productive water source.
The proportion of infected humans (PIH) followed the same trend as the distribution of EIR. The presence of a productive water source (Figure 2b) or a non-productive water source in patch 9 (Figure 2c) led to similar distributions of the proportion of infected humans, ranging from 85% to 90% in patches 2 and 9. Thus, productive and non-productive water bodies generate similar levels of increased risk of malarial infection. The sporozoite rate in unfed mosquitoes increases slightly with distance from a productive water source (Figure 2a). The presence of a non-productive water source at an intermediate distance results in a substantially increased sporozoite rate (Figure 2c). The difference between the two is the absence of young, non-infectious mosquitoes at the non-productive source.
Among the various scenarios, the overall maximum ratio of infective mosquitoes (fed plus unfed mosquitoes) to humans ranged from 0.85 (patch 2, Figure 2a) to 1.2 (patch 9, Figure 2b) at the maximum point for malaria transmission. In this model, the distributions of fed and unfed mosquitoes differed; overall, the distribution of fed and unfed mosquitoes both reflected the underlying distributions of productive water sources and non-productive water sources. Yet, the ratio of mosquitoes to humans was negatively correlated when water but no humans were available (patches 1 & 2; Figure 2a, b, c); the ratio of fed to unfed mosquitoes is high at the productive water source (Figure 2a; If = 0.78 in patch 1), and the unfed mosquitoes were mainly in the adjacent patch (Iu = 0.44 in patch 2). Thus, no infective unfed mosquitoes were present where only water but no humans were found.
Similar patterns emerged on the grid. Two areas were identified as high-risk zones. High values of daily EIR were observed in the patches next to the stream (ranging from EIR = 0.09 to EIR = 0.14). A second high-risk area was focused around the non-productive water source (EIR = 0.23, Figure 3a). Notably, the highest values of EIR were observed at the non-productive water source, not next to the stream where mosquitoes emerged. EIR decreased with the distance from water, whether it was a productive water source or non-productive water source (Figure 3a). Malaria prevalence also reflected EIR. Along the stream, malaria prevalence ranged from 81.8% to 87.2% and at the non-productive water source malaria prevalence was 92.1% (Figure 3b). This underlines a high level of spatial clustering in malaria risk distribution. Note in Figure 3 that the highest variation in malaria prevalence occurred as annual EIR ranged from 0 to 33 and prevalence varied from 0% to 82%; such ranges in EIR values were found over short spatial scales in the simulations, just two patches away from the productive water sources and one patch away from the non productive water source. For larger EIR values, (33 to 86), prevalence changed only 10%, ranging from 82% to 92%.
Figure 3 Malaria risk map in a 10 by 10 grid assumed to be a village: Productive water sources are located all along the left side of the grid and a non-productive water source is located in the centre of the grid. a) The map is based on annual EIR values. b) The map is based on the Proportion of Infected Humans (PIH).
The results of the multivariate sensitivity analysis on the linear array demonstrated that the risk of malaria varied, depending on the value of the parameters. The 5th and 95th percentile curves followed the same trends as the point estimate. In the vicinity of the non-productive water source (up to three patches away), malaria prevalence varied up to 49% (figure 4a). Because EIR is sensitive to the oviposition rate, increasing the resting time has a protective effect with respect to malarial infection (PRCCs = -0.18). Increasing the maximum number of patches flown through in search of an oviposition site (PRCCs = 0.27) and increasing lifespan (PRCCs = 0.28) increase the prevalence of malarial infection. All these correlation coefficients were significant (p < 0.0001, t-test). The univariate sensitivity analysis showed the great impact of the maximum mosquito flight distance; at small values, the second peak in the non-productive water source was substantially lower (PIH = 42.5% for w = 1 vs. PIH = 87.5% for w = 10) (Figure 4b). When mosquitoes flew a longer distance, they would bite along the way and a larger proportion would find and stay at a further water site. The impact of the mosquito lifespan and the oviposition rate had less impact on human prevalence (Figure 4c and 4d).
Figure 4 Proportion of infected humans according to various sets of oviposition parameters. (The number of empty patches a fed mosquito would fly over each day in order to oviposit, mortality rate of fed mosquitoes, time to oviposit): One productive water source is located in patch 1 and one non-productive water source in patch 9 (scenario c). a) Multivariate sensitivity analysis. The thick solid line represents the distribution of the PIH using the realistic values. The triangles represent for each patch the 95th and 5th percentile of the 10,000 simulations using the sets of simulated parameters. The squares represent the median of these 10,000 simulations. b) Univariate sensitivity analysis on the maximum number of patches w. The dashed lines represent the prevalence distribution for the extreme values (w = 1 and w = 17) c) Univariate sensitivity analysis on the time to oviposit Af-1. The dashed lines represent the prevalence distribution for two extreme values (Af-1 = 1 day and Af-1 = 3 days). d) Univariate sensitivity analysis on the mortality rate μ-1: the dashed lines represent the prevalence distribution for two extreme values (μ-1 = 5 days and μ-1 = 20 days).
Discussion
It has been demonstrated that the availability of water and associated mosquito oviposition behaviour can play an important role in determining the distribution of malaria risk. In some cases, proximity to water where mosquitoes oviposit increases the risk of malaria, whether or not the eggs develop into adults. In other words, a non-productive site for adult mosquito emergence can be a source for malaria. More generally, as would be expected, malaria prevalence would be higher close to water bodies. The transmission potential of mosquitoes is maximized when water and humans are both available.
Because mosquitoes return to water to oviposit, water bodies become a starting point in the search for a blood-meal host. Since mosquitoes fly until they find a host, EIR declines sharply away from water in the model and the heterogeneous distribution of larval habitat produces large variations in EIR over relatively short distances, in agreement with the results from field studies [40]. A review of the literature showed that adult mosquitoes may exhibit high dispersal rates between villages [41] and may fly up to 5 km, but half of the flights were within a 1 km radius [42]. An analysis encompassing surveys from all over Africa showed that annual EIR ranged from 0 to 702 and the malaria prevalence from 7% to 94.5% and that, as in this model, beyond a threshold, increases in EIR value did not affect malaria prevalence [43,44].
The parameter values were consistent with studies of Anopheles species, but substantial variation exists among species, locations and at the same location over time [1,7,45-49]. The aim of the research was to investigate the influence of oviposition behaviour on the spatial distribution of infective mosquitoes; substantial uncertainty remains about strategic aspects of mosquito behaviour, such as how mosquitoes locate and choose a place to oviposit. Many cues could make mosquitoes oviposit in non-productive water sources, including larval crowding and the ability of a mosquito to detect it, the inability to detect unsuitable habitat, habitat desiccation, wind, mosquito physiological status, and many other factors. The search for water in which to oviposit may be a sensitive point in the gonotrophic cycle because the mosquitoes are heavier, with higher energy expenditures and perhaps higher mortality. Thus, increases in flight distances for gravid mosquitoes may increase per-capita mortality rates and thus diminish transmission capacity. Another interesting result involved the effect of the maximum flight distances during this search for an oviposition site. For short flight distances, mosquito distributions reflected the distribution of larval habitat, true productive water sources. As flight distances increased, mosquito distributions resembled the distribution of water, including non-productive water sources as well as productive water sources (data not shown). However, increasing maximum flight distances beyond a threshold (about eight patches per day) had little impact on the distribution of EIR.
This research focused on the ecology and behaviour of Anopheles mosquitoes, and how the heterogeneous distribution of water bodies influences the heterogeneity in their biting. Mosquito populations fluctuate with weather and climate, increasing in the wet season and decreasing in the dry season [50]. Seasonality was ignored to better focus on the impact of oviposition behaviour on the risk of malaria transmission. For the same reason heterogeneous human populations were not considered [51]. Oviposition is one of many factors determining the distribution of risk, but it should be considered as a possible reason for mosquito aggregation, one that would interact with other factors such as seasonality and heterogeneous human distributions. For example, the distribution of oviposition habitat may become more heterogeneous during the dry season, leading to increased mosquito aggregation around water. The availability of larval habitat is sometimes correlated with household density as the number of breeding sites may increase with density up to some threshold [52]. Finally, mosquito memory may limit oviposition in unsuitable habitats [53]. It has been demonstrated that mosquito dispersal might be restricted by a tendency to return to known locations for oviposition, that is productive water sources. Nevertheless, even if vector learning counterbalances the possibility that mosquitoes oviposit in non-productive sites, short vector life spans and the ephemeral nature of suitable larval habitat make aggregation around non-productive sites a potentially important factor. In future research, model refinements could include improvements to the representation of vector biology by adding an explicit resting compartment and different mortality rates as functions of vector disease status. These refinements would further improve our understanding of how far mosquito dispersal can be observed and how large a control area should be explored.
Based in part on Macdonald's mathematical models, the Global Malaria eradication campaign focused on increasing adult Anopheles mortality using DDT and more specifically on reducing adult survival rates [47]. Some earlier workers had also emphasized the importance of distinguishing vector from non-vector species and identifying their actual breeding sites, so that targeted, sustainable anti-vector programs could replace ineffective or inefficient generalized anti-mosquito approaches. Ross used a mathematical model to conclude that, "...in order to counteract malaria anywhere we need not banish Anopheles there entirely...we need only to reduce their numbers below a certain figure" [54]. Though few of his contemporaries paid attention to this idea of threshold densities of Anopheles, some applied it in successful programs of environmental management [55]. Eliminating water in the neighbourhood of humans would force mosquitoes to commute longer distances, decreasing the human feeding rate and increasing mortality during the extrinsic incubation period, hence, decreasing vectorial capacity. Because a non-productive water body can be a source for malaria, an intervention that eliminates water where mosquitoes may oviposit, or fouls the water to deter oviposition, would be more effective for malaria control than using larvicide to reduce mosquito density. Even though larvicides may have adverse effects by killing larval predators [56], treating distant water sources with larvicide might provide a complementary control strategy. This concept is similar to zooprophylaxis [57]; it has been argued that cattle, treated with insecticide or not, provide efficient control if situated between humans and larval habitat and far enough from dwellings [58].
Conclusion
The approach previously described provides a framework for mapping the risk of malaria based on fine-grained maps of water and humans [59]. Such methods provide a tool for mapping risk and planning intervention. An important issue in developing these maps is to identify which biological details are necessary to include and which details can be omitted. These models suggest that malaria risk is highest in the vicinity of water where mosquitoes oviposit, a useful observation with great public health implications if true productive larval habitat is harder to identify.
Authors' contributions
ALM refined the model, performed the sensitivity analysis, carried out the simulations and wrote the manuscript. FEM actively participated in the follow-up of the study and helped to draft the manuscript. AF helped to draft the manuscript. DLS designed and supervised the study, built the model and helped to write the manuscript.
Acknowledgements
We would like to thank Rebecca Freeman Grais for her help in improving the manuscript.
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J NanobiotechnologyJournal of Nanobiotechnology1477-3155BioMed Central London 1477-3155-3-41592704710.1186/1477-3155-3-4Short CommunicationStability of beating frequency in cardiac myocytes by their community effect measured by agarose microchamber chip Kojima Kensuke [email protected] Tomoyuki [email protected] Kenji [email protected] Department of Life Sciences, Graduate school of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan2005 31 5 2005 3 4 4 19 11 2004 31 5 2005 Copyright © 2005 Kojima et al; licensee BioMed Central Ltd.2005Kojima et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
To understand the contribution of community effect on the stability of beating frequency in cardiac myocyte cell groups, the stepwise network formation of cells as the reconstructive approach using the on-chip agarose microchamber cell microcultivation system with photo-thermal etching method was applied. In the system, the shapes of agarose microstructures were changed step by step with photo-thermal etching of agarose-layer of the chip using a 1064-nm infrared focused laser beam to increase the interaction of cardiac myocyte cells during cultivation. First, individual rat cardiac myocyte in each microstructure were cultivated under isolated condition, and then connected them one by one through newly-created microchannels by photo-thermal etching to compare the contribution of community size for the magnitude of beating stability of the cell groups. Though the isolated individual cells have 50% fluctuation of beating frequency, their stability increased as the number of connected cells increased. And finally when the number reached to eight cells, they stabilized around the 10% fluctuation, which was the same magnitude of the tissue model cultivated on the dish. The result indicates the importance of the community size of cells to stabilize their performance for making cell-network model for using cells for monitoring their functions like the tissue model.
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Introduction
Development of reliable cell-based assay is important for high-speed, low cost drug screening. However, the conventional method using cells are still unstable and thus are still under trial to make reliable cell models showing the same extent of reliability as tissue/organ models. As heart is one of the most important organs for toxicology in drug screening, the properties of heart cells are examined and reported strenuously. For example, it has been reported that one beating cell can influence the rate of a neighbor with which it makes contact, and that a group of heart cells in culture, beating synchronously with a rapid rhythm, can act as pacemaker for a contiguous cell sheet from earlier tissue culture studies of cardiac myocyte cells [1]. Although these former results predicted that the importance of a rapidly beating region of tissue acts as pacemaker for a slower one and examined how the synchronization process of two isolated beating cardiac myocytes [2] and that the importance of the communication of each cells in the cell-network, the community size effect could not be measured successfully using the conventional cultivation method on the culture dish plate. As means of attaining the spatial arrangement of cardiac myocytes even during cultivation, we have developed a new single-cell based cultivation method and a system using agarose microstructures, based on 1064-nm photo-thermal etching [3-5]. Using this system, we measured the time course of synchronization process of adjacent two beating cardiac myocyte cells connected by 2-μm-width pathways, and found the synchronization of two cells occurred 90 min after their first physical contact [6,7].
This paper reports the cell network size effect (community effect) for stabilizing their beating intervals using our on-chip single-cell-based cultivation assay with stepwise modification of micorcultivation chamber structures during cultivation.
Results
The schematic drawing of the on-chip single-cell-based cultivation assay is illustrated on Figure 1. Our system consists of three parts: temperature-controlled cell cultivation part, in which single cardiac myocytes are arranged in each microchambers of agarose cell cultivation chip; photo-thermal etching system to fabricate both the microchambers and microchannels by melting of a porting of the 5-μm-thick agarose layer by the spot heating of a 1064-nm infrared focused laser beam; and image acquire/analysis system. Figure 2 summarizes the chip design and cell cultivation procedure on the chip. In this example, we arranged nine 30-μm-diameter microchambers on the chip and the adjacent chambers are connected by the microchannels, which are fabricated during cultivation by the photo-thermal etching. As the 1064-nm laser beam is not absorbed by either water or agarose, it melts a portion of the agarose on the chromium thin layer because only the chromium layer absorbs the beam. Using this non-contact etching, we can easily make microstructures such as holes and channels within only a few minutes without using any cast molding process. The melting of agarose by laser occurred as follows (see Figure 2). We have focused the 1064-nm infrared laser beam on the agarose layer on the glass slide to melt the agarose at the focal point and on the light pathway until the shape of the hole for cells formed (Figure 2a). When the focused beam was moved parallel to the chip surface, a portion of agarose around the focal spot of laser melted and diffused into water (Figure 2b). After the heated spot had been moved, a channel was created at the bottom of the agarose layer connecting the two adjacent holes (Figure 2c). A microscope observation confirmed that the melting had occurred, and then either the heating was continued until the spot size reached the desired one, or the heating position was shifted to achieve the desired shape. Individual cardiac myocytes were cultivated in each hole of the agarose microchambers on the chip as shown in Figure 2d. In our method we added micro channels one by one to connect neighbouring cardiac myocytes in adjacent microchambers during their cultivation. To improve the attachment of the cells to the bottom of the microchambers, collagen-type I (Nitta gelatin, Osaka, Japan) was coated on the chromium layer of the chip before coating of agarose layer (Figure 3).
Figure 1 Schematic drawing of the on-chip single-cell-based cardiac myocyte network cultivation assay. A phase contrast microscope was used to measure the contraction rhythm of the cardiac myocytes and to melt a portion of agarose layer on the chip for the stepwise network formation of cells in the microchambers. The spontaneous beating rhythm of cultured cardiac myocytes is evaluated by the image analysis system, in which the change of the size (cross-section of volume) of each cardiac myocyte is analyzed and recorded every 1/30 s.
Figure 2 Schematic drawing of the photo-thermal etching method (a-c), and the design cell cultivation chip (d).
Figure 3 Cross-sectional view of the cell cultivation chip.
A micrograph on Figure 4 shows the nine isolated cells cultivated in the nine-chamber agarose microcultivation chip (24 h following the beginning of the cultivation) and two isolated, independently beating cardiac myocytes coming into contact through the microchannel. During the cultivation, the beating frequency change of the cell indicated by the arrow was continuously observed.
Figure 4 Optical micrograph of 24-h cultivation of nine cardiac myocyte cells' network.
The time course change of the heart beating caused by the stepwise additional network formation was as shown in Figure 5. The frequency of isolated single cardiac myocyte cell's beating was fluctuated from the mean value (2.1 Hz, see Figure 5a). The beatings of the two-cell network (Figure 5b), in which one of the two cardiac myocytes was the same one as shown in Figure 5a. This two-cell network showed the much more stabilized beating than the isolated condition. We have further added more channels to the above two-cell network to form nine-cell network model. As shown in the graph in Figure 5c, the nine-cells network showed almost constant frequency of beating. The result of nine sets of different samples (one of the five sets was shown in Figure 5) summarized in Figure 6. As shown in the graph, the fluctuation of beating frequency was decreased according to the additional network formation of cardiac myocyte cells. In other words, the increase of network size improves the stability of the beating frequency. It should be noted that the magnitude of fluctuation for nine-cell network was about 10%, which was the same value of tissue culture sample of the same sample cultivated on the plate (data not shown). Moreover, we think we confirmed that the stepwise additional formation of channels during the cultivation by photo-thermal etching did not damage their beating ability just same as the stepwise formation of the neural network we have reported in previous papers [5,8].
Figure 5 Time courses of cardiac myocytes of isolated single cell (a), two-cells network (b), nine-cells network (c), respectively.
Figure 6 Cell network size dependence on the fluctuation of cardiac myocytes' beating frequency. In the graph, the mean values and standard deviations of nine sets of samples are indicated.
The above results indicate two facts. First, for the single cell based research as the cell-network model, the consideration of community size of cell group is important for acquiring the reliable, stable data. Second, the community size required for reliable measurement like tissue model is not so large, i.e. nine cells were enough in this case. That might mean that smaller than we expected number of cells is required to produce the same community effect as in the tissue model. And therefore the potential for creating individual-cell-based cell-network model might be practical for reliable drug screening assay especially for human organ model. Moreover, it should be noted that we succeeded in the separating of two factors affecting the beating synchronization in this system, gap-junction connection and physical stretching. In other words, using this system we can measure the effect of gap-junction connections on synchronization clearly. If we could not remove the effect of physical stretching caused by the physical contact of neighbouring cells, we could hardly clarify the effect of chemicals to inhibit gap-junction connections. Because the cluster of cells still synchronized by their physical stretching even after gap-junction was inhibited (data not shown). From this viewpoint, our system might be most beneficial in drug screening.
In conclusion, we applied the 1064-nm photo-thermal etching method and made the on-chip agarose single cell microcultivation system for generating cardiac myocyte networks of different size, which is important for understanding the community effect of rhythm synchronization. Using the system, we for the first time observed the differences in the synchronization process of cardiac myocyte cells and their dependence on the community size. This system can potentially be used in the biological/medical fields for cultivating next generation of networks from individual cultured cells and measuring their properties.
Materials and Methods
Ventricular myocytes were isolated from 1- to 3-day-old neonatal Wistar rats as described earlier [6,7]. Hearts were excised from rats anaesthetized with ethyl ether and transferred to phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) containing 0.9 mM CaCl2 and 0.5 mM MgCl2. after which ventricles were separated and minced into small fragments. Tissue fragments were further dissociated by incubating them twice with PBS containing 0.25% collagenase (Wako, Osaka, Japan) for 30 minutes at 37°C. The cell suspensions were transferred to a cell culture medium (DMEM [Invitrogen Corp., Carlsbad, CA USA] supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml Streptomycin) at 4°C. The cells were filtered through a 40-μm nylon mesh and were centrifuged at 180 g for 5 minutes at room temperature. The cell pellet was re-suspended in a HEPES buffer (20 mM HEPES, 110 mM NaCl, 1 mM NaH2PO4, 5 mM glucose, 5 mM KCl, and 1 mM MgSO4, pH 7.4). The cardiac myocytes present in the suspension were separated from other cells (i.e., fibroblasts and endothelial cells) by the density centrifugation method. The cell suspension was then layered onto 40.5% Percoll (Amersham Biosciences, Uppsala, Sweden) diluted in the HEPES buffer, which had previously been layered on 58.5% Percoll diluted in the buffer. The cell suspension was then centrifuged at 2200 g for 30 minutes at room temperature. Cardiac myocytes were retrieved from the interface of the 40.5% and 58.5% Percoll concentrations. Retrieved cells were then re-suspended in the cell culture medium. The 5-μl of the suspension, which was diluted to achieve a final concentration of 3.0 × 105 cells/ml, was plated into the chip and each cardiac myocyte was picked up by a micropipette and manually introduced into each microchamber in the chip. Then, it was incubated on a cell-cultivation microscope system at 37°C in a humidified atmosphere of 95% air and 5% CO2. It should be noted that, because the microchamber sidewalls were made of agarose, the cells could not easily pass over the chambers. A phase-contrast microscope was used both to measure the contraction rhythm (i.e. beating frequency) of the cardiac myocytes, and to record the shape of cell network in microchambers.
The spontaneous contraction rhythm of cultured cardiac myocytes was evaluated by a video-image recording method. Images of beating cardiac myocytes were recorded with a CCD camera through the use of a phase contrast microscope. The sizes (cross-sectional area of cell) of cardiac myocytes, which changed considerably with contraction, were also analyzed and recorded every 1/30 s by a personal computer with a video capture board and estimated their beating phenomenon by the change of their cross-sectional area sizes [6,7].
Authors' contributions
KK and TK carried out the microchamber design, cell preparation, single cell cultivation and observation, image analysis. They were equally contributed for this article. KY conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.
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| 15927047 | PMC1164432 | CC BY | 2021-01-04 16:38:05 | no | J Nanobiotechnology. 2005 May 31; 3:4 | utf-8 | J Nanobiotechnology | 2,005 | 10.1186/1477-3155-3-4 | oa_comm |
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Respir ResRespiratory Research1465-99211465-993XBioMed Central London 1465-9921-6-501593264410.1186/1465-9921-6-50ResearchMultiple exposures to swine barn air induce lung inflammation and airway hyper-responsiveness Charavaryamath Chandrashekhar [email protected] Kyathanahalli S [email protected] Hugh G [email protected] Philip [email protected] Baljit [email protected] Immunology Research Group and Departments of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, S7N 5B4, Canada2 Large Animal Clinical Sciences, University of Saskatchewan, Saskatoon, S7N 5B4, Canada3 Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, S7N 5B4, Canada2005 2 6 2005 6 1 50 50 2 10 2004 2 6 2005 Copyright © 2005 Charavaryamath et al; licensee BioMed Central Ltd.2005Charavaryamath et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Swine farmers repeatedly exposed to the barn air suffer from respiratory diseases. However the mechanisms of lung dysfunction following repeated exposures to the barn air are still largely unknown. Therefore, we tested a hypothesis in a rat model that multiple interrupted exposures to the barn air will cause chronic lung inflammation and decline in lung function.
Methods
Rats were exposed either to swine barn (8 hours/day for either one or five or 20 days) or ambient air. After the exposure periods, airway hyper-responsiveness (AHR) to methacholine (Mch) was measured and rats were euthanized to collect bronchoalveolar lavage fluid (BALF), blood and lung tissues. Barn air was sampled to determine endotoxin levels and microbial load.
Results
The air in the barn used in this study had a very high concentration of endotoxin (15361.75 ± 7712.16 EU/m3). Rats exposed to barn air for one and five days showed increase in AHR compared to the 20-day exposed and controls. Lungs from the exposed groups were inflamed as indicated by recruitment of neutrophils in all three exposed groups and eosinophils and an increase in numbers of airway epithelial goblet cells in 5- and 20-day exposure groups. Rats exposed to the barn air for one day or 20 days had more total leukocytes in the BALF and 20-day exposed rats had more airway epithelial goblet cells compared to the controls and those subjected to 1 and 5 exposures (P < 0.05). Bronchus-associated lymphoid tissue (BALT) in the lungs of rats exposed for 20 days contained germinal centers and mitotic cells suggesting activation. There were no differences in the airway smooth muscle cell volume or septal macrophage recruitment among the groups.
Conclusion
We conclude that multiple exposures to endotoxin-containing swine barn air induce AHR, increase in mucus-containing airway epithelial cells and lung inflammation. The data also show that prolonged multiple exposures may also induce adaptation in AHR response in the exposed subjects.
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Background
Respiratory diseases in agricultural workers are one of the earliest recognized occupational hazards [1]. Swine farmers work in confined buildings in close proximity to a large number of pigs and are exposed to toxic gasses such as ammonia and hydrogen sulfide, and to high levels of dust and endotoxins [2]. Exposure to such toxic bio aerosols including endotoxins in the barn air is a risk factor for the development of chronic respiratory symptoms and lung dysfunction [3-5]. Workers exposed to barn air report significantly higher frequencies of respiratory symptoms, cold, chest illness and pneumonia [2,3]. The severity of lung irritation and respiratory symptoms increases during winter and is also related to the number of working hours [6]. Single, 3–5 hour exposure of naïve, healthy, non-smoking subjects to swine barn air increases IL-6 in serum and IL-6 and IL-8 in nasal lavage and inflammatory cells in bronchoalveolar lavage fluid (BALF) [7,8]. Furthermore, pig barn dust stimulates IL-8 and IL-6 release from human bronchial epithelial cells in vitro [9]. Collectively, these data show that a single exposure to the barn air initiates acute lung inflammation.
Although swine barn workers are repeatedly exposed to barn air, majority of studies have focused on the acute pulmonary effects of single exposure [7,10]. Multiple exposures to barn air are linked to chronic lung inflammation including chronic bronchitis, decline in lung function and higher incidence of asthma [3,11,12]. Pig farmers with an average exposure history of 10.5 years and a daily exposure of 6.6 hours show significantly lower forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) compared to unexposed control subjects [3]. Interestingly, acutely exposed naïve volunteers, show significantly more lung dysfunction, AHR, increase in cytokine levels and inflammatory cell numbers in blood and nasal lavage compared to the pig barn workers repeatedly exposed to the barn air [7,13,11]. These data suggest induction of an adaptive response in subjects repeatedly exposed to the barn air.
There is paucity of data on in situ cellular and molecular changes following multiple exposures to pig barn air. This is largely because of lack of an animal model to investigate the physiological impact of exposure to barn air. Therefore, we decided to undertake an in vivo single and multiple exposure study using rats to characterize cellular and molecular responses. We hypothesized that single and multiple exposures to swine barn air will induce lung inflammation and a decline in lung function. The data show that single and multiple exposures cause increase in AHR, inflammatory cells in BALF, mucus cells in the airways and lung inflammation.
Methods
Rats and treatment groups
The experimental protocols were approved by the University of Saskatchewan Campus Committee on Animal Care and experiments were conducted according to the Canadian Council on Animal Care Guidelines. Specific pathogen-free, six-week-old, male, Sprague-Dawley rats (Charles River Laboratories, Canada) were maintained in the animal care unit of Western College of Veterinary Medicine. Rats were randomly divided into four groups (n = 6 each). All personnel involved in collection and analyses of samples were blinded to the treatment groups.
Exposure to swine barn air
We selected a regular commercial swine barn in the village of Aberdeen in Saskatchewan. The barn chosen for study had 60 dry sows and three boars. These pigs were fed with ground barley. Rat cages were hung from the barn ceiling at an approximate height of two meters above the floor. Groups of rats were exposed to barn air either for eight-hours for one-day, 5 days or for four cycles of 5 days (8 hours/day) each followed by 2 days in normal ambient air after every cycle. When rats were not exposed to the barn air, they were kept with the control animals in normal ambient air. Control rats were treated similarly except that they were not exposed to the barn air.
Barn air sampling for endotoxin analysis
We sampled the barn air twice weekly to determine endotoxin levels as described previously [14]. Briefly, we collected airborne barn dust onto a pre-weighted, binder-free glass fibre inline filter (SKC Edmonton, Canada) hung at the level of rat cages. Barn air was drawn through the sampler (DuPont Air Sampler) for eight hours on each sampling day. The average flow-rate of the sampler was noted before and after each sampling period. Filters were desiccated before and after sampling. After weighing, the filters were placed in 50-mL polypropylene centrifuge tubes and were stored at 4°C until endotoxin analysis.
Endotoxin analysis was performed as described elsewhere [14]. Briefly, the filters with collected dust were washed individually in centrifuge tubes with 10 mL of sterile pyrogen-free water (DIN 00624721; Astra Pharma Inc; Mississauga, ON, Canada) followed by incubation for one-hour at room temperature in a sonicating water bath. Serial two-fold dilutions of the supernatant fluids were analyzed for Gram-negative bacterial endotoxin using an end-point assay kit as recommended by the manufacturer (model QCL-1000; Cambrex Bioscience Inc.; Walkersville, MD). The endotoxin standard (Escherichia coli O111:B4) was used in duplicate at four concentrations (0.1 to 1.0 endotoxin units (EU)/mL) in each assay to generate the standard curve. The lower detection limit was 0.1 EU/mL, which is equivalent to 1.0 EU per filter. The sampling time and flow rate were used to calculate the concentration of endotoxin in air (EU/m3).
Viable microbial count
Viable microbial count was achieved using a six-stage viable cascade impactor (Graseby, Smyrna, GA). Air samples were collected from the vicinity of the rat cages hung from the ceiling of the barn by using a vacuum pump that was attached to the impactor capable of drawing air through the impactor at a rate of 1 ft3/ min (28.3 L/min). Six media plates of Tryptic Soy Agar with 5% sheep's blood were placed in the sampler and airborne microbes were directly collected onto 20 mL of media in 100 mm petri dishes. The air was drawn through the impactor for a duration of 15 seconds. The procedure was performed twice every week. The cascade impactor was cleaned thoroughly with 70% ethanol between each collection event. The plates were incubated at 37°C for 18–24 hours, and the colonies were counted using the positive-hole method correcting for microbial coincidence [15].
Measurement of airway hyper-responsiveness
AHR was measured in awake control and exposed rats in response to increasing concentrations of methacholine (Mch) using head-out whole body plethysmography [16]. Air was supplied to the head and body compartments of the plethysmograph through a small animal ventilator (Kent Scientific, Litchfield, CT) and changes in respiratory airflow were monitored using a flow sensor (TRS3300; Kent Scientific, Litchfield, CT) linked via a preamplifier and A/D board (Kent Scientific) to a computer-driven real-time data acquisition/analysis system (DasyLab 5.5; DasyTec USA, Amherst, NH). The compartment of the plethysmograph, which accommodates the animal's head, was connected to an ultrasonic nebuliser (UltraNeb 99; Devilbiss Co., Somerset, PA) to expose the rats to Mch (Sigma Chemical Co. St. Louis, MO) [17,18]. Each rat was sequentially exposed to aerosols of saline alone (Mch 0 mg/ml) and then increasing doses of Mch diluted in saline (0.75, 1.5 and 3.0 mg/mL) and Flow@50%Tve1 (lung airflow at 50% of the expiratory tidal volume) was noted for saline and each of the Mch concentrations.
Blood, bronchoalveolar lavage, tissue collection and processing
At the end of the exposure period, rats were euthanized (1 mg xylazine and 10 mg ketamine / 100 g) and blood, BALF and lung samples were collected. Blood was collected by cardiac puncture for differential and total leukocyte counts. BALF was collected by washing the whole lung with 3 ml of ice cold Hanks Balanced Salt Solution (Sigma Chemicals Co., St. Louis, MO). Three pieces from each lung lobe (left and right) were fixed in 4% paraformaldehyde for 16 hours and embedded in paraffin for light microscopy. Haematoxylin and eosin stained sections were used for histopathological evaluation of pulmonary inflammation.
Quantification of mucus-producing cells
Mucus-producing goblet cells were quantified in lung sections stained with Periodic-acid Schiff (PAS) reagent [19]. Images were captured with the 20× objective lens of an Olympus microscope (Olympus BH2) connected to a digital camera (DVC Digital Camera, Diagnostic Video Camera Company, Austin, TX 78736-7735). The images were analysed using image analyses software (Northern Eclipse, version 6; Empix Imaging Inc., Mississauga, ON, Canada). Only those bronchi with a length to width ratio of less than 2.5 were selected for counting PAS-positive cells so as to minimize the error that might arise from tangential sectioning [20]. The PAS-positive goblet cells were counted manually and normalized to the length of the bronchial epithelial perimeter on the basal side, and expressed as the number of PAS-positive cells per mm of basement membrane.
Immunohistochemistry
Lung sections were processed for immunohistochemistry as described previously [21]. Briefly, the sections were deparaffinized, hydrated and incubated with 5% hydrogen peroxide for 30 minutes to quench endogenous peroxidase, treated with pepsin (2 mg/ml in 0.01 N HCl) for 45 minutes to unmask the antigens and blocked with 1% bovine serum albumin for 30 minutes. Sections were incubated with primary antibodies against rat macrophage (1:400; ED-1, Serotec Inc. NC, USA) or monoclonal mouse anti-human smooth muscle actin (1:50; clone 1A4; DAKO A/S, Denmark), followed by appropriate biotinylated or horseradish peroxidase (HRP)-conjugated secondary antibodies (1:150; DAKO A/S, Denmark). Sections incubated with biotinylated antibodies were incubated with HRP conjugated streptavidin (1:300, DAKO A/S, Denmark) before color development. The reaction was visualized using a color development kit (VECTOR-VIP, Vector laboratories, USA). Controls consisted of staining without primary antibody or with isotype matched immunoglobulin instead of primary antibody.
Quantification of macrophages and airway smooth muscle
ED-1 positive macrophages in the septa were counted in 20-high power fields (using 40× objective covering an area of 9.6 mm2). For smooth muscle quantification, a method described by Leigh et al. [22] was followed with a slight modification. A line was drawn along the outer border of the positively stained smooth muscle area and total stained area within that circle was measured using Northern Eclipse image analyses software. Next, a similar line was drawn along the inner border of the airway smooth muscle area to demarcate and measure the stained area. Stained area within the line drawn along smooth muscle inner border was deducted from the stained area within line drawn along smooth muscle outer border, to obtain the total stained area of airway smooth muscle. This total stained area of airway smooth muscle was normalized to the length of the outer perimeter of the airway smooth muscle, and results were expressed as, smooth muscle stained area in mm2 per mm of airway smooth muscle perimeter.
Statistical analyses
All data were expressed as mean ± SD. Group differences were examined for significance using one-way analysis of variance or two-way repeated measures analysis of variance with Fishers LSD as post hoc test (Sigma Stat Version 2.0, SPSS Inc., Chicago, IL 60611). Significance was established at P < 0.05.
Results
Barn air characterization
The mean endotoxin concentration in the swine barn air for the period of exposure was 15361.75 ± 7712.16 EU/m3 of air. The amount of endotoxin in air samples from the room where control animals were kept (normal ambient air) was below the level of detection. The levels of endotoxin in the barn air in our study are much higher than those reported by other researchers [23,3]. The total viable aerobic bacterial counts in the barn air during the exposure period are shown in Table 1. Air samples collected from the room where control rats were kept did not yield any bacterial colonies.
Table 1 The total, respirable and non-respirable aerobic viable bacterial count (CFU/m3 of air sampled) from the barn air
Classification Viable aerobic bacterial count × 104 (CFU/m3 of sampled air)*
Total 12.10 ± 8.47
Respirable 4.85 ± 4.97
Non-respirable 7.26 ± 7.50
* Viable bacterial counts are expressed as Mean ± SD.
Airway hyper-responsiveness (AHR)
Inhalation of increasing concentrations of Mch caused decrease in airflow (Flow@50%Tve1) indicating airway reactivity and broncho-constriction. The data showed group differences in percent decrease in Flow@50%Tve1 (Figure 1; P < 0.001). Both 1- and 5-day exposed rats showed increased AHR compared to controls (P < 0.001) and 20-day exposed (P < 0.05). However, there were no differences in AHR between the control and 20-day exposed (P = 0.207) and 5-day and 1-day (P = 0.249) exposed rats.
Figure 1 Airway hyper-responsiveness. Airway hyperresponsiveness to methacholine challenge in rats was measured using a whole-body head-out plathysmograph. Compared to controls, both 1-day and 5-day (P < 0.001) exposed rats showed increased airway hyperresponsiveness. Compared to 20-day exposed rats, 5-day (P = 0.001) and 1-day (P = 0.014) exposed rats showed increased airway hyper-responsiveness There was no difference between control and 20-day exposed (P = 0.207) and 1-day and 5-day exposed (P = 0.249) rats. *: Significantly different from other groups as indicated by line/s.
BALF cell counts
There were differences in total leukocyte counts in BALF among the four groups (Figure 2A; P < 0.001). The one day exposure group had higher BALF total leukocytes compared to the control, 5-day or 20-day exposed rats (P < 0.001). The 20-day exposed animals contained higher numbers of total leukocytes than control (P = 0.01) and those exposed for 5 days (P = 0.008). BALF total leukocytes were not different between control and 5-day exposed rats (P = 0.932).
Figure 2 Total and differential leukocytes in the bronchoalveolar lavage fluid. Bronchoalveolar lavage was performed on the whole lung using 3 ml of cold HBSS. Cells were counted using a hemocytometer. Cytospins were prepared from BAL fluid and cells were differentiated with Wright's staining. 2A. BALF total leukocyte counts. BALF total leukocytes were different among the four groups (P < 0.001). Compared to controls, 5-day and 20-day exposed, 1-day exposed rats showed increased numbers of BALF total leukocytes (P < 0.001). Rats exposed for 20 days showed increased numbers of BALF total leukocytes when compared to controls (P = 0.01) and 5-day (P = 0.008) exposed rats. 5-day exposed rats did not differ from controls in their BALF total leukocyte numbers (P = 0.932). ** Significantly different from control, 5-day and 20-day exposed rats and * significantly different from control, 1-day and 5-day exposed rats. 2B. BALF absolute neutrophil counts. BALF absolute neutrophil counts were different among the groups (P < 0.001). 1-day exposed rats showed higher BALF absolute neutrophils when compared to control, 5-day and 20-day exposed rats (P < 0.001). 20-day exposed rats showed higher BALF absolute neutrophil count when compared to control rats (P = 0.022). There was no difference between control and 5-day exposed (P = 0.538) and 20-day and 5-day exposed (P = 0.119) rats. ** Significantly different from control, 5-day and 20-day exposed rats and * significantly different from control. 2C. BALF absolute macrophage counts. BALF absolute macrophage count was different among the four groups (P < 0.001). BALF absolute macrophage count was higher in 1-day exposed when compared to control, 5-day and 20-day exposed rats (P < 0.001). 20-day exposed rats showed higher BALF absolute macrophage count when compared to control and 5-day (P < 0.001) exposed rats. There was no difference between control and 5-day exposed rats (P = 0.789). ** Significantly different from control, 5-day and 20-day exposed rats and * indicates significantly different from control and 1-day and 5-day exposed rats. D. BALF absolute lymphocyte count (Figure 2D). BALF absolute lymphocyte count was different among the four groups (P < 0.001). BALF absolute lymphocyte count was higher in 1-day exposed when compared to control, 5-day and 20-day exposed rats (P < 0.001). * Significantly different from other three groups.
The increased BALF total leukocytes in single exposure group, compared to control, 5- and 20-day exposed rats, were characterised by increased absolute neutrophil, macrophage and lymphocyte numbers (Figure 2B-D, P < 0.001). Increased BALF total leukocytes in 20-day exposed rats were characterized by increased absolute neutrophil (from controls, P = 0.022) and macrophage (control and 5-day exposed rats, P < 0.001) numbers. BALF absolute eosinophil numbers did not differ among the four groups (P = 0.178).
Blood cell counts
There was no difference among the groups for total leukocyte counts (Figure 3A; P = 0.090). However, the absolute neutrophil numbers were different among the four groups (Figure 3B; P < 0.001). Rats exposed for 20 days showed higher absolute neutrophil numbers compared to the control and those exposed for 1 or 5 days (P < 0.001). Furthermore, rats exposed for 1 day showed higher blood absolute neutrophils when compared to 5-day exposed rats (P = 0.038). Blood absolute monocyte numbers did not differ among the four groups (Figure 3C; P = 0.122). Blood absolute lymphocyte numbers were different among the four groups (Figure 4D; P < 0.001). Compared to 20-day exposed, control (P = 0.003), 1-day (P < 0.001) and 5-day (P = 0.011) exposed rats showed increased numbers of blood absolute lymphocytes.
Figure 3 Total and differential leukocyte count in blood. Blood total leukocytes were counted using hemocytometer and smears were differentiated with Wright's stain. 3A. Blood total leukocyte count did not differ among the groups (Figure 3A; P = 0.090). 3B. Blood absolute neutrophils count was different among the four groups (Figure 3B; P < 0.001). 20-day exposed rats showed higher blood absolute neutrophils count when compared to control, 1-day and 5-day exposed rats (P < 0.001). 1-day exposed rats showed higher blood absolute neutrophil count when compared to 5-day exposed rats (P < 0.038). Both 1-day (P = 0.073) and 5-day exposed rats (P = 0.678) did not differ from controls. ** Indicate significantly different from control, 1-day and 5-day exposed rats and * indicate significantly different from 20-day and 5-day exposed rats. 3C. Blood absolute monocyte count did not differ among the four groups (Figure 3C; P = 0.122). 3D. Blood absolute lymphocyte count was different among the four groups (Figure 4D; P < 0.001). Compared to 20-day exposed, control (P = 0.003), 1-day (P < 0.001) and 5-day (P = 0.011) exposed rats showed increased numbers of blood absolute lymphocytes. * indicates significantly different from other three groups.
Figure 4 Histopahtological evaluation of lung sections. Histopathological changes in the lungs of swine barn air exposed and control rats were evaluated using hematoxylin and eosin stained sections. Control rat lungs (A) showed no inflammatory cell infiltration. Among the exposed groups, 1-day (B), 5-day (C) and 20-day exposed rats (not shown) showed peribronchiolar neutrophilic (C; arrows) and 5-day (D) and 20-day exposed (not shown) showed eosinophilic (D; arrows and inset) infiltration. Bronchus-associated lymphoid tissue (BALT) in control (E), 1-day and 5-day exposed (both not shown) appeared normal and had no germinal centers, whereas 20-day exposed rat lungs had activated BALT with germinal centers (F; outlined in black line) containing several mitotic cells (F; inset). Original magnification A-C: ×400; D-F: ×100; Insets: ×1000
Histopathology
Lung sections from control rats showed normal histology (Figure 4A) while those exposed for 1 day, 5 (Figure 4B-C) or 20 days (not shown) showed neutrophil infiltration into the lung tissue. Lung sections from 5-day (Figure 4D) and 20-day (not shown) exposed rats manifested perivascular and peribronchial eosinophil infiltration. Bronchus-associated lymphoid tissue (BALT) showed germinal centres and mitotic cells indicating BALT activation in rats exposed for 20 days (Figure 4F) compared to the controls (Figure 4E) or those subjected to 1 and 5 exposures (data not shown).
Mucus cell quantification
Because PAS method stains mucus as pink, it is commonly used as a method to identify mucus-containing cells (Figure 5). Morphometric data revealed more PAS-positive mucus-containing goblet cells in the airways of rats exposed for 5 or 20 days compared to the controls (5-day: P = 0.040; 20-day: P < 0.001) and 1-day (5-day: P = 0.007; 20-day: P < 0.001) exposed rats (Figure 5A-D). Furthermore, rats exposed 20 times contained more airway mucus cells compared to the 5-day exposure group (P < 0.001). There was no difference between control and 1-day exposed rats (P = 0.435).
Figure 5 Quantification of mucus producing cells in the airways. Mucus producing goblet cells in the airways were quantified using PAS staining. Control rats showed no mucus producing cells in the bronchioles (A). 5-day exposed and 20-day exposed rats showed large number of mucus producing cells (B&C; arrows). Quantification of PAS-positive cells showed a significantly higher number of cells in 5-day and 20-day exposed rat lungs compared to the controls (5-day: P = 0.040; 20-day: P < 0.001) and one-day (5-day: P = 0.007; 20-day: P < 0.001) exposed rats (Figure D). Also, the increase in mucus producing cells was higher in 20-day exposed compared to 5-day exposed rat lungs (P < 0.001). Number of mucus producing cells did not differ between control and 1-day exposed rats (P = 0.435). *: Significantly different from control, 1-day and 20-day exposure. **: Significantly different from control, 1-day and 5-day exposure. The bars represent mean ± SD. Original magnification A-C; ×400
Quantification of ED-1 positive macrophages
The numbers of macrophages in the alveolar septa, stained with ED-1 antibody were not different among the four groups (Figure 6, P = 0.350).
Figure 6 Quantification of septal macrophages in the lung. Macrophages were stained using ED-1 antibody. Lungs from control (A), 1-day (not shown in picture), 5-day exposed (B) and 20-day exposed (C) rats appeared to have similar numbers of septal macrophages. To confirm this we quantified ED-1 positive cells in the septum. D: Is a scatter plot showing number of ED-1 cells in the septum, in different groups. The horizontal bars in each group represent the mean for that particular group. There was no difference between the groups (P = 0.350). Original magnification A-C; ×400
Immunohistochemical quantification for smooth muscle actin (SMA)
We used anti-human SMA antibody, which cross reacts with rat tissue to stain smooth muscles around the bronchi, bronchioles and blood vessels. Morphometric analyses showed no differences in smooth muscle area among the groups (Figure 7, P = 0.681).
Figure 7 Airway smooth muscle quantification. The staining pattern for smooth muscle in controls (A), 1-day (not shown in picture), 5-day exposed (B) and 20-day exposed (C) rat lungs appeared similar. D. The stained area of smooth muscle around the bronchioles was measured using image analyses software. The area of smooth muscle was not significantly different between the groups (P = 0.681). Original magnification A-C; ×400
Discussion
We report in vivo and in situ data using an animal model on the effects of single and multiple exposures to the swine barn air. The data show that exposures to swine barn air induce an initial increase in AHR in one and five day exposed rats followed by an adaptive response in 20-day exposed rats; the 20-day group resembled the controls. Swine barn exposure induced lung inflammation in all the exposed groups characterized by infiltration of inflammatory cells, activation of BALT in 20-day exposed rats and an increase in mucus cells in the airway epithelium of 5- and 20-day exposed rats.
Our data show that one and five exposures to barn air induce significantly greater AHR in rats compared to 20 exposures and the unexposed. The AHR observed after 20 exposures was not different from controls. The precise mechanisms of increased AHR following one or five exposures to the barn air and an apparent adaptive response after 20 exposures remain incompletely understood. Previously, it was speculated that similar airway responses in the barn workers are initiated by the endotoxin present in the barn air [7,24]. It is likely that high levels of endotoxin in the barn air observed in our study are partially contributing to lung dysfunction induced in the exposed rats. Endotoxin in house dust has also been identified as a cause of lung dysfunction, which is characterized by increased AHR and inflammation [25]. Notwithstanding the cause of AHR following exposure to the highly complex barn air, there was amelioration of AHR in rats exposed for 20 days in conjunction with persistent inflammation. Previous data from a mouse model of allergic and IL-6 induced lung inflammation have shown dissociation between intensity of AHR and the lung inflammation [26,27]. Thus, our observations show that multiple exposures to barn air, which contains many toxic aerosols including endotoxins and ammonia, initially show an increase in AHR followed by an adaptive response. These data from exposed rats parallel the observations from barn workers who showed initial increase in AHR and decreased FEV1, FVC and mid-expiratory flow (FEV 25–75) followed by an adaptation indicated by less severe AHR [28,29]. Based on the similarity in lung responses following exposure to the barn air, the rat may be a good model to investigate in vivo and in situ cellular and molecular aspects of lung dysfunction in pig barn workers.
Rats, following single and 20 exposures, demonstrated more neutrophils and macrophages in their BALF. Rats exposed 20 times showed activation of BALT compared to the control and those exposed for 1 or 5 times indicating a progression towards chronic inflammation. BALT activation similar to that observed in our study has been reported in chronic bacterial infection [30,31], and following exposure to endotoxin and diesel exhaust [32,33]. Lung sections from all the exposed groups contained perivascular and peribronchial infiltration of inflammatory cells. It is well established that inflammatory cells are recruited in response to expression of adhesion molecules and chemoattractants on activated cells [34]. We believe that high levels of endotoxins measured in our study, in addition to other toxic aerosols such as ammonia, in the barn air may have activated expression of adhesion molecules and chemoattractants, such as IL-8, to promote recruitment of inflammatory cells [35-38].
Lung sections from rats exposed to the barn air for 20 days contained more mucus-containing goblet cells in the airway epithelium compared to the controls, 1 day and five day exposure group. Chronic LPS exposure [39] and many chronic respiratory diseases [40] present mucus hyper-secretion as a hallmark feature of airway inflammation. Such an increased mucus production in the airways is associated with reduced airway caliber, occlusion of small airways, reduced FEV1 [40], impaired gas exchange and compromised muco-ciliary clearance [41]. Our experiments do not identify the causative agent or the mechanisms of increase in mucus-containing goblet cells in the lungs of exposed rats. However, there are some possibilities. First, neutrophilic inflammation, such as one observed in the rats exposed to the barn air, has been shown to increase expression of epidermal growth factor and mucus synthesis [42]. Second, elastase released from activated neutrophils is known to stimulate degranulation of goblet cells and secretion of mucus [43]. Third, eosinophil recruitment, such as that observed in the lungs of 5- and 20-day exposed rats, is associated with goblet cell hyperplasia and increased mucus production in asthma and chronic obstructive pulmonary disease [44-46]. Lastly, chronic exposures to endotoxin, similar to those in our study, increase PAS-positive mucus cells [47,39]. These data show a causal relationship among exposures to the barn air, increased AHR, neutrophil and eosinophil recruitment, activation of BALT and goblet cell hyperplasia in the exposed rats.
Although we observed higher levels of endotoxins in the barn air, our study does not address precise mechanisms of BALT activation in the exposed rats. We believe that the inflammatory and increased AHR outcomes in our study are due to a combined effect of exposure to endotoxins and other toxicants such as ammonia [35,36,48] in the barn air. Although swine barn air contains both gram-positive and gram-negative bacteria [49,50], high levels of endotoxin in the study appear to be an indirect evidence for the presence of high-density of gram-negative bacteria in the barn air. We recorded higher levels of endotoxin in the barn air compared to those previously reported [3,23], which may be an outcome of husbandry practices as well as reduced ventilation in the winter season to conserve heat.
Conclusion
Our data show that, single and multiple exposures to endotoxin rich-swine barn air induce lung inflammation characterized by infiltration of inflammatory cells, increased mucus positive-epithelial cells and activation of BALT in 20-day exposed rats. Furthermore, single and five exposures increased AHR. Because the barn air, in addition to endotoxins, contains dust, ammonia, microorganisms, aeroallergens [51], CO2, moulds [52], H2S [53], microorganisms and associated products such as bacterial cell wall, pig dander, fecal material and feed materials [54], more in vivo animal studies and detailed characterization of the barn air are needed to precisely identify the causative agents and their respective contributions to lung dysfunction and specific interactions of host genome and the environment.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
CC carried out the experiment, AHR, immunohistochemistry, image analyses, statistical analyses and drafted the manuscript. KSJ helped during the experiment, did histopathological evaluation, prepared figures and participated in manuscript preparation. HT participated in the statistical analyses. PW performed endotoxin analysis and helped to calculate total bacterial count in the barn air. BS conceived of the study, participated in its design and coordination as well as manuscript preparation. All authors have read and approved the final manuscript.
Acknowledgements
The work was supported through a grant from Lung Association of Saskatchewan to Dr. B. Singh and other financial support was provided by the Canadian Network for Bacterial Pathogens of Swine. Dr. C. Charavaryamath is a recipient of a Graduate Merit Scholarship from College of Graduate Studies and Research, and a Founding Chairs Fellowship from Institute of Agricultural, Rural and Environmental Health, University of Saskatchewan. Dr. K. Janardhan is a recipient of Graduate Merit Scholarship from the college of Graduate Studies and Research, University of Saskatchewan. The authors thank Ms. Jayda Cleave and Ms. Shelly Kirychuk for technical assistance and Dr. John Gordon for the use of whole body plethysmograph in his laboratory.
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| 15932644 | PMC1164433 | CC BY | 2021-01-04 16:23:26 | no | Respir Res. 2005 Jun 2; 6(1):50 | utf-8 | Respir Res | 2,005 | 10.1186/1465-9921-6-50 | oa_comm |
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Respir ResRespiratory Research1465-99211465-993XBioMed Central London 1465-9921-6-541594386710.1186/1465-9921-6-54ResearchRespiratory rehabilitation after acute exacerbation of COPD may reduce risk for readmission and mortality – a systematic review Puhan Milo A [email protected] Madlaina [email protected] Thierry [email protected] Johann [email protected] Horten Centre, University of Zurich, Switzerland2 Respiratory Division, Respiratory Rehabilitation, and Faculty of Kinesiology and Movement Sciences, Katholieke Universiteit Leuven, Leuven, Belgium2005 8 6 2005 6 1 54 54 17 2 2005 8 6 2005 Copyright © 2005 Puhan et al; licensee BioMed Central Ltd.2005Puhan et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Acute exacerbations of chronic obstructive pulmonary disease (COPD) represent a major burden for patients and health care systems. Respiratory rehabilitation may improve prognosis in these patients by addressing relevant risk factors for exacerbations such as low exercise capacity. To study whether respiratory rehabilitation after acute exacerbation improves prognosis and health status compared to usual care, we quantified its effects using meta-analyses.
Methods
Systematic review of randomized controlled trials identified by searches in six electronic databases, contacts with experts, hand-searches of bibliographies of included studies and conference proceedings. We included randomized trials comparing the effect of respiratory rehabilitation and usual care on hospital admissions, health-related quality of life (HRQL), exercise capacity and mortality in COPD patients after acute exacerbation. Two reviewers independently selected relevant studies, extracted the data and evaluated the study quality. We pooled the results using fixed effects models where statistically significant heterogeneity (p ≤ 0.1) was absent.
Results
We identified six trials including 230 patients. Respiratory rehabilitation reduced the risk for hospital admissions (pooled relative risk 0.26 [0.12–0.54]) and mortality (0.45 [0.22–0.91]). Weighted mean differences on the Chronic Respiratory Questionnaire were 1.37 (95% CI 1.13–1.61) for the fatigue domain, 1.36 (0.94–1.77) for emotional function and 1.88 (1.67–2.09) for mastery. Weighted mean differences for the St. Georges Respiratory Questionnaire total score, impacts and activities domains were -11.1 (95% CI -17.1 to -5.2), -17.1 (95% CI -23.6 to -10.7) and -9.9 (95% CI -18.0 to -1.7). In all trials, rehabilitation improved exercise capacity (64–215 meters in six-minute walk tests and weighted mean difference for shuttle walk test 81 meter, 95% CI 48–115).
Conclusion
Evidence from six trials suggests that respiratory rehabilitation is effective in COPD patients after acute exacerbation. Larger trials, however, are needed to further investigate the role of respiratory rehabilitation after acute exacerbation and its potential to reduce costs caused by COPD.
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Introduction
Acute exacerbations of chronic obstructive pulmonary disease (COPD) represent a major burden for patients and health care systems. For patients, acute exacerbations are a common reason for hospital admissions and severely affect health-related quality of life (HRQL) [1] and prognosis[2]. Mortality rates during hospitalisations are around 10% [3,4] and during the year following a hospitalisation may be as high as 40% [3,5].
From the health care provider's perspective, COPD is resource consuming [6]. A small proportion of COPD patients of around 10% suffering from acute exacerbations accounts for over 70 percent of costs caused by COPD because of emergency visits and hospitalisations [6-8]. The readmission rate is typically high in these high-risk patients. A recent large study found a readmission rate of 63% during a mean follow-up of 1.1 year with physical inactivity amongst the significant predictors for readmissions[9].
Recent position papers of the American College of Physicians and American College of Chest Physicians provided recommendations on the management of acute exacerbations [10,11]. However, a weakness of these papers was that they did not provide recommendations how future exacerbations and hospitalisations could be prevented despite being one of the main goals of COPD management [11,12]. One solution that has been adopted in clinical practice is to provide rehabilitative care after treatment of acute exacerbation including physical exercise, patient education focusing on self-management strategies and psychosocial support. The rationale to offer rehabilitation in patients recently treated for acute exacerbation is to enhance HRQL as in stable COPD patients [13], but also to modify factors associated with increased risk for post-exacerbation morbidity and mortality. A recent study showed that exacerbations results in acute muscle deconditioning and weakness[14]. Hence patients with frequent exacerbations have more pronounced skeletal muscle weakness and a more limited six minute walking distance [15], which is in turn a risk factor for exacerbations and mortality [3,16].
Thus respiratory rehabilitation may have the potential to reduce hospital admissions by improving exercise capacity. It is hence surprising, and in contrast to the large body of evidence supporting respiratory rehabilitation in stable patients [13,17], that the effects of respiratory rehabilitation in patients after acute exacerbation has never been studied systematically. Therefore, our aim was to conduct a systematic review of all randomized controlled trials that compared respiratory rehabilitation after acute exacerbation and usual care.
Methods
Identification of studies
We used five strategies to identify studies including electronic databases, consultations with experts from North America and Europe, our own files, bibliographies of articles that met the inclusion criteria and conference proceedings of the International Conference of the American Thoracic Society and the Congress of the European Respiratory Society.
An information specialist conducted electronic database searches in MEDLINE (Ovid version, New York, New York, from inception to April 2005), EMBASE (DataStar version, Cary, North Carolina from inception to April 2005), PEDRO (online version, University of Sydney, Australia, April 2005) and the Cochrane Central Register of Controlled Trials (Oxford, United Kingdom, 2005, Issue 1). We did not restrict the search to COPD patients with exacerbation only because exacerbation is not indexed as a Medical subject heading term and we feared to miss relevant studies with a narrow search. We used a broad search strategy using the terms "lung diseases obstructive", "chronic obstructive lung disease", "chronic obstructive pulmonary disease", "rehabilitation", "exercise", "exercise movement techniques", "physical endurance", "muscle training", "kinesiotherapy", "clinical trial", "controlled study" and "epidemiologic methods". A detailed search strategy is available on request. We also searched the Science Citation Index database (Web of Science, Thomson ISI, Philadelphia, Pennsylvania) and the "related articles" function of PubMed (National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894) by entering all included studies.
Inclusion criteria
We included randomized controlled trials comparing respiratory rehabilitation of any duration after acute exacerbation of COPD with conventional care. Respiratory rehabilitation programmes needed to include at least physical exercise. We included studies if more than 90% of study participants had COPD. Main outcome measure was unplanned hospital admissions and secondary outcomes included exacerbations, outpatient visits, dyspnea, HRQL as measured by disease-specific or generic questionnaires, functional and maximum exercise capacity, mortality and adverse events during rehabilitation. We did not apply any language restrictions.
Study selection
The bibliographic details of all retrieved articles were stored in a Reference Manager file (Professional Edition Version 10, ISI ResearchSoft, Berkeley, California). We removed duplicate records resulting from the various database searches. Two members of the review team (MAP, MS) independently scrutinized the titles and abstracts of all identified citations (see Figure 1) and ordered the full text of any article that was deemed potentially eligible by one of the reviewers. The two reviewers evaluated the full text of all retrieved papers, made a decision on in- or exclusion and discussed the decisions. Any disagreement was resolved by consensus with close attention to the inclusion/exclusion criteria. We recorded the initial degree of discordance between the reviewers and corrected discordant scores based on obvious errors. We resolved discordant scores based on real differences in interpretation through consensus or third party arbitration.
Figure 1 Study flow from identification to final inclusion of studies.
Data extraction and quality assessment
We performed the data extraction using pilot-tested data forms. One reviewer extracted details about study patients, interventions and outcome measures as well as the results in a predefined data form and the second reviewer checked the data extraction for accuracy. We contacted all authors of the primary studies to obtain missing information. Two reviewers independently evaluated the quality of included trials using a detailed list of quality items assessing components of internal validity (Table 2) [18]. We did not rate the two items "blinding of patients" and "blinding of persons who implements intervention" because patients and treatment providers cannot be blinded in studies comparing respiratory rehabilitation and usual care.
Table 2 Quality assessment
Study Prognostically homogenous study population Concealment of random allocation Prestratification on prognostically relevant variables Description of randomisation procedure Registration of loss to follow-up Registration of co-interventions for each group Blinding of outcome assessors Check success of blinding
Behnke [19, 20] +/- - - - + +/- - -
Kirsten 1998 [22] +/- - - - + +/- - -
Man 2004 [24] +/- + + +/- + - - -
Murphy 2005 [21] +/- + - - + - - -
Nava 1998 [23] +/- - - +/- + - - -
Troosters [25, 26] +/- + - - + - - -
+: Fulfilled; +/-: Partially fulfilled; -: Not fulfilled or no information provided
Methods of analysis and synthesis
We summarized the results of the data extraction and assessment of study validity in structured tables. We pooled trial results using fixed effects models if there was no significant heterogeneity (p ≤ 0.1 with Q statistic for continuous and Cochran chi-squared test for binary outcomes). In anticipation of significant heterogeneity we established a priori hypotheses to explain differences in outcomes across studies. First, heterogeneity may arise from the setting patients were recruited (in- or outpatient treatment of exacerbation), second from different lengths of follow-up, third from different length of the intervention and finally from differences in the methodology of the intervention. Pooled risk ratios and 95% confidence intervals (CIs) were computed by calculating weighted mean differences and pooled risk ratios using STATA (version 8.2, Stata Corp., College Station, Texas).
Results
We show the study selection process and agreement on study inclusion in Figure 1. Out of the 22 potentially relevant articles, we included seven reports (Table 1). Two articles were based on the same trial. One reported the results after six [19] and the other one after 18 months [20]. In five trials, patients were recruited after inpatient care and in one trial [21] after hospital at home treatment for acute exacerbation. Two trials reported on the short-term benefit of inpatient rehabilitation programs [22,23] and four trials had rehabilitation programs of six weeks to six months duration [20,21,24,25]. One trial was published as an abstract only [25], but additional information was available from an earlier publication[26] and from the author. Altogether 140 patients were randomized to the rehabilitation intervention, and 90 were randomized into respective control groups.
Table 1 Characteristics of included studies
Study Population Intervention Follow up Outcomes
Behnke 2000 [19] and 2003 [20] 26 COPD patients (mean age 67 years, 77% males, mean FEV1 = 36% predicted) after inpatient treatment for acute exacerbation. Rehabilitation: Within 4–7 days after admission, inpatient respiratory rehabilitation with endurance exercise (5 walking sessions/day for 10 days), followed by six months of supervised home-based endurance exercise (3 walking sessions/day for 6 months)
Usual care: Standard inpatient care without exercise and standard community care with respirologist. 18 months CRQ, Transition dyspnea index, 6 MWT, hospital readmission, mortality
Kirsten 1998 [22] 29 COPD patients (mean age 64 years, 90% males, mean FEV1 = 36% predicted) after inpatient treatment for acute exacerbation. Rehabilitation: Within 6–8 days after admission, inpatient respiratory rehabilitation with endurance exercise (5 walking sessions/day for 10 days).
Usual care: Standard inpatient care without exercise. 11 days Transition dyspnea index, 6 MWT
Man 2003 [24] 42 COPD patients (mean age 70 years, 41% males, FEV1 = 39% predicted) after inpatient treatment for acute exacerbation. Rehabilitation: Multidisciplinary outpatient respiratory rehabilitation (within 10 days of discharge) with endurance and strength exercise and patient education for 12 weeks (2 sessions/week).
Usual care: Standard community care with respirologist 12 weeks CRQ, SGRQ, Short form survey 36, shuttle walk test, hospital readmission, hospital days, emergency admissions, mortality
Murphy 2005 [21] 26 COPD patients (mean age 66 years, 65% males, mean FEV1 = 40% predicted) after home for hospital treatment for acute exacerbation. Rehabilitation: Supervised home-based respiratory rehabilitation with endurance and strength exercise for 6 weeks (2 supervised sessions/week and daily unsupervised sessions).
Usual care: Standard community care with respirologist 6 months SGRQ, EuroQol, MRC dyspnea scale, shuttle walk test, 3-minute step test, hospital readmission
Nava 1997 [23] 70 COPD patients (mean age 66 years, 73% males, mean FEV1 = 32% predicted, 76% needed mechanical ventilation) admitted to inpatient care for treatment of acute exacerbation. Rehabilitation: Within 3–5 days after admission, inpatient respiratory rehabilitation with four steps of increasing intensity.
Step I, if unable to walk: Mobilisation and strength training for lower extremities.
Step II, if able to walk: Endurance exercise (walking)
Step III, if possible: Endurance exercise (cycling and stair climbing) and respiratory muscle training
IV, if possible: Endurance exercise (cycling at highest tolerated intensity, 2 sessions/day for 3 weeks)
Usual care: Only steps I and II. 6 weeks Dyspnea on exertion, 6 MWT, mortality
Troosters 2002 [25, 26] 48 COPD patients (mean age 62 years, 85% males, FEV1 = 39% predicted) after inpatient treatment for acute exacerbation. Rehabilitation: Outpatient respiratory rehabilitation with endurance and strength exercise for 6 months (3 sessions/week in first 3 months, then 2/week).
Usual care: Standard community care with respirologist. 6 months (6 MWT) and 4 years (survival) 6 MWT, mortality
6-MWT: 6-minute walk test; CRQ: Chronic Respiratory Questionnaire; SGRQ: St. Georges Respiratory questionnaire; MRC: Medical Research Council
Initial agreement of reviewers on quality assessment was 85% for all items (chance corrected kappa = 0.70). All disagreement could be resolved by consensus. The quality of trials was moderate (Table 2). Three trials provided details about the randomisation procedures and three trials about concealment of random allocation, while in none of the trials the outcome assessors were blinded.
Effect on hospital admissions
Figure 2 shows the effect of respiratory rehabilitation on unplanned hospital admissions for each study [20,21,24] and the pooled relative risk ratio of 0.26 (0.12–0.54). The other trials included either only inpatients [22,23] or did not record hospital admissions during the follow-up [25].
Figure 2 Effect of respiratory rehabilitation on unplanned hospital admissions. Boxes with 95% confidence intervals represent point estimates for the risk ratio.
Effect on HRQL
Three trials assessed HRQL using the Chronic Respiratory Questionnaire (CRQ) [20,24] and the St Georges Respiratory Questionnaire (SGRQ) [21,24] (Figure 3). With both instruments, the trials found large effects exceeding the minimal important difference of 0.5 on the CRQ and of 4 on the SGRQ. Weighted mean differences (expressed as points change on a scale from 1 to 7) on the CRQ were 1.37 (95% CI 1.13–1.61) for the fatigue domain, 1.36 (0.94–1.77) for emotional function and 1.88 (1.67–2.09) for mastery. Weighted mean differences for the SGRQ total score, impacts and activities domains were -11.1 (95% CI -17.1 to -5.2), -17.1 (95% CI -23.6 to -10.7) and -9.9 (95% CI -18.0 to -1.7). For the CRQ dyspnea and SGRQ symptoms domain, results were too heterogeneous to be pooled (Q = 6.44, p = 0.01 for CRQ dyspnea domain and Q = 3.50, p = 0.06 for SGRQ symptoms domain), but all studies showed a consistent effect in favor of the rehabilitation intervention.
Figure 3 Effect of respiratory rehabilitation on Health-related quality of life as assessed by the Chronic Respiratory Questionnaire (CRQ) and St. Georges Respiratory Questionnaire (SGRQ). Boxes with 95% confidence intervals represent point estimates for the difference between respiratory rehabilitation and usual care.
Man and Murphy also used generic HRQL instruments and found improvements by respiratory rehabilitation of 10.6 (-0.3 to 21.6) and 20.1 (3.3 to 36.8) on the physical composite and mental composite score of the Short-Form Survey 36 [24] and of 0.18 (95% CI 0.04 to 0.32) with the EuroQol score [21].
Effect on dyspnea
In the trial by Behnke [20], the mean difference between groups on the transition dyspnea index was 6.9 (3.9 to 9.9) at the end of the treatment period and 8.6 (6.3–10.9) after 18 months. Kirsten[22] found significant differences in Transition dyspnea index scores after a short inpatient rehabilitation (p < 0.05, no additional data available) and Nava [23] also observed a significant effect of rehabilitation on dyspnoea (difference between groups 17 mm on visual analogue scale after a 50 meter walk, p < 0.01). Murphy [21] used the Medical Research Council dyspnea scale and also found that respiratory rehabilitation decreased dyspnea by 0.3 although this did not reach statistical significance (95% CI -0.92 to 0.32).
Effect on exercise capacity
All trials showed a significant benefit of respiratory rehabilitation on the six-minute walking distance (Figure 4). We did not pool the results of the six-minute walking tests because of statistically significant heterogeneity (Q = 28.33, p < 0.001), which could not be explained by our a priori defined sources for heterogeneity. The trials reported by Behnke [19] and Kirsten[22] were conducted in the same institution and showed much larger effects (mean effects of 215 and 158 meters on the six minute walking test) compared to the trials of Nava [23] (68 meters) and Troosters [25] (64 meters). All studies showed a consistent benefit in favor of the rehabilitation group, which exceeded the minimal clinically important difference of 53 meters. The meta-analysis of the shuttle walk tests results showed a weighted mean difference of 81 meters (95% CI 48 to 115) between the rehabilitation and usual care groups.
Figure 4 Effect of respiratory rehabilitation on six-minute walking and shuttle walk distance. Boxes with 95% confidence intervals represent point estimates for the difference between respiratory rehabilitation and usual care.
Effect on mortality
The individual study relative risks for mortality ranged from 0.40 (0.18–0.86) to 1.00 (0.07–15.04, Figure 5). The pooled risk ratio was 0.45 (0.22–0.91). Although no significant heterogeneity was present, it should be noted that the length of follow-up differed substantially between these studies. We did not include one trial [23] in the primary meta-analysis because severity of disease of included patients differed considerably from those of the other studies. For this trial a mortality of 20% for patients of either group (12/60 in rehabilitation group and 4/20 in control group) was observed while staying in the respiratory intensive care unit with a mean survival of 18.1 days (SD 7.2) for patients with and 12.4 days (SD 11.1) for patients without rehabilitation (p > 0.05). Of the 12 patients of the rehabilitation group who died, only five started a walking training (stage 2, Table 1). If this trial is included in the meta-analysis the pooled risk ratio is 0.59 (0.34–1.05) favoring the rehabilitation group.
Figure 5 Effect of respiratory rehabilitation on mortality. Boxes with 95% confidence intervals represent point estimates for the risk ratio.
Adverse events
Two trials explicitly recorded adverse events. Neither Man [24] nor Behnke [19] observed adverse events during the rehabilitation.
Discussion
The meta-analyses showed that respiratory rehabilitation after acute exacerbation of COPD reduced the risk for hospital admissions and mortality and led to large improvements of HRQL and exercise capacity.
Strengths of this systematic review include the extensive literature search, rigorous adherence to a predefined protocol and contacts to authors of the included trials who all provided additional information about their data. A limitation is the small number of patients included in the trials and methodological shortcomings that limit conclusions.
The effect of respiratory rehabilitation after acute exacerbation appears to be large. For HRQL and exercise capacity, the effects were well above the threshold for the minimal important difference for the CRQ (0.5 point difference [27]), St. Georges Respiratory Questionnaire (4 points [28]), SF-36 (5 points[29]) and Six-minute walking distance (around 53 meters [30]). In addition, the number of unplanned hospital admissions and mortality was reduced substantially. When one assumes that respiratory rehabilitation improves activity level in patients with COPD, it seems plausible that rehabilitation reduces readmission rate as inactivity has been shown to be a predictor of readmissions[9].
Compared to respiratory rehabilitation in stable COPD patients [13], its effects tend to be even larger after acute exacerbation. Several factors may contribute to this. First, as mentioned above, exacerbations lead to significant reductions in muscle function[14] and quality of life [1]. This initial deterioration may render patients more likely to improve from respiratory rehabilitation. Second, since patients were hospitalized, there may be a deficiency in self-management, or education. This may be partially targeted with the rehabilitation intervention, and patient education, as an additional part of multidisciplinary rehabilitation programs, may be of particular benefit to modify behavior. Indeed, a recent study showed impressive results of a patient management program including home exercises for COPD patients after acute exacerbation [31]. The mean number of hospital admissions per patient was reduced from 1.6 to 0.9 in the year following a hospital admission due to acute exacerbation. It is well known from earlier studies that the recovery period is long even in patients who have no further exacerbations and that another exacerbation within 6 months limits recovery markedly [32]. Our meta-analyses showed that respiratory rehabilitation during the recovery period is superior compared with usual care to improve prognosis and HRQL.
A word of caution is needed when interpreting the current analysis. A clear limitation of the trials is their relatively small sample size. All trials, in particular the trials reported by Behnke [20] and Kirsten[22] showed large effects of respiratory rehabilitation on HRQL and exercise capacity. Small trials tend to overestimate the effect of an intervention compared to large trials [33-36]. This phenomenon can partly be attributed to a publication bias, that is, the fact that small trials are more likely to be published if they show statistically significant treatment effects [37]. On the other hand, methodological shortcomings of small trials such as inadequate generation of the randomisation code, insufficient concealment of random allocation and lack of blinding contribute to discrepancies between the results of single large trials and pooled estimates based on small trials[35]. In our systematic review, the trials had methodological limitations and it cannot be excluded that the estimates provided by the meta-analyses represent overestimations of the effect of respiratory rehabilitation after acute exacerbation.
Larger trials seem justified to challenge the data presented in this article. Such trials should assess the effect of respiratory rehabilitation on unplanned out- and inpatient care but also include data on patient-important outcomes such as HRQL. Conducting trials on respiratory rehabilitation after acute exacerbation is, however, challenging. First, recruitment of patients is difficult because not all of them may want to be randomly allocated to respiratory rehabilitation or usual care in a situation of poor health status. Second, one needs to take into consideration that exercise capacity is particularly low after acute exacerbations[14] so that the exercise program should be designed carefully. Strength exercise and tolerable whole body exercise modalities such as interval exercise may be particularly suitable for these patients [38,39]. Third, the definition of usual care raises a number of difficulties. Patients willing to participate in the trial are likely to have a preference for respiratory rehabilitation. If they are randomized to the control group, they might ask for respiratory rehabilitation at any time during the follow-up. Given the clear benefits of this intervention in stable patients, confirmed in meta-analyses [13], patients should not be refrained from rehabilitative strategies. It would perhaps be ethically justifiable to conduct a large rehabilitation trial in places where respiratory rehabilitation is currently not readily available to the general patient. This appears to be the case in many countries including Switzerland [40], the UK [41] and Canada [42]. These countries are just few examples of countries where the lack of access to rehabilitation has been pointed out as an important caveat in health care. In these places patients could be randomized to additional respiratory rehabilitation or standard treatment by general practitioners and respirologists because respiratory rehabilitation can be offered to a small proportion of COPD patients only. Alternatively relatively short term studies (3–6 months follow-up) could be conducted with re-admission as a primary end point. It has been shown that re-admission occurs often soon after discharge [43,44]. Obviously, such studies could never address mortality as a primary end point, due to a lack of events. Whatever design investigators choose, a careful discussion of ethical and methodological issues is necessary before conducting large trials.
The present data show that respiratory rehabilitation has the potential to reduce the large COPD-related costs due to hospital admissions. It may not only reduce the number of acute exacerbations but also their severity. Patients may learn to notice imminent exacerbations and seek medical attention earlier leading to a shift from inpatient to the less costly outpatient treatment of acute exacerbations. The significant reduction in hospital readmissions is suggestive of a beneficial cost-benefit balance. However, larger trials should provide the final evidence base for formal cost analyses to test the hypothesis that respiratory rehabilitation after acute exacerbation is cost effective.
The data presented in this review are the first to show a survival benefit of respiratory rehabilitation in patients at risk. Although the results should be interpreted with caution, as mentioned above, this study provides the most solid evidence currently available that mortality is reduced. In summary, current evidence suggests that respiratory rehabilitation reduces unplanned hospital admissions and mortality and improves HRQL and exercise capacity when initiated immediately after acute exacerbations.
Abbreviations
COPD: Chronic obstructive pulmonary disease
HRQL: Health-related quality of life
CI: confidence interval
CRQ: Chronic Respiratory Questionnaire
SD: Standard deviation
Contributions
Protocol writing: Puhan, Scharplatz, Steurer
Acquisition of data: Puhan, Scharplatz
Analysis and interpretation of data: Puhan, Scharplatz, Troosters, Steurer
Drafting of manuscript: Puhan
Critical revision of manuscript for important intellectual content: Puhan, Scharplatz, Troosters, Steurer
Conflict of interest
The author(s) declare that they have no competing interests.
Funding
Helmut Horten Foundation; Zurich Lung League. Thierry Troosters is a postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.
Acknowledgements
We thank Dr. Pius Estermann, information specialist at the hospital library, University Hospital, Zurich, Switzerland, for performing the literature searches.
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| 15943867 | PMC1164434 | CC BY | 2021-01-04 16:23:26 | no | Respir Res. 2005 Jun 8; 6(1):54 | utf-8 | Respir Res | 2,005 | 10.1186/1465-9921-6-54 | oa_comm |
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J Transl MedJournal of Translational Medicine1479-5876BioMed Central London 1479-5876-3-201588820410.1186/1479-5876-3-20MethodologySimultaneous assessment of cytotoxic T lymphocyte responses against multiple viral infections by combined usage of optimal epitope matrices, anti- CD3 mAb T-cell expansion and "RecycleSpot" Bihl Florian K [email protected] Elisabetta [email protected] John V [email protected] Hannah S [email protected] Leah M [email protected] Caitlyn [email protected] Todd J [email protected] Johnson T [email protected] Nicole [email protected] Pietro [email protected] Christian [email protected] Partners AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, USA2 Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, USA3 Dipartimento di Cardioangiologia ed Epatologia, Ospedale S. Orsola-Malpighi, Università degli Studi di Bologna, Italy2005 11 5 2005 3 20 20 9 3 2005 11 5 2005 Copyright © 2005 Bihl et al; licensee BioMed Central Ltd.2005Bihl et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The assessment of cellular anti-viral immunity is often hampered by the limited availability of adequate samples, especially when attempting simultaneous, high-resolution determination of T cell responses against multiple viral infections. Thus, the development of assay systems, which optimize cell usage, while still allowing for the detailed determination of breadth and magnitude of virus-specific cytotoxic T lymphocyte (CTL) responses, is urgently needed. This study provides an up-to-date listing of currently known, well-defined viral CTL epitopes for HIV, EBV, CMV, HCV and HBV and describes an approach that overcomes some of the above limitations through the use of peptide matrices of optimally defined viral CTL epitopes in combination with anti-CD3 in vitro T cell expansion and re-use of cells from negative ELISpot wells. The data show that, when compared to direct ex vivo cell preparations, antigen-unspecific in vitro T cell expansion maintains the breadth of detectable T cell responses and demonstrates that harvesting cells from negative ELISpot wells for re-use in subsequent ELISpot assays (RecycleSpot), further maximized the use of available cells. Furthermore when combining T cell expansion and RecycleSpot with the use of rationally designed peptide matrices, antiviral immunity against more than 400 different CTL epitopes from five different viruses can be reproducibly assessed from samples of less than 10 milliliters of blood without compromising information on the breadth and magnitude of these responses. Together, these data support an approach that facilitates the assessment of cellular immunity against multiple viral co-infections in settings where sample availability is severely limited.
Cytotoxic T CellsHIVEBVCMVHCVHBVCTLepitopepeptidecell expansionanti-CD3ELISpotpeptide matrix
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Introduction
Cell-mediated immunity is considered critical for the prevention and control of many viral infections [1-6]. The approaches developed to detect these responses in vitro have evolved over the years and have provided quantitative and qualitative information on virus-specific T cells for a number of viral infections. These assays include, besides others, lymphoproliferative assays using 3H-thymidine incorporation or CFSE staining, limiting dilution precursor-frequency assays for the enumeration of CTL precursor frequencies, intracellular cytokine staining (ICS) and enzyme-linked immunospot (ELISpot) assays [7-10]. Although these assays differ in their minimal cell requirements, the detailed, simultaneous analysis of anti-viral immunity against multiple viral infections is often limited by cell availability, regardless of the assay employed.
The ELISpot assay has become widely used for rapidly assessing cellular immune responses to extensive numbers of antigens while using relatively few cells. A number of studies have also employed peptide matrix approaches, where every antigenic peptide is tested in two peptide pools, so that responses to reactive pools sharing a specific peptide can help to identify the targeted peptide [9,11]. This has reduced the required cell numbers significantly, so that for instance HIV-specific responses can generally be comprehensively assessed using less than 15 × l06 cells [9]. However, despite such advances, the simultaneous enumeration of virus-specific immunity to multiple viral infections still exceeds the required sample size that can routinely be obtained. Sample size may not be of great concern when assessing CTL mediated immune responses against single, small genome viruses such as HIV and HCV, which can be tested in a comprehensive manner using overlapping peptide sets spanning the entire expressed viral genome [9,12]. Nevertheless, such comprehensive approaches are not feasible for larger viruses, such as DNA-based herpesviruses like EBV, CMV and KSHV [4,13]. Instead, immune analyses need either to be restricted to a selected number of specific viral proteins, or to the use of previously defined, optimal CTL epitopes. Responses against such optimally defined epitopes can account for a significant part of the total virus-specific immune responses, especially when they represent immunodominant epitopes covering the most immunogenic proteins of specific viral genomes. For well-studied viruses such as HIV, HCV, EBV and CMV, large sets of such optimally defined CTL epitopes, restricted by common HLA alleles, have been described in the past [14-17], and provide a valuable alternative to measure pathogen-specific CTL responses without the need to synthesize comprehensive peptide sets spanning the entire viral genomes.
The present study describes an algorithm by which matrices of optimally defined CTL epitopes derived from five different human viral infections are used in the same ELISpot assay. As not all wells of the ELISpot plate contain antigens to which the tested PBMCs will respond, there are consistently some wells with cells that have not been stimulated during this first assay. Theoretically, these cells could be recovered from the ELISpot plate before developing it and re-used in subsequent analyses. Indeed, others have suggested the use of "recycled" cells for DNA isolation[18], however, to our knowledge, no data exist on re-using these cells in functional assays. Since the peptide matrix approach is ideally followed by the subsequent confirmation of single targeted peptides present in two corresponding peptide pools, recycled cells from unstimulated ELISpot wells could be used for these assays. Although this second step could be achieved using in vitro expanded cells, for instance by anti-CD3 monoclonal antibody (mAb) stimulation, expanded cells may lose some of the responses compared directly to cells tested ex vivo [19,20]. In addition, the absolute and relative magnitude of responses may be distorted during cell expansion and assays can only be run after prolonged in vitro culture[21,22] Therefore, as long as functionality of recycled cells in secondary assays can be ensured, they may provide a simple way to complete initial ELISpot screenings, yielding reliable information on the magnitude of specific CTL responses. The feasibility of this approach was tested and it was shown that combined use of optimal epitope matrices, in vitro T cell expansion and RecycleSpot can provide relevant immune data on multiple viral infections even when cell availability is severely limited.
Materials and methods
Isolation of fresh PMBCs from whole blood
Whole blood was collected using Citrate Vacutainer tubes (BD, Franklin Lakes, NJ) and peripheral blood mononuclear cells (PBMC) were isolated by Histopaque (Histopaque® 1077, Sigma, St. Louis, MO) density centrifugation as described [9]. Fresh PBMC were either used directly after isolation, after in vitro expansion or after freezing and thawing with and without subsequent in vitro expansion. For in vitro use, cells were re-suspended in R10 medium (RPMI 1640 containg 10% heat inactivated FCS (both Sigma), 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomucin and 10 mM HEPES (all Mediatech, Hemdon, VA)) at a concentration of 1 × 106 cells/ml. Cells were thawed using R10 medium containing 50 U/ml DNAse (Deoxyribnuclease I, RNase-free, Sigma), washed twice in the same medium, re-suspended in R10 and incubated at 37°C with 5% CO2 for 3–4 hours before they were counted and re-suspended in R10 at 1 × 106 cells/ml. The thawed cells were then either used directly in ELISpot assays or expanded.
For in vitro expansion, 1 to 5 × 106 PBMC were added to 25 ml culture flasks in 10 ml R10 supplemented with 1 μl of the anti-CD3 specific monoclonal antibody (mAb) 12F6 [23]. Cells were fed twice a week using R10 supplemented with 50 U/ml of recombinant Interleukin 2 (IL-2) for 2 weeks. Before use in ELISpot assays, cells were washed twice in R10 medium and incubated overnight at 37°C with 5% CO2 in the absence of IL-2. This overnight starving step was necessary to eliminate background in the subseqnet ELISpot assay, which was, in our hands, not an issue, regardless of how long the in vitro culture had been maintained.
Design of Optimal Peptide Matrix
A total of 416 optimal epitopes from five different viruses were assembled in 98 different peptide pools and used in 5 peptide matrices each containing peptides from a single virus. The number of pools and total number of peptides contained in each virus-specific peptide matrix are summarized in Table 1. Each peptide was present at a final concentration of 200 μg/ml in the peptide pools. Detailed lists of all optimal epitopes included in this study, along with their sequence and HLA restriction, are given in Tables 2 through 6.
Table 1 Virus specific peptide matrix design using previously defined HLA class I restricted CTL epitopes
Virus Optimal epitopes No. of peptide pools Max. no. of peptides per pools
HIV 173 29 14
EBV 91 23 12
CMV 38 13 7
HCV 77 19 11
HBV 37 14 8
Table 2 Optimal HIV-derived HLA class I restricted CTL epitopes
Protein HLA Restriction Sequence Position
gp120 A02 RGPGRAFVTI 311–320
gp120 A03 TVYYGVPVWK 37–46
gp120 A11l SVITQACPK 199–207
gp120 A24 LFCASDAKAY 53–62
gp120 A29 SFEPIPIHY 209–217
gp120 A30 HIGPGRAFY 310–318
gp120 A32 RIKQIINMW 419–427
gp120 B07 RPNNNTRKSI 303–312
gp120 B08 RVKEKYQHL 2–10
gp120 B1516/Cw04 SFNCGGEFF 379–387
gp120 B3801 MHEDIISLW 104–112
gp120 B35 VPVWKEATTTL 42–52
gp120 B35 DPNPQEVVL 77–85
gp120 B44 AENLWVTVY 30–38
gp120 B51 LPCRIKQII 416–424
gp120 B55 VPVWKEATTT 42–51
gp120 A33 VFAVLSIVNR 187–196
gp120 A33 EVAQRAYR 320–327
gp41 A01 RRGWEVLKY 787–795
gp41 A02 SLLNATDIAV 818–827
gp41 A0205 RIRQGLERA 335–343
gp41 A03/A30 RLRDLLLIVTR 775–785
gp41 A23/A24 RYLKDQQLL 591–598
gp41 A30 IVNRNRQGY 704–712
gp41 A30 KYCWNLLQY 794–802
gp41 A6802 IVTRIVELL 782–790
gp41 B07 IPRRIRQGL 843–851
gp41 B08 YLKDQQLL 591–598
gp41 B08 RQGLERALL 848–856
gp41 B14 ERYLKDQQL 589–597
gp41 B2705 GRRGWEALKY 791–799
gp41 B35 TAVPWNASW 611–619
gp41 B4001 QELKNSAVSL 810–819
gp41 Cw3/Cw15 RAIEAQQHL 46–54
p17 A02 SLYNTVATL 77–85
p17 A03 KIRLRPGGK 18–26
p17 A03 RLRPGGKKK 20–28
p17 A03 RLRPGGKKKY 20–29
p17 A11 TLYCVHQRI 84–92
p17 A24 KYKLKHIVW 28–36
p17 A30 RSLYNTVATLY 74–86
p17 B08 GGKKKYKL 24–31
p17 B08 ELRSLYNTV 74–82
p17 B2705 IRLRPGGKK 19–27
p17 B35 WASRELERF 36–44
p17 B35 NSSKVSQNY 124–132
p17 B4001 IEIKDTKEAL 92–101
p17 B4002 GELDRWEKI 11–19
p24 A0207 YVDRFYKTL 164–172
p24 A11 ACQGVGGPGHK 349–359
p24 A24/B44 RDYVDRFFKTL 296–306
p24 A25 QAISPRTLNAW 145–155
p24 B07 SPRTLNAWV 148–156
p24 B07/B42/B81/Cw8 TPQDLNTML 48–56
p24 B07 GPGHKARVL 223–231
p24 B07 HPVHAGPIA 84–92
p24 B08 EIYKRWII 260–267
p24 B08 DCKTILKAL 329–337
p24 B14 DRFYKTLRA 298–306
p24 B1501 GLNKIVRMY 267–277
p24 B18 FRDYVDRFYK 293–302
p24 B2703 RRWIQLGLQK 260–269
p24 B2705 KRWIILGLNK 265–274
p24 B35 NPVPVGNIY 245–253
p24 B35 PPIPVGDIY 254–262
p24 B39 GHQAAMQML 193–201
p24 B4001 SEGATPQDL 176–184
p24 B4002 KETINEEAA 70–78
p24 B4002 AEWDRVHPV 78–86
p24 B44 AEQASQDVKNW 174–184
p24 B44 EEKAFSPEV 28–36
p24 B52 RMYSPTSI 143–150
p24 B53 TPYDINQML 48–56
p24 B53/B57 QASQEVKNW 176–184
p24 B57 ISPRTLNAW 15–23
p24 B57 KAFSPEVIPMF 30–40
p24 B57 TSTLQEQIGW 108–118
p24 B57 KAFSPEVI 30–37
p24 B58 TSTLQEQIGW 108–117
p24 B58 TSTVEEQIQW 108–117
p24 Cw0I VIPMFSAL 36–43
p24 A25 ETINEEAAEW 71–80
p24 A26 EVIPMFSAL 35–43
p15 A02 FLGKIWPSYK 1–10
p15 B14 CRAPRKKGC 42–50
p15 B4001 KELYPLTSL 33–41
p15 B4002 TERQANFL 64–71
Protease A6802/A74 ITLWQRPLV 3–11
Protease A6802 DTVLEEMNL 30–38
Integrase A30 KIQNFRVYY 219–227
Integrase A03/A11 AVFIHNFKRK 179–188
Integrase B1503 RKAKIIRDY 263–271
Integrase B42 VPRRKAKII 260–268
Integrase B57 KTAVQMAVF 173–181
RT A26 ETKLGKAGY 604–612
RT A02 ALVEICTEM 33–41
RT A02 VIYQYMDDL 179–187
RT A02 ILKEPVHGV 309–317
RT A03 ALVEICTEMEK 33–43
RT A03 GIPHPAGLK 93–101
RT A03/A1 1 AIFQSSMTK 158–166
RT A03 QIYPGIKVR 269–277
RT A03 KLVDFRELNK 73–82
RT A03 RMRGAHTNDVK 356–366
RT A11 IYQEPFKNLK 341–350
RT A11 QIIEQLIKK 80–88
RT B51 TAFTIPSI 128–135
RT B57 IVLPEKDSW 244–252
RT B58 IAMESIVIW 375–383
RT B81 LFLDGIDKA 715–723
RT B1503 VTDSQYALGI 651–660
RT A30 KQNPDIVIY 173–181
RT A30 KLNWASQIY 263–271
RT A30 RMRGAHTNDV 356–365
RT A32 PIQKETWETW 392–401
RT B08 GPKVKQWPL 18–26
RT B1501 LVGKLNWASQIY 260–271
RT B1501 IKLEPVHGVY 309–318
RT B35 TVLDVGDAY 107–115
RT B35 VPLDEDFRKY 118–127
RT B35 NPDIVIYQY 175–183
RT B35 HPDIVIYQY 175–183
RT B4001 IEELRQHLL 202–210
RT B42 YPGIKVRQL 271–279
RT B51 EKEGKISKI 42–50
Vpr A02 AIIRILQQL 59–67
Vpr B07/B81 FPRIWLHGL 34–42
Vpr B51 EAVRHFPRI 29–37
Vpr B57 AVRHFPRIW 30–38
Tat A6801 ITKGLGISYGR 39–49
Tat B1503 FQTKGLGISY 38–47
Tat B53 EPVDPRLEPW 2–11
Tat Cw12 CCFHCQVC 30–37
Vif A03 RIRTWKSLVK 17–26
Vif A03 HMYISKKAK 28–36
Vif A03 KTKPPLPSVKK 158–168
Vif B07 HPRVSSEVHI 48–57
Vif B18 LADQLIHLHY 102–111
Vif B57 ISKKAKGWF 31–39
Nef A02 PLTFGWCYKL 136–145
Nef A02 VLEWRFDSRL 180–189
Nef A03/A11 QVPLRPMTYK 73–82
Nef A03/A11 AVDLSHFLK 84–92
Nef A11 PLRPMTYK 75–82
Nef A24 RYPLTFGW 134–141
Nef A33 TRYPLTFGW 133–141
Nef B07 FPVTPQVPLR 68–77
Nef B07 FPVTPQVPL 68–76
Nef B07 TPQVPLRPM 71–79
Nef B07 RPMTYKAAL 77–85
Nef B07 TPGPGVRYPL 128–137
Nef B07 RQDILDLWIY 106–115
Nef B08 WPTVRERM 13–20
Nef B08 FLKEKGGL 90–97
Nef B1501 TQGYFPDWQNY 117–127
Nef B1501 RMRRAEPAA 19–27
Nef B1503 WRFDSRLAF 183–191
Nef B18/B53 YPLTFGWCY 135–143
Nef B2705 RRQDILDLWI 105–114
Nef B35 VPLRPMTY 74–81
Nef A01/A29/837/857 YFPDWQNYT 120–128
Nef B40 KEKGGLEGL 92–100
Nef B42 TPGPGVRYPL 128–137
Nef B53 YPLTFGWCF 135–143
Nef B57 HTQGYFPDWQ 116–125
Nef B57 HTQGYFPDW 116–124
Nef Cw07 RRQDILDLWIY 105–115
Nef Cw7 KRQEILDLWVY 105–115
Nef Cw8 AAVDLSHFL 83–91
Rev A03 ERILSTYLGR 57–66
Rev B57/B58 KAVRLIKFLY 14–23
Rev Cw05 SAEPVPLQL 67–75
Vpu A33 EYRKILRQR 29–37
All epitopes were referred from the Los Alamos HIV Immunology Database 2004 [24].
Table 6 Optimal HBV-derived HLA class I restricted CTL epitopes
Protein HLA Restriction Sequence Position Reference
Core A2 FLPSDFFPSV 18–27 [84]
Core A2 CLTFGRETV 107–115 [85]
Core A2 VLEYLVSFGV 115–124 [85]
Core A2/A24 EYLVSFGVW 117–125 [86, 87]
Core A2 ILSTLPETTV 139–148 [86]
Core A33/A68 STLPETTVVRR 141–151 [88]
Core A2 AILSKTGDPV 152–161 [89]
Env A2 LLDPRVRGL 131–139 [85]
Env A2 VLQAGFFLL 177–185 [90]
Env A2 FLLTRILTI 183–191 [91]
Env A2 SLNFLGGTTV 201–210 [92]
Env A2 FLGGTPVCL 204–212 [89]
Env A2 LLLCLIFLL 250–258 [86]
Env A2 LLCLIFLLV 251–259 [92]
Env A2 LLDYQGMLPV 260–269 [92]
Env A2 LVLLDYQGML 269–278 [85]
Env A2 VLLDYQGML 270–278 [85]
Env A2 LLDYQGMLPV 271–280 [85]
Env A2 WLSLLVPFV 335–343 [92]
Env A2 LLVPFVQWFV 338–347 [92]
Env A2 GLSPTVWLSV 348–357 [92]
Env A2 SIVSPFIPLL 370–379 [89]
Env A2 LLPIFFCLWV 378–387 [92]
Env A2 ILSPFFFLPLL 382–390 [85]
x-Protein A2 VLCLRPVGA 15–23 [93]
x-Protein A2 TLPSPSSSA 36–44 [93]
x-Protein A2 HLSLRGLFV 52–60 [93]
x-Protein A2 VLHKRTLGL 92–100 [93]
x-Protein A2 AMSTTDLEA 102–110 [93]
x-Protein A2 CLFKDWEEL 115–123 [93]
Pol A24 LYSSTVPVF 62–70 [90]
Pol A2 GLSRYVARL 455–463 [90]
Pol A2 YMDDVVLGA 551–559 [91]
Pol A2 FLLSLGIHL 575–583 [90]
Pol A24 KYTSFPWLL 756–764 [87]
Pol A2 ILRGTSFVYV 773–782 [91]
Pol A2 SLYADSPSV 816–824 [91]
ELISpot assay
96-well polyvinylidene plates (Millipore, Bedford, MA), pre-coated overnight with 2 μg/ml of anti-interferon gamma (IFN-γ) mAb 1-D1K (Mabtech, Stockholm, Sweden), were washed six times with sterile phosphate buffered saline (DPBS, no Ca & Mg, Mediatech) containing 1% fetal calf serum (FCS) before use. After washing, 30 μl of R10 were added to each well to avoid drying of the membrane, and 100,000 to 200,00 cells per well were added in 100 μl R10. 100,00 cells/well were used to detect responses to HIV, CMV and EBV, whereas responses to HCV and HBV were tested using 200,000 cells/well. Each peptide was added at a final concentration of 14 μg/ml (both single peptides as well as pools). As a negative control, cells were incubated in medium alone, and PHA was added at a concentration of 1.8 μg/ml to serve as a positive control. Plates were incubated for 16 h at 37°C with 5% CO2 before being developed. After washing six times with PBS, 100 μl of biotinylated anti-IFN-γ mAB 7-B6-1 (0.5 μg/ml, Mabtech) were added and plates were incubated for 1 hour at room temperature (RT). The plates were washed again and incubated with a 1:2000 dilution of streptavidin-coupled alkaline phosphatase (Streptavidin-ALP-PQ Mabtech) for 1 hour at RT in the dark. After washing the plates again, IFN-γ production was detected as dark spots after a short incubation of 10–20 minutes with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (BioRad, Hercules, CA). The color reaction was stopped by washing plates with tap water and the plates were air-dried before counting using a AID ELISPOT Reader Unit (Autoimmun Diagnostika GmbH, Strassberg, Germany). Results were expressed as spot forming cells (SFC) per million input cells. Thresholds for positive responses were determined as either 5 spots (50 SFC/106 input cells) or as the mean plus 3 standard deviations of negative control wells, whichever was higher.
RecycleSpot
After overnight incubation in a primary ELISpot assay, cells from all wells of the ELISpot plate were transferred to a 96-well round-bottom plate and incubated at 37°C with 5% CO2 while developing the ELISpot assay. Cells from wells without any spots (including negative control wells) were then pooled, counted and used for secondary ELISpot assays. In control experiments, cells corresponding to wells with positive responses were also pooled, washed extensively (>5 times) and re-used in subsequent, secondary ELISpot assays as well. Cells from positive control wells (PHA stimulated) were not used for subsequent assays.
Results
Design of the optimal epitope matrix for five viral infections
To simultaneously test CTL responses against five different viruses with a limited number of PBMCs, a peptide matrix approach was used that included all previously published, well-defined CTL epitopes in HIV, HCV, HBV, EBV and CMV. The total number of described CTL epitopes for these viruses varied from 37 described optimal epitopes in HBV to more than 170 optimal epitopes in HIV. A list of all the optimal epitopes included in the present study is given in Tables 2 through 6, totaling 416 well-defined, HLA class I-restricted CTL epitopes. The included HIV epitopes were derived from the annually updated list of HIV CTL epitopes at the Los Alamos National Laboratory HIV immunology database[24]. For all the other pathogens, the epitopes listed were those for which, to the best of our knowledge, at least one publication existed showing CTL activity against this epitope in at least one infected individual. While the optimal epitopes in HIV, HCV and HBV cover large parts of their respective viral genomes, the epitopes defined in EBV and CMV represent only a portion of the proteins expressed by these viruses. Given the approximately 100 open reading frames in these large-genome viruses, complete representation of all viral proteins can hardly be achieved and most studies on these pathogens have thus focused on a relatively small number of viral proteins, especially concentrating on those containing serological determinants and those characterized by specific viral gene expression patterns. Thus, described EBV and CMV encoded CTL epitopes are derived from eleven and four different viral proteins respectively, whereas the known HIV, HCV and HBV epitopes cover all the viral proteins in these small-genome pathogens.
As the number of described optimal CTL epitopes varies between pathogens, separate peptide matrices were designed for each virus (Table 1). Importantly, the first set of pools ("protein pools") was designed so that the pools contained all the epitopes derived from the same viral protein, whereas the second half of matrix peptide pools contained the epitopes in a non-protein specific composition ("random pools"). This matrix design allowed assessment of the virus specific immune response at different levels of resolution including i) a "total virus" specific response by adding up all the protein pool specific or random peptide pool specific responses, ii) a "protein" specific responses by focusing on single pools containing all the epitopes of a given protein; and iii) upon single peptide confirmation, on a single epitope level, by comparing responses in pools containing the same epitope. Together, the epitope matrix design facilitated the assessment of T cell responses to more than 400 CTL epitopes from five different viruses simultaneously, using less than 10 × 106 PBMCs while still allowing determination of breadth and magnitude of virus-, protein-, and epitope-specific responses for each virus separately.
Moreover, since each epitope is tested twice in different pools, it should reflect the same magnitude of response in each pool, thus the matrix approach provides its own internal control. Additionally, "protein pools" and "random pools" should theoretically yield the same total magnitude of responses since they, as a whole, contain the same set of peptides. To test this, and rule out the possibility that peptide compositions in the different pools interfered with the detection of specific responses, the magnitude of all "protein pool" and "random pool" specific responses were compared in 19 subjects infected with EBV (n = 19) and co-infected with CMV (n = 14), HIV (12), and HCV (9). These analyses showed a statistically highly significant correlation between total magnitudes of responses detected by either set of peptide pools, indicating that the peptide mixtures in the pools sharing a specific response did not significantly impact the detection of the targeted epitope (Figure 1). Of note, for all four viruses analyzed, the "random pools" detected a slightly higher, statistically not significant total virus-specific response than the "protein pools". This is likely due to the presence of highly reactive epitopes which, when tested in the same peptide pool, can exceed the upper detection limit of the ELISpot assay and may thus underestimate the total virus-specific magnitude of responses. This may be more likely for epitopes in "peptide pools" than "random pools" if some proteins elicit generally stronger immune responses than others. A protein pool accumulating strongly reactive epitopes would result in fewer spots than the total of the respective "random pools" containing these epitopes equally distributed and fully quantitative.
Figure 1 Comparable magnitude of responses detected by "protein" and "random" peptide pools: The magnitude of CTL responses was determined by adding magnitudes for all "protein" or "random" pools for each virus. Responses on the Y-axes represent the total of all virus specific "random pools", the X-axes indicate total responses detected using the "protein pools". Data from 12 HIV-, 19 EBV-, 14 CMV-, 9 HCV-infected individuals were tested against either set of peptide pools for A) HIV, B) HCV, C) EB V, and D) CMV and compared using the non-parametric Wilcoxon matched pairs test.
Cells from negative ELISpot wells can be used in secondary ELISpot assays (RecycleSpot)
In order to maximize cell use in samples with limited cell availability, we investigated whether cells from initial ELISpot matrix screens could be re-used in subsequent functional assays. Specifically, cells from wells that did not respond to peptides added in the first assay as well as the cells in the negative control wells may be used for secondary ELISpot assays. To assess the feasibility of this strategy, all wells from the initial ELISpot plate were transferred to a 96-well plate and incubated at 37°C with 5% CO2 while the ELISpot plate was developed. Cells from negative ELISpot wells were then used to confirm the identity of the epitope(s) targeted in the matrix peptide pools. In separate experiments, cells from initially positive wells were also tested in subsequent assays to determine if continuing IFN-γ production in these cells would prevent them from being used in further ELISpot assays. The analyses also compared ELISpot results in plates that were either undisturbed, or from which cells were transferred for later use.
Representative RecyleSpot assays using PBMC and recovered cells from initial ELISpot assays from three individuals are shown in Figure 2. In all cases, negative wells from initial peptide matrix ELISpot assays were re-used to reconfirm the identity of the presumed, single targeted epitope shared by the two pools. Further, initially positive pools were re-tested to assess whether recycled cells responded with a different magnitude compared to the initial assay. The data show that sufficient cells were recovered from initial assays to perform reconfirmations of single targeted epitopes in the RecycleSpot, and that background activity and magnitude of responses were not significantly different between the first and the subsequent assays. RecycleSpot assays that used initially positive wells, or mixtures of initially positive and negative wells, showed high background in the secondary assay, indicating ongoing IFN-γ production and thus precluding these cells from use in the RecycleSpot (data not shown). No effects on the quality and the number of spots between the manipulated and non-manipulated wells were observed, indicating that harvesting cells from the ELISpot plate did not negatively interfere with the quality of the assay, at least when cells are removed by careful pipetting using a 12-channel pipetor Furthermore, RecycleSpot assays were performed using both fresh and frozen/thawed cells and showed that HIV-and EBV-specific responses were maintained in recycled cells in both cases (data not shown). Together, the data indicate that RecycleSpot can provide sufficient numbers of cells from initial assays and that these cells maintain functional capacity for use in subsequent assays, without raising background activity. Also, the data show that re-using the cells form negative wells after an overnight incubation did not reduce the magnitude of responses to a statistically significant level.
Figure 2 RecycleSpot using recycled cells for the de-convolution of positive peptide pools: Wells of primary ELISpot and secondary RecycleSpot are shown. Line A shows the data from the initial ELISpot assay, including two positive wells indicating cellular response to EBV peptide pools, three negative and one positive control wells. Line B shows the same outline as in A, this time with recycled cells in a secondary ELISpot analysis and, one separate well, using the predicted targeted epitopes from the matrix analysis. The numbers indicate the spot forming cells per million PBMC.
In vitro expanded T cells mount responses detected in fresh ex vivo PBMC samples
Even though rational optimal epitope matrix design and RecycleSpots may help in reducing the required cell numbers for in vitro analyses, cell availability may still be limiting in settings where only very small biological samples can be obtained. In such instances, investigators have resorted to the use of in vitro expanded cells [19,20,25]. However, despite its potential usefulness in situations of small sample size (e.g. tissue biopsies or small volume peripheral blood samples), relatively little is known on how in vitro expansion impacts magnitude and breadth of detectable responses [20,25]. Furthermore, CTL responses to pathogens like HIV, for which a defect in their proliferative capacity has been shown, may be severely distorted by in vitro expansion, even when stimulated unspecifically [7]. To address this issue and to investigate whether stimulation of PBMC with an anti-CD3 mAb (12F6) expands CTL of different specificity equally well, we tested cells either directly or after expansion against peptide sets of described HIV- and EBV-specific epitopes restricted by the individual's HLA alleles.
These analyses included twelve subjects, of which seven were tested for responses to HIV and EBV epitopes, while the remaining five were tested for EBV-specific responses only (Figure 3).
Figure 3 In vitro expansion of thawed cells increases the magnitude and breadth of HIV and EBV specific responses: Thawed PBMC from 12 individuals were tested against HIV and EBV peptide pools (n = 7 subjects) or against EBV peptide pools only (n = 5). Cells were used either directly after thawing or after thawing and a subsequent two-week in vitro expansion using the anti-CD3 mAb 12F6. A) The breadth of the detected responses (number of peptide pools reacting) and B) the total magnitude (sum of all positive peptide pools) is compared between the two cell preparation using the non-parametric Wilcoxon matched pairs test. C) PBMC from 5 EBV infected individuals were used either directly after isolation of after a two-week in vitro expansion or as frozen/thawed cells with and without in vitro expansion, and compared for the breadth (number of pools recognized) and D) total magnitude of the EBV specific responses.
In a first analysis, frozen PBMC were either tested directly or after a 2-week stimulation using 12F6 and the number of targeted HIV or EBV epitopes were compared, resulting in 19 data points (seven individuals tested for HIV and EBV responses and five subjects tested for EBV-specific responses). Flow cytometry in nine individuals showed preferential expansion of CD8 T cells, as CD4 expressing T cells ranged between 0.5% and 14% only, independent of HIV infection and starting CD4 T cell counts (data not shown). The Elispot results revealed no difference in the breadth of responses (number of targeted epitopes) between the directly tested and the expanded cells, as a median of 6.4 and 6.9 positive responses were detected for HIV and EBV, respectively (Figure 3A). The recognition of HIV- and EBV-derived epitopes was equally frequent by the two different cell preparations (data not shown). When the magnitude of responses was compared between directly used and expanded cells, expanded cells responded with a slightly higher magnitude than unexpanded cells. This trend was more prominent when HIV and EBV responses were analyzed separately. The HIV responses in directly tested cells showed a median of 185 SFC/106 PBMC, as compared to 285 SFC/106 PBMC in expanded cells (p = 0.0005); whereas the median EBV-specific responses had a magnitude of 170 SFC/106 in unexpanded PBMC compared to 190 SFC/106PBMC in expanded cells (p > NS).
Moreover, to determine whether freshly isolated cells could also be expanded without drastic changes in their response patterns, PBMC from five EBV-infected subjects were tested directly after isolation, or after freezing, and with or without in vitro expansion. In agreement with the data from frozen samples, no significant difference in the number of pools targeted or the median magnitude of these responses was observed (Figure 3 C and 3 D). Despite concordance among the response patterns between the different cell preparations that was as low as 80%, the overall breadth and magnitude of these responses did not change. In addition, when comparing the magnitudes of the responses between each other, the relative magnitude of the responses was maintained between the four different cell preparations (data not shown). Combined, the data demonstrate that anti-CD3 expanded cells maintain their specificity and relative magnitudes when compared to unexpanded cells (both when used fresh or after thawing) indicating that in vitro expansion could be employed when the breadth, but not the absolute magnitude of responses, is being assessed. This was the case for the assessment of HIV- as well as the EBV-specific responses, suggesting that cells specific for HIV do not significantly differ from EBV specific cells in their ability to undergo in vitro expansion using a non-antigenic stimulus.
Discussion
Cell availability can severely hamper in vitro analyses of antigen specific immune responses, hence approaches which optimize cell use are urgently needed. This is especially true for assays requiring extensive sets of antigens to be tested while only a limited number of cells can be obtained. However, logistic considerations may prevent repetitive sample collection for larger trials, and re-use of fresh or frozen samples could provide more effective ways to perform necessary analyses. The present study introduces a novel approach by which some of the sample limitations can be overcome, and may prove helpful in routine laboratory tests that currently do not make optimal use of available cells. This may not only facilitate currently performed assays, but may open possibilities to expand analyses to simultaneous assessment of even larger sets of antigens and additional functional aspects.
In the present study, we have designed and tested an approach that allows the assessment of the CTL mediated immunity against five different viral infections, including HIV, HCV, HBV, EBV and CMV. We provide an up-to-date listing of currently determined viral epitopes for which the minimal length and HLA restriction have been established. In the case of the small genome viruses HIV and HCV, these optimal epitopes represent a large portion of the respective immune targets [26]. Although they do not include all responses detected in OLP screenings, our comparative analyses of HIV-specific responses from 100 individuals detected by either overlapping peptide (OLP) sets or optimal epitopes show that on average 68% of the observed OLP responses are covered by previously established HIV optimal epitopes (data not shown).
The present data also show that PBMC recycled from negative wells from an ELISpot assay can be re-used for subsequent functional assays. Depending on the analyses performed in the subsequent assay, such as reconfirmation of single epitope responses predicted in the initial matrix analyses, relatively small numbers of cells maybe required. Thus, although individuals with broad responses in the initial ELISpot assay will not yield many negative wells from which to recycle cells, the wells with non-targeted peptide in addition to the negative control wells often provide sufficient quantities of recycled cells to complete the matrix based analyses. Since the responses in the RecycleSpot are not significantly diminished as compared to the initial assay (Figure 2), the magnitude of responses in the subsequent assay can still provide adequate data at the single epitope level.
In vitro expanded cells have been used in a number of studies where cell availability has been the limiting factor[21,22]. However, no study has directly compared for instance biopsy and PBMC-derived responses in a systematic manner and on a single epitope level, and it is unclear whether the in vitro expansion provides identical data. In the present report, we have compared the response patterns to EBV- and HIV-derived antigens in directly ex vivo and in vitro expanded PBMC preparations. No significant differences were observed, although some responses are lost or gained upon expansion. As no difference in the concordance between EBV- and HIV-specific responses was observed, the data indicate that responses to both viruses are equally well expandable in vitro using an antigen-unspecific stimulus, despite the ongoing viral replication in most HIV infected subjects tested here.
Thus, optimal epitope matrices, RecycleSpot and in vitro expansion of cells can be combined to achieve maximal information on an extensive set of antigens, even if sample availability is limited. As a practical approach, expanded cells from frozen PBMC aliquots can be used initially to screen a large number of antigens to determine the approximate breadth of responses within the set of antigens used. Subsequent studies using unexpanded cells and antigen matrices in conjunction with RecycleSpot would then allow determination of the true breadth and, more importantly, the true magnitude of these responses while requiring minimal cell numbers. Furthermore, cells can be successfully recovered from the RecycleSpot once more to be used for genetic analyses such as HLA typing. This combined approach should facilitate future work in settings in which cell availability is of constant concern.
Table 3 Optimal EBV-derived HLA class I restricted CTL epitopes
Protein HLA Restriction Sequence Position Reference
BMLF1 A1 LVSDYCNVLNKEFT 25–39 [27]
BMLF1 A2 GLCTLVAML 280–288 [28]
BMLF1 B18 DEVEFLGHY 397–405 [28]
BMLF1 n.d.* KDTWLDARM 265–273 [29]
BMLF1 A24 DYNFVKQLF 320–328 [30]
BHRF A2 LLWAARPRL 204–212 [31]
BZLF1 B7 LPCVLWPVL 44–52 [13]
BZLF1 B8 RAKFQLL 190–197 [32]
BZLF1 Cw6 RKCCRAKFKQLLQH 186–201 [1]
BMRF1 Cw6 YRSGIIAW 268–276 [16]
BMRF1 Cw3 FRNLAYGRTCVLGK 86–100 [16]
BRLF1 A2 YVLDHLIVV 109–117 [33]
BRLF1 A2 RALIKTLPRASYSSH 225–239 [27]
BRLF1 A3 RVRAYTYSK 148–156 [1]
BRLF1 A11 ATIGTAMYK 134–142 [16]
BRLF1 A24 DYCNVLNKEF 28–37 [27]
BRLF1 A24 TYPVLEEMF 198–206 [30]
BRLF1 B61 QKEEAAICGQMDLS 529–543 [1]
BRLF1 Cw4 ERPIFPHPSKPTFLP 393–407 [1]
gp110 A2 ILIYNGWYA 106–114 [1]
gp110 B35 VPGSETMCY 544–552 [1]
gp110 B35 APGWLIWTY 190–198 [1]
gp85 A2 TLFIGSHVV 420–428 [1]
gp85 A2 LMPIIPLINV 542–550 [1]
gp85 A2 SLVIVTTFV 225–233 [1]
gp350 A2 VLQWASLAV 863–871 [1]
gp350 A2 VLTLLLLLV 871–879 [34]
gp350 A2 LIPETVPYI 152–160 [34]
gp350 A2 QLTPHTKAV 67–75 [34]
EBNA1 A2 FMVFLQTHI 562–570 [13]
EBNA1 B7 RPQKRPSCI 72–80 [35]
EBNA1 B7 IPQCRLTPL 528–536 [35]
EBNA1 B53 HPVGEADYF 407–415 [35]
EBNA2 A2/B51 DTPLIPLTIF 42–50 [36]
EBNA3A A2 SVRDRLARL 596–604 [37]
EBNA3A A3 RLRAEAQVK 603–611 [38]
EBNA3A A24 RYSIFFDY 246–253 [37]
EBNA3A A29 VFSDGRVAC 491–499 [16]
EBNA3A A30 AYSSWMYSY 176–184 [1]
EBNA3A B7 RPPIFIRRL 379–387 [39]
EBNA3A B7 VPAPAGPIV 502–510 [16]
EBNA3A B8 QAKWRLQTL 158–166 [37]
EBNA3A B8 FLRGRAYGL 325–333 [40]
EBNA3A B35 YPLHEQYGM 458–466 [37]
EBNA3A B46 VQPPQLTLQV 617–625 [41]
EBNA3A B62 LEKARGSTY 406–414 [16]
EBNA3A n.d.* HLAAQGMAY 318–326 [16]
EBNA3B A1l NPTQAPVIQLHAVY 101–115 [40]
EBNA3B A1l AVFDRKSDAK 399–408 [16]
EBNA3B A1l LPGPQVTAVLLHEES 481–495 [40]
EBNA3B A1l DEPASTEPVHDQLL 551–563 [40]
EBNA3B Al1 IVTDFSVIK 416–424 [40]
EBNA3B A24 TYSAGIVQI 217–225 [16]
EBNA3B A27 RRARSLSAERY 243–253 [42]
EBNA3B B35 AVLLHEESM 488–496 [1]
EBNA3B B44 VEITPYKPTW 657–666 [16]
EBNA3B B58 VSFIEFVGW 279–287 [43]
EBNA3B B62 GQGGSPTAM 831–839 [16]
EBNA3C B7 QPRAPIRPI 881–889 [39]
EBNA3C B27 RRIYDLIEL 258–266 [44]
EBNA3C B27 HRCQAIRK 149–157 [16]
EBNA3C B27 FRKAQIQGL 343–351 [16]
EBNA3C B27 RKIYDLIEL 258–266 [45]
EBNA3C B27 RRIFDLIEL 258–266 [45]
EBNA3C B27 LRGKWQRRYR 249–258 [44]
EBNA3C B37 LDFVRFMGV 285–293 [46]
EBNA3C B39 HHIWQNLL 271–278 [16]
EBNA3C B44 KEHVIQNAF 335–343 [47]
EBNA3C B44 EENLLDFVRF 281–290 [40]
EBNA3C B44 EGGVGWRHW 163–171 [48]
EBNA3C B62 QNGALAINTF 213–222 [49]
EBNALP A2 SLREWLLRI 284–292 [43]
LMP1 A2 YLQQNWWTL 159–167 [50]
LMP1 A2 YLLEMLWRL 125–133 [50]
LMP1 A2 LLVDLLWLL 167–175 [50]
LMP1 A2 TLLVDLLWL 166–174 [50]
LMP1 A2 LLLIALWNL 92–100 [50]
LMP1 B51 DPHGPVQLSYYD 393–404 [51]
LMP2 A2 FLYALALLL 356–364 [52]
LMP2 A2 LLWTLWLL 329–337 [53]
LMP2 A2 CLGGLLTMV 426–434 [54]
LMP2 A2 LTAGFLIFL 453–461 [53]
LMP2 A11 SSCSSCPLSKI 340–350 [53]
LMP2 A23 PYLFWLAAI 131–139 [55]
LMP2 A2 LLSAWILTA 447–455 [43]
LMP2 A24 TYGPVFMCL 419–427 [53]
LMP2 A24 IYVLVMLVL 222–230 [30]
LMP2 A25 VMNSNTLLSAW 442–451 [16]
LMP2 A27 RRRWRRLTV 236–244 [44]
LMP2 B40 IEDPPFNSL 200–208 [53]
LMP2 B63 WTLWLLI 331–338 [1]
*not determined
Table 4 Optimal CMV-derived HLA class I restricted CTL epitopes
Protein HLA Restriction Sequence Position Reference
pp65 B35 IPSINVHHY 123–131 [56]
pp65 B35 DDVWTSGSDSDEELV 397–411 [57]
pp65 B35 VFPTKDVAL 187–195 [17]
pp65 B38 PTFTSQYRIQGKL 367–379 [17]
pp65 B7 TPRVTGGGAM 417–426 [57]
pp65 B7 RPHERNGFTVL 265–275 [17]
pp65 A1 YSEHPTFTSQY 363–373 [17]
pp65 A1101 SVLGPISGHVLK 13–24 [17]
pp65 A2402 FTSQYRIQGKL 369–379 [17]
pp65 A68 FVFPTKDVALP 186–196 [17]
pp65 A2 NLVPMVATV 495–503 [57]
pp65 A2 VLGPISGHV 14–22 [58]
pp65 A2 MLNIPSINV 120–128 [58]
pp65 B44 EFFWDANDIY 512–521 [57]
pp65 A2402 VYALPLKML 113–121 [59]
pp65 A2402/Cw0401 QYDPVAALF 341–349 [60, 61]
pp65 B5201 QMWQARLTV 155–163 [62]
pp65 A0207 RIFAELEGV 522–530 [61]
pp65 A1101 ATVQGQNLK 501–509 [61]
pp65 B1501 KMQVIGDQY 215–223 [61]
pp65 B4001 CEDVPSGKL 232–240 [61]
pp65 B40 HERNGFTVL 267–275 [61]
pp65 B4006 AELEGVWQPA 525–534 [61]
pp65 B4403 SEHPTFTSQY 364–373 [61]
pp65 B5101 DALPGPCI 545–552 [61]
pp65 Cw0102 RCPEMISVL 7–15 [61]
pp65 Cw0801 VVCAHELVC 198–206 [61]
pp65 Cw1202 VAFTSHEHF 294–302 [61]
pp65 A33 SVNVHNPTGR 91–100 [63]
pp150 A0301 TTVYPPSSTAK 945–955 [17]
pp150 A68 QTVTSTPVQGR 792–802 [17]
IE B7 CRVLCCYVL 309–317 [64]
IE A2 YILEETSVM 315–323 [65]
IE B18 ELKRKMIYM 199–207 [65]
IE B18 CVETMCNEY 279–287 [65]
IE B18 DEEDAIVAY 379–387 [65]
IE B18 SDEEEAIVAYTL 378–389 [56]
GB A2 FIAGNSAYEYV 618–628 [66]
Table 5 Optimal HCV-derived HLA class I restricted CTL epitopes
Protein HLA Restriction Sequence Position Reference
Core B60 GQIVGGVYLL 28–37 [67]
Core A0201 YLLPRRGPRL 35–44 [68]
Core B7 GPRLGVRAT 41–49 [69]
Core B44 NEGCGWAGW 88–96 [70]
Core A0201 DLMGYIPLV 132–140 [71]
Core A0201 ALAHGVRAL 150–158 [15]
Core A0201 LLALLSCLTV 178–187 [72]
Core A11 MSTNPKPQK 1–9 [73]
P7 A29 FYGMWPLLL 790–798 [15]
P7 Cw7 FYGMWPLL 790–797 [15]
E1 A0201 ILHTPGCV 220–227 [74]
E1 B35 NASRCWVAM 234–242 [25]
E1 A0201 QLRRHIDLLV 257–266 [74]
E1 A23 FLVGQLFTF 285–293 [15]
E1 A0201 MMMNWSPTT 322–330 [15]
E1 A0201 SMVGNWAKV 363–371 [74]
E1 B35 CPNSSIVY 207–214 [15]
E2 A0201 SLLAPGAKQNV 401–411 [74]
E2 B53 CRPLTDFDQGW 460–469 [69]
E2 B51 YPPKPCGI 489–496 [73]
E2 B60 GENDTDVFVL 530–539 [75]
E2 B50 CVIGGAGNNT 569–578 [73]
E2 A0201 RLWHYPCTV 614–622 [76]
E2 A11 TINYTIFK 621–628 [69]
E2 B60 LEDRDRSEL 654–662 [75]
E2 A2402 EYVLLLFLL 717–725 [77]
E2 B57 NTRPPLGNWF 541–550 [15]
NS2 A29 MALTLSPY 827–834 [25]
NS2 A25 SPYYKRYISW 832–841 [78]
NS2 A23 YISWCLWWL 838–845 [69]
NS3 A24 AYSQQTRGL 1031–1039 [79]
NS3 A0201 CINGVCWTV 1073–1081 [68]
NS3 A0201 LLCPAGHAV 1169–1177 [68]
NS3 A0201 LLCPSGHAV 1169–1177 [68]
NS3 A11 TLGFGAYMSK 1261–1270 [80]
NS3 A0201 ATLGFGAYM 1260–1268 [81]
NS3 A0201 TLHGPTPLL 1617–1625 [81]
NS3 A0201 TGAPVTYSTY 1287–1296 [79]
NS3 A2402 TYSTYGKFL 1292–1300 [77]
NS3 B35 HPNIEEVAL 1359–1367 [82]
NS3 B8 HSKKKCDEL 1395–1403 [69]
NS3 A0201 KLVALGINAV 1406–1415 [68]
NS3 B8 LIRLKPTL 1611–1618 [75]
NS3 A11 TLTHPVTK 1636–1643 [80]
NS3 A68 HAVGLFRAA 1175–1184 [15]
NS3 A0201 GLLGCIITSL 1038–1047 [81]
NS4 A2402 FWAKHMWNF 1760–1768 [77]
NS4 B35 IPDREVLY 1695–1712 [15]
NS4 A24 VIAPAVQTNW 1745–1754 [15]
NS4 B57 LTTSQTLLF 1801–1809 [15]
NS4B A25 EVIAPAVQTNW 1744–1754 [78]
NS4B A25 ETFWAKHMW 1758–1766 [78]
NS4B A0201 SLMAFTAAV 1789–1797 [68]
NS4B A0201 LLFNILGGWV 1807–1816 [72]
NS4B A0201 ILAGYGAGV 1851–1859 [72]
NS4B B37 SECTTPCSGSW 1966–1976 [78]
NS4B B38 AARVTAIL 1941–1948 [75]
NS5 A2 VLSDFKTWL 1987–1995 [83]
NS5 B35 EPEPDVAVL 2162–2170 [15]
NS5 B57 LGVPPLRAWR 2912–2921 [15]
NS5A B60 HEYPVGSQL 2152–2160 [75]
NS5A B35 PCEPEPDVAVL 2161–2171 [75]
NS5A B38 NHDSPDAEL 2218–2226 [75]
NS5A A2 SPDAELIEANL 2221–2231 [75]
NS5A A25 ELIEANLLW 2225–2233 [78]
NS5A A0201 ILDSFDPLV 2252–2260 [68]
NS5A B60 REISVPAEIL 2267–2275 [80]
NS5B A3 SLTPPHSAK 2510–2518 [80]
NS5B A3 RVCEKMALY 2588–2596 [69]
NS5B A2 ALYDWTKL 2594–2602 [78]
NS5B B57 KSKKTPMGF 2629–2637 [80]
NS5B A0201 GLQDCTMLV 2727–2735 [72]
NS5B B38 HDGAGKRVYL 2794–2804 [80]
NS5B A25 TARHTPVNSW 2819–2828 [78]
NS5B A2402 RMILMTHFF 2841–2849 [77]
NS5B A2402 CYSIEPLDL 2870–2878 [77]
NS5B A31 VGIYLLPNR 3003–3011 [80]
Acknowledgements
This work was supported by a grant of the Swiss National Science Foundation to FKB (SNF-PBSKB-102686) and by the Solid Organ Transplantation in HIV: Multi-Side Study (AI052748) funded by the National Institute of Allergy and Infectious Diseases.
==== Refs
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-3-341595815810.1186/1477-7819-3-34Case ReportLong term remission of metastatic placental site trophoblastic tumor (PSTT): Case report and review of literature Behtash Nadereh [email protected] Fatemeh [email protected] Malihe [email protected] Gynecology Oncology, Tehran University of Medical Sciences, Tehran, Iran2005 15 6 2005 3 34 34 1 5 2005 15 6 2005 Copyright © 2005 Behtash et al; licensee BioMed Central Ltd.2005Behtash et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Placental site trophoblastic tumor (PSTT) is a rare and unique form of gestational trophoblastic disease (GTD). This tumor represents a neoplastic transformation of intermediate trophoblastic cells. We document a case of long term remission in a patient with metastatic PSTT.
Case presentaion
A 27-year-old patient with metastatic PSTT was treated with combination therapy (chemotherapy and surgery). Patient is alive after 10 years without any evidence of recurrence. Literature on PSTT was searched using Medline and cross references, and pertinent articles were reviewed.
Conclusion
With surgery and chemotherapy it is possible to achieve long-term remission in metastatic PSTT. Only a handful of previously reported cases with prolonged remission had been treated with the described combined chemotherapy and surgical approach. We suggest that this approach may be recommended for metastatic PSTT.
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Background
Placental Site Trophoblastic Tumor (PSTT) is a rare form of gestational trophoblastic disease (GTD) and was described by Kurman et al [1]. In 1976, it was thought to be an exaggerated expression of the invasive nature of normal trophoblastic tissue which did not assume the characteristics of a malignant tumor. Twigg's et al, described a patient with trophoblastic pseudotumor which was progressive and fatal in 1981 [2]. Scully and Young in 1981 described additional 14 cases of which two died from metastatic disease [3].
Pathologically, the tumor is characterized by mononuclear intermediate trophoblastic cells with occasional multinuclear intermediate trophoblastic cells and occasional multinuclear giant cell which infiltrate both myometrium and blood vessels [4]. Immunohistochemical staining reveals many prolactin cells and few gonadotropin producing ones. Thus gonadotropin levels may be normal to elevated [5].
The etiology, epidemiology and risk-factors of PSTT are poorly understood. Presenting symptoms generally include irregular bleeding or amenorrhea and rarely nephrotic syndrome, sepsis, and erythrocytosis [6], or the metastatic sites may be the presenting symptoms. PSTT presents with metastases in about 10% of the cases [7] and metastases develop in an additional 10% during follow-up [8].
Although the majority of patients with non metastatic PSTT are cured by hysterectomy, a number of cases require aggressive treatment with chemotherapy and/or radiation.
This paper presents one case of PSTT treated by combined treatment modalities. Hysterectomy was performed in this case at a time when the exact diagnosis was not apparent.
We also reviewed the literature for the different strategies in treatment of the metastatic disease.
Case Presentation
In Sep. 1994, a 27-year-old woman (gravida 3, para 3) was referred to Gynecology Oncology Unit in Vali-e-Asr Hospital, Tehran, Iran. Her previous pregnancy was terminated at 40 weeks, with vaginal delivery in May 1991. After a few months of abnormal uterine bleeding, she underwent dilatation and curettage and then a total abdominal hysterectomy (2 months before referral). Histology of the resected specimen revealed choriocarcinoma.
Upon her first visit to our clinic, she was in respiratory distress, and had cough with bloody sputum. Physical examination revealed no abnormal finding except wheezing. Chest X-ray and computerized tomographic (CT) scan showed multiple metastatic nodules in both lungs parenchyma (Figure 1). Abdominal and pelvic CT was normal. Histology was reviewed by two expert pathologists in our center, and PSTT was confirmed (Figure 2). Serum β-hCG level was 210 mIU/ml. Serial serum β-hCG ranged between 170 – 250, and increased after 3 courses of combination chemotherapy with methotrexate, actinomycin and cyclophosphamide.
Figure 1 Chest X-ray and Computerized tomographic scan showing multiple metastatic nodules in the lung
Figure 2 Photomicrograph showing proliferating intermediate trophoblast with scares cytotophoblastic and systrophoblastic elements
Three years later, in november 1994, the patient was given three cycles of EMA chemotherapy. However, due to plateau titer of β-hCG the chemotherapy regimen was changed to EMA-CE. Reassessment of staging work-up was performed using pelvic ultrasonography that revealed a hypoechoic mass in right ovary (42 mm). At the end of six courses of EMA-CE chemotherapy, serum β-hCG remained at 50 mIu/ml.
In May 1995, patient underwent laparotomy and right oophrectomy one month following which the serum β-hCG was negative. Patient is on regular follow-up (clinical exam, β-hCG tests, pelvic and abdominal sonography and chest CT) and has shown no signs of local or systemic recurrence.
Discussion
PSTT is a rare form of GTD with unpredictable malignant potential and highly variable clinical course [9]. It could also present as a fulminant metastatic disease, resistant to conventional treating modalities. PSTT accounts for 3.1/1000 to 2/100 of all trophoblastic diseases [10,11]. Rate of PSTT to choriocarcinoma has been reported to be 1/138 [10]. The disease is usually seen in young women, although cases have been reported in post-menopausal women as well. The mean age at diagnosis is 31 to 33 years, and it can appear following any type of pregnancy [10-12]. The antecedent pregnancy is a full term normal in 53% of the cases [11,12], or a molar pregnancy seen in 21% of the cases [11,13]. The mean interval from the last pregnancy and diagnosis of PSTT can vary from several weeks up to 15 years [14].
Unlike choriocarcinoma, the level of serum β-hCG in PSTT correlate neither with tumor burden, nor with the malignant behavior. Thus β-hCG appears to have no predictive value and the disease may still progress even if levels are not raised [14,23]. The range of serum β-hCG concentrations at diagnosis in 79% of the patients is below 1000 and in 58% lower than 500 [11].
In NETDC report, most patients (12 from 13 patients) had β-hCG levels under 500 mIU/ml [12]. PSTT clearly present a wide spectrum of clinical course. The most frequent presenting symptom is vaginal bleeding (79%) [11]. Ninty-two percent of Feltmate series presented with amenorrhea or abnormal bleeding [12].
Outcome of PSTT as reported in literature is highly variable [9]. All cases of metastasis to vital organs, such as the brain, result in mortality despite all forms of treatment.
PSTT is usually confined to uterus at the time of diagnosis. In cases with distant spread of disease, metastases predominantly occur in the vagina and in lung as in our case. Brain metastases have been detected in more than half of PSTT patients [17,18]. Lung metastases have been reported in nearly one third of the patients in Charing Cross series [11]. In this series all the seven deaths in patients with lung metastasis occurred in patients that presented 4 years or more after last pregnancy [11].
Extrauterine spread of the disease appears to be the most useful prognostic factor for progression [19,20]. The interval from the last known antecedent pregnancy appears to be a second major prognostic variable in PSTT. In a multivariate analysis, the risk for unfavorable behavior of the disease increased considerably with the length of this interval [9,21]. Diagnosis less than 2 years from the antecedent pregnancy, and the disease localized to the uterus are associated with better outcome [9,11]. How et al, found that likelihood for fatal outcome was 14 times higher if the mitotic count was well above 5 [20].
In contrast to choriocarcinoma, PSTT is relatively resistant to chemotherapy. Consequently surgery is the mainstay of treatment. In most series more than one treatment modalities have been used [9,11,14,19].
Conservative surgery in form of dilatation and curettage is justified only if the fertility is to be preserved [22]. Further, as PSTT is generally resistant to chemotherapy, there are only few long term survivors, with metastatic PSTT despite intensive multimodal therapy [9,23]. Local uterine resection may be considered in such patients and in those who wants to retain fertility. When local resection is considered, ultrasound, MRI, and/or PET scan may identify the site of residual tumor.
In other cases, hysterectomy followed by adjuvant chemotherapy is primary treatment- chemotherapy within one week of hysterectomy, results in lower recurrence rates [14]. The most recent data from different centers, suggest that EMA/EP is the most effective treatment for metastatic or recurrent PSTT [9].
Conclusion
PSTT is a disorder of uncertain outcome, some of the patients do well while others have poor outcome. Combined treatment have been reported in 26% – 55% of the cases. Second look surgery, and multiple courses of combined chemotherapy may be necessary for remission.
Competing interests
The author(s) declare that they have no competing interest
Authors' contributions
NB carried out the surgery and participated in drafting the manuscript.
FG carried out the chemotherapy and follow-ups.
MH participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
Patient consent was obtained for publication of her case records and photographs
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BMC Emerg MedBMC Emergency Medicine1471-227XBioMed Central London 1471-227X-5-31592151710.1186/1471-227X-5-3Research ArticleReporting of unintended events in an intensive care unit: comparison between staff and observer Capuzzo Maurizia [email protected] Imad [email protected] Matilde [email protected] Vanna [email protected] Marco [email protected] Raffaele [email protected] Department of Surgical, Anaesthetic and Radiological Sciences, Section of Anaesthesiology and Intensive Care, University Hospital of Ferrara, Corso Giovecca 203 44100 Ferrara, Italy2005 27 5 2005 5 3 3 25 11 2004 27 5 2005 Copyright © 2005 Capuzzo et al; licensee BioMed Central Ltd.2005Capuzzo et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
In order to identify relevant targets for change, it is essential to know the reliability of incident staff reporting. The aim of this study is to compare the incidence and type of unintended events (UE) reported by facilitated Intensive Care Unit (ICU) staff with those recorded concurrently by an observer.
Methods
The study is a prospective data collection performed in two 4-bed multidisciplinary ICUs of a teaching hospital. The format of the UE reporting system was voluntary, facilitated and not necessarily anonymous, and used a structured form with a predetermined list of items. UEs were reported by ICU staff over a period of 4 weeks. The reporting incidence during the first fourteen days was compared with that during the second fourteen. During morning shifts in the second fourteen days, one observer in each ICU recorded any UE seen. The staff was not aware of the observers' study. The incidence of UEs reported by staff was compared with that recorded by the observers.
Results
The staff reported 36 UEs in the first fourteen days and 31 in the second.. The incidence of UE detection during morning shifts was significantly higher than during afternoon or night shifts (p < 0.001). Considering only working day morning shifts, the rate of UE reporting by the staff per 100 patient days was 26.9 (CI 95% 16.9–37.0) in the first fourteen day period and 20.3 (CI 95% 10.3–30.4) in the second. The rate of UE detection by the observers was 53.1 per 100 patient days (CI 95% 40.6–65.6), significantly higher (p < 0.001) than that reported concurrently by the staff. There was excellent agreement between staff and observers about the severity of the UEs recorded (Intraclass Correlation Coefficient 0.869). The observers recorded mainly UEs involving Airway/mechanical ventilation and Patient management, and the staff Catheter/Drain/Probe and Medication errors (p = 0.025).
Conclusion
UE incidence is strongly underreported by staff in comparison with observers. Also the types of UEs reported are different. Invaluable information about incidents in ICU can be obtained in a few days by observer monitoring.
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Background
Voluntary and anonymous Critical Incident Reporting (CIR) in Intensive Care Units (ICUs) is a technique for collecting information about any unintended event or outcome that reduced or could have reduced the safety margin for the patient. CIR has been used in single adult [1-5] as well as neonatal-pediatric [6] ICUs, and implemented at a national level in Australia [7-9].
It has been claimed that the rate of critical incidents could be a marker of the quality of care [5]. Unfortunately, CIR does not provide an objective numerator and there is no unequivocal denominator. Taking patient days as denominator, Bracco and co-workers [4] reported 777 critical incidents (241 of them were human errors) in 2810 patient days, giving a rate of 27.4 critical incidents (8.6 human errors) per 100 patient days, while Flaatten and Hevroy [10] found 2.7 errors per 100 patient days and, more recently, Osmon and co-workers [11] reported 8.93 medical errors per 100 ICU days. On the other hand, a pharmacist identified 187 medication administration errors in 851 patients [12], and an observer detected 132 medication errors in 88 patient days of observation [13]. Moreover, it has been shown that ICU staff reported only 61% of the errors detected by an external observer [14]. In some cases, such differences may reflect different definitions, but in others [3-6] they suggest that the real size of the patient safety problem in ICU is unknown.
The ultimate goal of incident reporting is to implement strategies to prevent recurrence [15]. Therefore, to ensure that relevant targets for change are recognized, it is essential to know the reliability of staff reporting.
The aim of this study is to compare the incidence and type of unintended events reported by facilitated ICU staff with those recorded concurrently by an observer.
Methods
Design
The study lasted for 28 days in 2003, from 24 November (7.00 am) to 22 December (7.00 am). Staff reporting of UEs on structured forms was active throughout the period. The UEs reported by the staff during the first fourteen days were compared with those reported during the second fourteen to detect any change in staff reporting.
During morning shifts (7.00 am-14.00 pm) in the second fourteen days (weekends and holidays were excluded), one observer in each ICU who had no other duties recorded any UE seen. The observations were performed concurrently in two ICUs and compared with the incidence of UE reporting by staff over the same period of time, to measure the reliability of spontaneous reporting.
The Ethics Committee of the Hospital approved the study.
Setting
The study was performed in two four-bed multidisciplinary ICUs located in different structures of the same 916-bed teaching hospital. Each ICU consists of a wide room with four beds allowing direct patient observation, with sliding curtains between beds.
Both ICUs have the same nurse-to-patient ratio, which is 1:2 (excluding 1 daytime unit sister per each ICU, with organizational and administrative tasks), and three nurse shifts: morning (7.00–14.00), afternoon (14.00–22.00) and night (22.00-7.00). In the ICUs, medical care is provided by specialists in anaesthesia and intensive care according to national rules. The ICUs have one overall medical director. A full time specialist and a resident doctor manage each ICU in the daytime (8.00–20.00), while a specialist and a resident doctor cover both ICUs at night (20.00-8.00).
Procedure
Before the study was initiated, several meetings with the research team were held to familiarize the ICU staff with the concept of CIR, to describe the non-punitive nature of the study, and to develop a structured form with a predetermined list of UEs for data collection. The form was tested in both ICUs, modified according to staff comments (excluding items never used and adding space for possible items not previously considered) and then implemented. The list of the items in the structured reporting form is given in the Table 1. The staff was instructed to report any UE irrespective of whether it was on the predetermined list, and to use the appropriate space in the form if the item was not listed.
Table 1 List of the items in the structured reporting form.
ICU where the UE has occurred Shift of UE detection: Morning
Reporter's qualification: Nurse, Physician Afternoon
Date of detection Night
Type of UE: Problems with airway/mechanical ventilation Type of UE: Problems with catheter/drain/probes
Accidental extubation Unplanned removal
Unplanned reintubation Dislodgement
Tracheal tube obstruction Inappropriate opening
Tracheal cuff leakage Inappropriate disconnection
Incorrect ventilator setting
Ventilator auto cycling Type of UE: Problems with medication
Turn off of heated humidifier Prescription error
Turn off of ventilator alarms Transcription error
Wrong dose
Type of UE: patient management: Wrong route of administration
Delayed treatment
Incorrect patient positioning Type of UE: unit management
Documentation lacking Organization
Documentation reported incorrectly or inaccurately Communication
Equipment failure
Turn off of oxygen saturationalarm
Other problem:................................
Severity of the unintended event
Intercepted by the staff
Self resolving
Minor
Serious
During the period of the study, the Director of the Unit enhanced reporting by emphasizing its relevance during ward rounds, encouraging staff to fill in the form immediately after discovering any UE, and discussing relevant (minor or serious) reports weekly with the whole ICU staff. Reporting was facilitated by the cooperative attitude of all the staff and by allowing but not requiring anonymity. The format of UE reporting system was voluntary, non-punitive, facilitated and not necessarily anonymous.
Blank forms were available on the nursing desk and completed ones were stored in a free deposit box, which was emptied every evening by those responsible for the study, who first analysed the reports. A poster with guidelines for reporting, definitions of UEs, some examples and the name of the person responsible for the research was displayed on the wall next to the forms.
During morning shifts in the second fourteen day period of the study, two residents attending the Specialist School in Anaesthesia and Intensive Care of the University (one in each ICU) acted as observers. They had already performed a one-year training period in general ICU and, before the beginning of the study, they had studied CRI methodology, analyzed precise definitions of variables in the literature and received instruction, with examples, about the definitions of UEs used in the present study. The observers, like the staff, were instructed to report any UE irrespective of whether it was in the predefined list, and to use the appropriate space in the form if the item was not listed. The observers used the same rules and structured forms as the staff, but they filled in a new form for each UE in a separate room and retained it in a separate box until the end of the study. The staff was not aware of the observers' study; the presence of the observers was explained by the collection of data for a different study.
Definitions
For the purpose of the study, any unintended event (UE) that reduced or could have reduced the safety margin for the patient while in ICU was considered. UEs occurring during transport or in other areas of the hospital were not considered. The following information, collected for each UE (items listed in the Table 1), was analyzed: date and shift of detection, reporter's qualification (nurse or physician), type and severity. The type of UE was categorized as follows [8]: Airway/mechanical ventilation, Catheter/Drain/Probe, Medication error, Patient management, Unit management, and Other (with space for details). UE severity was classified according to the reporter's opinion of the outcome and defined as follows:
• intercepted and spontaneously rectified by the staff before the patient could be affected (for example: a doctor prescribed a wrong dose of a drug, the nurse realized that it was a mistake and informed the doctor, who corrected the error so that the patient received the correct dose);
• self-resolving without specific treatment at the time of the event without harm to the patient (for example: accidental removal of a nasogastric tube that was no longer necessary, or alarms turned off after the end of nursing activities or physician round);
• minor at the time of the event but requiring transient increase in surveillance or adjustment of treatment (for example: unplanned intubation after planned extubation, without major physiological complications);
• serious, i.e. life-threatening or responsible for an increased length of hospitalization.
Data analysis
Statistical analysis was carried out using a software package (SigmaStat 2.0) and p values less than 0.05 were considered significant. Proportions were reported with 95% Confidence Intervals (CI) [16] and compared by a z test.
For the purpose of the study, the 4-week period was split into two intervals of fourteen days: the first (A) included 10 working and 4 non-working (weekend) days, and the second (B) included 9 working and 5 non-working days (4 weekend days and 1 national holiday). The following comparisons were performed: 1) between UEs reported by the staff during periods A and B, to exclude any effect of the presence of the observer; 2) between number and type of UEs reported by staff and observers during period B, to measure the reliability of spontaneous reporting; 3) between the severity scores given by staff and observer to the same UE, using an Intraclass Correlation Coefficient (ICC). The ICC assesses agreement, ranging from 0 (no agreement) to 1 (scores identical). Values higher than 0.80 indicate good agreement.
Results
Patient characteristics
During the period of study 31 patients were admitted to or already present in the ICU. The median age was 68 y (range 28–92), median duration of mechanical ventilation was 3 days (range 0–21), and median length of stay in ICU was 6 days (range 1–43). The median values of SAPS II [17] and APACHE II [18] were 37 (range 7–67) and 15 (range 7–31) respectively. One patient died in the ICU and 10 patients died in hospital. The characteristics of the ICU patients present during periods A and B are shown in table 2.
Table 2 Patients present in the ICU during the first and second 14 day periods.
Period of fourteen days first second
Number of patients present in the period 18 20
Age (years): median 74 73
Type of ICU admission
Surgical planned 6 8
Surgical unplanned 7 7
Medical 5 5
SAPS II at ICU admission: median 40 39
APACHE II at ICU admission: median 18 15
Number of ICU days (hours/24) in the study 110 99
Number of ventilation days (hours/24) in the study 71 59
Available ICU bed days 112 112
The total number of patients surveyed was 31. Seven patients (median values of age 76 y, SAPS II 47, APACHE II 19) were present in both periods. Numbers of ICU and ventilation days are given as exact numbers: counted hours divided by 24.
Bed day occupation
The exact number of bed days occupied (patient days), computed as sum of the hours spent by each patient in the ICU during the period of study divided by 24, was 209 over the whole 4 week period. The numbers of patient days during the working day morning shifts of periods A and B were 78 and 64, respectively; the numbers of bed days available were 80 and 72, respectively; and bed occupancies were 98% and 89%, respectively.
Unintended events reported by the staff
The staff reported 36 UEs in period A and 31 in period B. There was no significant difference in incidence (33 vs 31/100 patient days) between the two periods (p = 0.872), or between the number of UEs reported in periods A and B by physicians and nurses (31 and 25 versus 5 and 6, respectively; p = 0.786). Of the 67 UEs reported by the staff, 52 occurred during working and 15 in non-working days, with incidences of 36 and 22/100 patient days, respectively (p = 0.061). Considering the 4 weeks as a whole, the incidence of UEs detected during morning shifts (22/100 patient days) was significantly higher(p < 0.001) than in afternoon (6/100 patient days) or night (4/100 patient days) shifts.
Of the 67 UEs reported by staff, 8 were classified as intercepted by the staff (for instance, 5 medication errors in drug prescription were rectified before administration), 45 as self-resolving, 13 as minor and 1 as serious. The last was a ventricular fibrillation, treated without sequelae. The patient's serum potassium level was low according to a laboratory test on a blood sample taken in the morning. Potassium chloride 40 mEq was prescribed to be added to the parenteral bag. The nurse injected the drug into the bag without stopping the infusion pump. In 1 min, the patient, who was intubated but alert, became agitated, so the physician and the nurse hastened to him. Afterwards, the patient lost consciousness with the EGC monitor showing ventricular fibrillation; defibrillation was performed in less than 30 s.
Unintended events detected by observer
In the working day morning shifts of period B, observers detected 34 UEs in 64 patient days. These UEs were classified as intercepted by the staff (3 cases), self-resolving (25), and minor (6). The types of UEs detected by observers were as follows: 13 were in the Airway/mechanical ventilation category, 3 Catheter/Drain/Probe, 5 Medication errors, 12 Patient management and 1 Unit management.
Comparison between staff and observer reports
In the working day morning shifts of period A, the rate of UEs per 100 patient days was 26.9 (21/78), with CI 95% = 16.9–37.0. In the working day morning shifts of period B, the observers detected 21 UEs not recorded by the staff. No UE was reported by staff and missed by observers. The rate of UEs per 100 patient days was 20.3 (CI 95% = 10.3–30.4) according to the staff and 53.1 (CI 95% = 40.6–65.6) according to the observers (p < 0.001). Of the UEs detected by the ICU observers, the staff reported 37.5% in one ICU and 38.5% in the other.
The staff and the observer recorded the same severity in 11 of the 13 UEs that were recorded by both (1 intercepted by the staff, 7 self resolving and 3 minor); of the remainder, the observer scored one UE higher than the staff (minor vs self-resolving) and the other lower (self-resolving vs minor). The ICC for the staff and observer severity ratings of the 13 UEs was 0.869, showing excellent agreement.
The types of UEs reported by the staff during the whole study period (4 weeks), and those reported during period B (morning shifts in working days) by the staff and the observers, are shown in table 3. Of the UEs reported by observers, 6 out of the 13 Airway/mechanical ventilation events involved turning off ventilator alarms and 5 out of the 12 patient management events involved saturation alarms being switched off. The incidences of the 5 types of UE recorded by the observers were significantly different (p = 0.025) from those recorded by staff over the whole study period. No difference was found in the incidences of the 5 types of UE reported by staff during the 4-week study period or in the working day morning shifts of period B (p = 0.265).
Table 3 Types of unintended events according to the time of reporting and reporters.
Period of fourteen days all first second
Days all working working
Shifts all morning morning
Reporter staff staff observer
N. of events concerning
Airway/mechanical ventilation 13 (19%) 2 (15%) 13 (38%)
Catheter/Drain/Probe 18 (27%) 1 (8%) 3 (9%)
Medication errors 19 (28%) 3 (23%) 5 (15%)
Patient management 13 (19%) 6 (46%) 12 (35%)
Unit management 4 (6%) 1 (8%) 1 (3%)
Total number of events 67 13 34
Statistical significance: staff (entire period, first column) vs staff (first fourteen days, second column) p = 0.265 (chi square 5.222); staff (entire period, first column) vs observers (last column) p = 0.025 (chi square 11.127).
Discussion
This study demonstrates that the rate of spontaneous reporting of UEs by ICU staff is less than half an observer's reporting rate. Therefore, information obtained by CIR markedly underestimates the real UE frequency. Even more important, the type of UEs spontaneously reported by staff is different from that recorded by an observer using the same criteria as the staff. If we had had to prioritize changes on the basis of staff reporting in our setting (Table 3, first column), we would have devoted more attention to the Catheter/Drain/Probe and Medication error categories. In contrast, Airway/mechanical ventilation and Patient management were the most frequent types of UE recorded by observers. Therefore, spontaneous staff reporting does not appear reliable enough to mirror the truth.
Generally, 12% (8 of 67) of UEs reported by the staff and 9% (3 of 34) of those detected by observers were intercepted by the staff, suggesting a measure of the efficiency of control in our setting. Most of the UEs recorded by observers involved latent errors in alarm settings, i.e. potential problems within the system [19]. Surprisingly, staff members were not aware of this kind of problem and only the observer reports allowed us to identify it.
In this study, most UEs (83%) were reported by physicians. This could be due to the facilitated format of reporting, i.e. the enhancement of immediate reporting by the attending physicians, with stronger emphasis on the report than on the anonymity; and to the cooperative attitude of all the staff, which allowed physicians to record on the form an event discovered by a nurse. The lack of double reports indicates that nurses and doctors, attending and resident, were informed about UEs already reported; the report forms remained in an open box throughout the day of the event. On the other hand, nurses reported 43% of critical incidents [3] and 59% of medical errors [11]. Moreover, nurses reported 74% of incidents in the Australian Incident Monitoring Study published in 1996 [7] and 49% of the incidents collected in 2003, by facilitated incident monitoring, in an ICU where CIR had been used for more than 5 years [5]. This finding and our results suggest that physician reporting increases when it is facilitated and, possibly, not anonymous.
The fact that the highest frequency of UEs was discovered in morning shifts agrees with the findings of Donchin and coworkers [14], who studied the diurnal distribution of errors, and Frey and coworkers, who analyzed critical incidents in pediatrics [6]. Other authors found the highest incidence of critical incidents [3] or errors [10] in afternoon shifts. Our finding is consistent with the high number of activities performed, especially by physicians, during the morning. Nevertheless, in the present study, the time of UE reporting was taken as the time of UE detection, and we cannot exclude the possibility that some UEs reported in morning shifts had actually occurred during other shifts.
The study has some limitations. The use of a predefined list of items could have induced both staff and observers to pay attention mainly to the listed items, even though space was allowed for additional ones. Therefore, we can not exclude the possibility that other UEs were missed. Nor can we exclude the possibility that the staff reported fewer events in period B than in period A due to a lessening of the initial enthusiasm, although there was no statistically significant difference. However, the lower number of patient days in working day morning shifts during period B (64 vs 78 in period A) could explain the lower number of events reported in terms of both direct (reduced number of patients at risk, fewer procedures) and indirect (reduced nursing workload) effects. Another limitation is that nursing workload during the study period was not measured. We did not investigate inter-observer reliability, but agreement in responses to questions concerning adverse event reporting has been shown to be good [20]. Indeed, the staff of the two ICUs reported similar percentages of the events recorded by the observers. This finding supports our general conclusion that the incidence of unintended events is markedly underestimated by spontaneous reporting. Finally, no attempt was made to investigate whether the UEs reported were preventable, considering that it has already been shown that most incidents are preventable [3,5].
Conclusion
Our study demonstrates that the incidence of UEs is markedly underestimated by spontaneous staff reporting in comparison with observers' recording. Even more important, the type of UE reported is different. One final implication of this study, performed in a single centre and over a short period of time, is of general value: invaluable information about incidents in ICU can be obtained in a few days by observer monitoring.
List of abbreviations used
Intensive Care Units ICU
unintended events UE
Critical Incident Reporting CIR
Simplified acute physiology score SAPS II
Acute Physiology And Chronic Health Evaluation APACHE II
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MC conceived and proposed the study, participated in the design and in meetings to prepare the staff, performed the statistical analysis and draft the manuscript. IM and MC participated in the designing and preparing the structured form, collected data as observers, participated in the analysis and in drafting the article. VV and MV participated in meetings with the staff to prepare the structured form, stimulated the staff to reporting, discussed the reports with the staff and participated in drafting the article. RA (Director of the Unit) participated in the design, enhanced reporting at round visits, and critically revised the article. At the time of the study, MC, IM, MC and RA were completely aware of the study (comparison between staff and observers), while VV and MV were informed only about the staff data reporting.
All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This study was presented, in part, at the 17th Congress of the European Society of Intensive Care Medicine (ESICM) in Berlin, 10–13 October 2004.
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| 15921517 | PMC1165974 | CC BY | 2021-01-04 16:31:03 | no | BMC Emerg Med. 2005 May 27; 5:3 | utf-8 | BMC Emerg Med | 2,005 | 10.1186/1471-227X-5-3 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1598491210.1371/journal.pbio.0030247Research ArticleEcologyEvolutionGenetics/Genomics/Gene TherapyOtherHomo (Human)Traces of Archaic Mitochondrial Lineages Persist in Austronesian-Speaking Formosan Populations mtDNA Variation in Indigenous TaiwaneseTrejaut Jean A [email protected]
1
¤Kivisild Toomas
2
Loo Jun Hun
1
Lee Chien Liang
1
He Chun Lin
1
Hsu Chia Jung
1
Li Zheng Yuan
1
Lin Marie [email protected]
1
1 Transfusion Medicine Laboratory, Mackay Memorial Hospital, Taipei, Taiwan,2 Estonian Biocentre, Tartu, EstoniaPenny David Academic EditorMassey UniversityNew Zealand8 2005 5 7 2005 5 7 2005 3 8 e24729 4 2005 11 5 2005 Copyright: © 2005 Trejaut et al.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Mitochondrial DNA Provides a Link between Polynesians and Indigenous Taiwanese
Genetic affinities between aboriginal Taiwanese and populations from Oceania and Southeast Asia have previously been explored through analyses of mitochondrial DNA (mtDNA), Y chromosomal DNA, and human leukocyte antigen loci. Recent genetic studies have supported the “slow boat” and “entangled bank” models according to which the Polynesian migration can be seen as an expansion from Melanesia without any major direct genetic thread leading back to its initiation from Taiwan. We assessed mtDNA variation in 640 individuals from nine tribes of the central mountain ranges and east coast regions of Taiwan. In contrast to the Han populations, the tribes showed a low frequency of haplogroups D4 and G, and an absence of haplogroups A, C, Z, M9, and M10. Also, more than 85% of the maternal lineages were nested within haplogroups B4, B5a, F1a, F3b, E, and M7. Although indicating a common origin of the populations of insular Southeast Asia and Oceania, most mtDNA lineages in Taiwanese aboriginal populations are grouped separately from those found in China and the Taiwan general (Han) population, suggesting a prevalence in the Taiwanese aboriginal gene pool of its initial late Pleistocene settlers. Interestingly, from complete mtDNA sequencing information, most B4a lineages were associated with three coding region substitutions, defining a new subclade, B4a1a, that endorses the origin of Polynesian migration from Taiwan. Coalescence times of B4a1a were 13.2 ± 3.8 thousand years (or 9.3 ± 2.5 thousand years in Papuans and Polynesians). Considering the lack of a common specific Y chromosomal element shared by the Taiwanese aboriginals and Polynesians, the mtDNA evidence provided here is also consistent with the suggestion that the proto-Oceanic societies would have been mainly matrilocal.
An extensive phylogenetic analysis of mtDNA from nine Taiwanese tribes reveals an unambiguous genetic link between aboriginal Taiwanese and Polynesian populations, to the exclusion of mainland Asians.
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Introduction
Present day Taiwan is a home to heterogeneous groups of people. The main part of the Taiwanese population is composed of the Minnan (73.5%) and Hakka (17.5%) who descend from immigrants from the Fujian and Guangdong provinces of Southeast China during the last 400 years. After World War II, migration from different provinces of China brought a present-day share of 7.5% to 13% to Taiwan [1]. Only 1.5% of today's population of Taiwan is represented by Austronesian speakers—Atayalic, East Formosan, Puyuma, Paiwan, Rukai, Tsouic, Bunun, Western plains, Northwest Formosan, and Malayo-Polynesian languages [2]—who are generally considered indigenous to the country. According to the year 2000 census, Yami with 4,050 individuals and Amis with 146,796 individuals represent the smallest and the largest tribal populations of Taiwan respectively.
The archaeological record suggests that a substantial cultural change occurred in Taiwan approximately in the sixth millennium BC [3]. Whether or not the transition to Neolithic technology in Taiwan corresponded to a substantial gene flow from China is unclear. It is also not clear whether there were one or multiple waves of Neolithic migrations to northern and southern Taiwan from southeast China [4–6]. There is archaeological evidence for the occupation of caves in southern Taiwan by humans by at least 15 thousand YBP [4]. Yet, the bulk of archaeological material started to accumulate during the Neolithic period.
Three different views have been put forward to explain the origin of present day Austronesian-speaking tribes in Taiwan, including (a) origin from expanding regions of early rice cultivation in central China [7], (b) origin in insular Southeast Asia or (c) local settlement after the rise of the sea levels [8]. During the massive immigration of Han speakers to the western plains of Taiwan, most tribes took refuge in remote regions such as the central mountain ranges or the east coast of Taiwan. It is believed that this geographical isolation has largely contributed toward maintaining their culture and languages until the present day in contrast to the plain tribes that are characterized by high levels of admixture [9].
The role of Taiwanese indigenous populations in the Austronesian settlement of island Southeast Asia and Polynesia has come under intense discussion during the last two decades, in particular among geneticists working with mitochondrial DNA (mtDNA), Y chromosome, and HLA loci [9–21]. Although the landscape of possible migration out of Taiwan and interaction scenarios is quite complex [22], two opposing models, the “express train” [23] and the “entangled bank” models [24], emerging from among other alternatives [8,16,25] have commonly been tested with genetic data. Proponents of the express train model [23] hold that the Polynesian islands were settled in a relatively short period with a migration originating from southern China to Taiwan. According to this model, the Austronesian migration from Taiwan was coupled by the spread of Lapita culture, whose supposed precursor pottery in Taiwan is dated to at least 6000 YBP [26]. The entangled bank model [24], along with the “slow boat” model [8,16,25], on the other hand, proposes that the ancestors of Polynesians did not move rapidly through Southeast Asia and Melanesia. According to these two models, the interaction with Melanesians before colonizing the Pacific was the major factor determining the genetic composition of the Polynesian ancestors. The proponents of the intermediary slow boat model [8,16,25], also popularly introduced in the book Eden in the East [27], propose a date and a location in Southeast Asia for the origin of the Polynesian expansion and question the Taiwanese genetic contribution to the ancestors of Polynesians.
Previous mtDNA studies [13,28] on four tribal groups (Amis, Atayal, Bunun, and Paiwan) and on the Taiwanese general population [12,29] have revealed their common ancestral origins with populations of China and/or Southeast Asia. Tajima et al. [9] characterized two hyper-variable segment I (HVS-I) lineage groups as unique to Taiwanese aboriginals, accounting for 22% of their mtDNA variation, and estimated their origin to approximately 11,000–26,000 years ago. Three groupings of aboriginal Taiwanese populations were suggested by this study [9]: southern (Rukai and Paiwan), northern (Atayal and Saisiat), and central-eastern (Amis, Bunun, Tsou, Puyuma, and Yami). However, the data did not conclusively show whether these three distinctive clusters reflected three different migrations to Taiwan or whether they could be explained by the effect of drift and long-term isolation of the island's populations.
A common HVS-I motif 16189–16217–16261, classified within haplogroup B4a [30], is shared between Taiwanese and Polynesian populations. This motif was considered first as the genetic link supporting Polynesian origins in Taiwan [10,13]. This root haplotype of B4a from which the Polynesian motif derives by a single HVS-I mutation is, however, widely spread in East Asia. Besides Taiwan and Southeast Asia, B4a occurs frequently in southern China and has been observed as far inland as among Mongolians [21,30–35]. Its deep time depth, 40,400 ± 9,600 years in China [34], significantly predates the timeframe that is relevant to the peopling of Polynesia. Therefore, this general genetic link between Taiwan and Polynesia has now been refuted as being supportive of Polynesian origins specifically in Taiwan within a recent timeframe as implicated by the fast train model [8].
An overwhelming majority of mainland East Asian mtDNA lineages are nested within haplogroups A, G, M, N9, and R9 [30,31,34–38]. Although previous mtDNA research on Taiwanese aboriginal populations has focused only on hyper-variable displacement loop (D-loop) data, we attempt here to define the distribution of these haplogroups and their region-specific twigs in nine Taiwan indigenous tribes (Figure 1). Using the coalescent approach, we examine the hypothesis that the distinct mtDNA variation and genetic structure seen in present-day Taiwanese mountain tribes might be the result of a Neolithic migration or of a long-term separation from their common ancestors with the modern Chinese populations of mainland East Asia. Finally, to determine if Taiwanese B4a lineages share any coding region variants with Polynesian B4a lineages [39], we determined the complete mtDNA sequence for eight different B4a lineages from indigenous Taiwanese.
Figure 1 Geographic Distribution of Nine Indigenous Tribes of Taiwan
Results
Haplogroup Structure and Distribution in Taiwanese Aboriginal Populations
Ninety-six distinct haplotypes were identified by 81 variable sites of the mtDNA control region in 640 Taiwanese aboriginal samples representing all nine mountain tribes (Figure S1). Among these, 39 individuals possessed a unique HVS-I haplotype. From the 96 haplotypes, 30 were shared by at least two different tribes. Haplotype sharing occurred mostly between adjacent tribes (Figures 2 and S1). Phylogenetic analysis using control and coding region information clustered the observed haplotypes into 20 distinct haplogroups and subgroups whose distribution among tribes was compared to available information from other Asian populations (Table 1). Four basic haplogroups—B, E, R9, and M7–accounted for more than 90% of the variation observed in aboriginal Taiwanese. In comparison, the combined frequency of these haplogroups in China averaged less than 40%, with southern Chinese having significantly higher frequency than northern Chinese (Table 1). Among these four basic haplogroups, haplogroup E was nearly absent in continental Asia. On the other hand, haplogroups A, D4, G, and M8-M10 accounted approximately for 41% of the sequences from the mainland, whereas it was rare or absent in Taiwanese Aborigines. Only two unique D4 haplotypes were observed in the Atayal and Saisiat populations and a single G1a lineage in Tsou (Table 1; Figures 2 and S1).
Figure 2 Tree Drawn from a Median-Joining Network of 96 mtDNA Haplotypes Observed in Nine Indigenous Taiwanese Populations
The tree is based on sequences of HVS-I (16024–16390) and a coding region segment covering 9,793 to 10,899 bps. Haplogroup defining HVS-II mutations were manually added after the generation of the network. Additional coding region mutations, ascertained through complete mtDNA sequencing of an individual of each subclade of the haplogroup defined by the mutation, were used to generate the network and are shown in blue. All nps are numbered according to reference sequence [71]. Mutations in italic indicate back conversions. Nucleotide change is specified only for transversions. Node areas are proportional to haplotype frequencies of the pooled nine tribes. The population codes are as follows: A, Atayal; B, Bunun; M, Amis; P, Paiwan; R, Rukai; S, Saisiat; U, Puyuma; T, Tsou; and Y, Yami. CRS = Cambridge reference sequence [70].
Table 1 Haplogroup Frequencies in Taiwan, East Asia, and Oceania
Principal Components Analysis
Principal components (PC) analysis using haplogroup frequencies revealed a high level of differentiation between Taiwanese aborigines as compared with other Asian populations (Figure 3). In particular, Taiwanese aboriginal populations appeared closer to island Southeast Asian populations (Luzon, Philippines, Moluccas, and Indonesia) than to populations from mainland East Asia (Fujian, South Vietnam, Malaysia, and Thailand). The three southernmost populations of Taiwan (Puyuma, Paiwan, and Rukai) and, more distantly, Yami from Orchid Island clearly differentiated the southern populations from the northern and central populations of Taiwan from which the Bunun sample emerged as an outlier. Although the Amis population clustered closely with northern and central tribes in the first two dimensions of the PC analysis, their haplogroup structure and relatively high frequency of B4a, D5, and M7c clades at the same time showed an affinity toward southern tribes of Taiwan. The grouping of Taiwanese indigenous populations revealed by our analysis differs from the population tree drawn from pairwise nucleotide differences by Tajima et al. [20] in which Puyuma and Yami, for example, clustered with central and eastern populations.
Figure 3 Principal Components Analysis
Principal components map obtained from a matrix of haplogroup frequencies in nine Taiwan indigenous tribes (Table 1), northern and southern Chinese [34], Taiwan urban population (Minnan and Hakka) [29], Japan [73], Korea [33], Luzon [12], Moluccas [14], Indonesia, Ryukyu [42] Malaysia, South Vietnam, the Philippines [20], and Thai [45,46]. Austronesian-speaking populations are represented by circles, non–Austronesian-speaking populations by stars.
Tribes of Northern and Central Taiwan
Haplogroups B4b, B5a2, E, F4b, and M7b covered more than 80% of the mtDNA variation observed in Atayal, Saisiat, and Bunun populations (north and central Taiwan; see Figure 2). The frequency of these haplogroups elsewhere in Taiwan was significantly lower (24%; p < 0.0001). Haplogroup E was most divergent and frequent in the Saisiat population in the northern mountain ranges of Taiwan.
Two subclades of haplogroup B5a can be defined on the basis of complete sequence information [37,38], available restriction fragment length polymorphism (RFLP) and HVS-I information from Southeast Asia [11,30,33,34,40,41], and our data: The loss of a HaeIII site at nucleotide position (np) 6957 in association with 16266A allele defines the major subclade B5a1 spread in Southwest China, Thailand, Vietnam, and Nicobar Islands whereas the presence of +11146 DdeI site at np 11146 defines a B5a2 clade that is predominantly in association with 16266G allele. Within the B5a2 clade, available D-loop information allows us to postulate the presence of two further branches that are highly region specific in Southeast and East Asia: HVS-I motif 16140–16189–16266G-16362 (B5a2a), which so far has been observed exclusively in Taiwan [9,28], whereas another motif, 16140–16187–16189–16266A/G, characterizes all B5a variants observed so far in Korean, Japanese, and Han lineages from northeast China [33,34,40,42]. In Taiwanese aboriginals, B5a2 lineages were found all over the island whereas their frequency, likely due to drift, was the highest in north-central regions, among the Tsou and Saisiat (see Figure 2).
Over one quarter of Taiwanese from northern and central mountain regions (Atayal, Saisiat, and Bunun) belonged to a novel subclade of haplogroup F4 [38,39,43,44] called F4b because of its distinctive mutation motif 10097C-16218–16311 (see Figure 2). Considering available mtDNA datasets for Southeast Asia, haplogroup F4b has a marginally low frequency (<1%) in China [30,31,34] and was not found among 519 Thai samples [45,46], in which the overall haplogroup F frequency was one of the highest in Asia (22%).
Most northern Taiwanese aboriginals from the M7b clade possessed a combination of four HVS-I substitutions (at nps 16086–16129–16324), which defines the clade M7b3 [47]. HVS-I sequences with a similar motif have been observed previously only in one Hui, one Uighur, one Dai, and one Mongolian sequence [32,43,47,48]. Consistent with the study of Tajima et al. [9] in which the HVS-I–defined cluster C4 corresponds to our M7b3, we found no exact matches to the Taiwanese M7b3 sequences in the available literature. Two Taiwanese complete sequences with M7b3 HVS-I motif [39], one matching our haplotype 56, the other deriving from haplotype 60 by a single HVS-I mutation, shared seven coding region mutations (at nps 1664, 4454, 6351, 9468, 10497, 12121, and 14115) that have not been seen in other M7b sequences from China or Japan [37,38,49]. Furthermore, the Taiwanese M7b3 sequences lacked a mutation at np 12811 that is shared by sister clades M7b1 and M7b2 [37]. Interestingly, all Taiwanese aboriginal populations lacked the M7b2 subclade that occurs with remarkable haplotype diversity (h = 1) and frequency (8%) in neighboring Ryukyu island populations [42]. Also, subclade M7b1, which is common in southern Chinese [30] and the non-indigenous Taiwanese population (9% frequency; [42]) was found at low frequency only among the Amis. Interestingly, all five Amis M7b1 sequences derived from their ancestral haplotype by a substitution at np 16126 that was not observed among any other 28 M7b1 samples from Asia [30]. This observation makes unlikely the possibility that admixture with Han populations from China would be the source of the M7b1-16126C lineages seen in Amis.
Highly homogeneous haplogroup B4b lineages were concentrated in the northern and central regions of Taiwan, likely involving a founder effect in Bunun. This clade is frequent in southern China where an exact match for the dominant B4b haplotype is also observed among the Taiwanese [30].
Haplotype 30 (see Figure S1), observed in six Saisiat and three Atayal samples within haplogroup Y, included only a match with two Han Chinese from Shanghai [40]. Similarly, exact haplotype matches with Han Chinese from China were found among tribal samples grouped in haplogroups B4b and D4, which were observed only in the northern-central parts of Taiwan, in the Atayal, Saisiat, and Bunun populations. Haplogroups B4b and D4 could represent recent admixture mediated through urban populations. Nonetheless, this appears to be quite unlikely for the Bunun who have historically been one of the most isolated tribes in Taiwan, who practiced head hunting until the early 20th century. The high incidence of two specific B4b haplotypes among them, in contrast, can be well explained by drift operating in a strictly endogamous small community.
Thirteen Tsou sequences, one Paiwan, and one Bunun sample shared a characteristic haplogroup R9c substitution pattern [50] in the control region (see Figure 2). This haplogroup has been observed at low frequency in mainland Southeast Asia [34,50,51].
Yami and Tribes of Southern and Southeastern Taiwan
Haplogroups B4a, D5, F3b, M7c, and N9a characterized 72.2% of the mtDNA variation of the populations of south and southeast Taiwan. Two distinct subclades of M7c were observed. The first subclade, M7c1c [30], occurs frequently all over the southeast coast, and is most frequent among Puyuma (29%). The second subclade, M7c1a, is seen in Amis (8%) and one Tsou sample. Haplotype diversity (h = 0.59) of M7c1 in Taiwan is likely reduced by genetic drift and founder effects among the Puyuma and Yami tribes. Similarly, without any downstream variation, only the founder HVS-I haplotype (16223–16257A) of haplogroup N9a was observed in Atayal, Amis, and Puyuma populations. Subclade B4c1b, frequent in southern Taiwan, has been previously detected at low frequency in China, Japan, Malaysia, Vietnam, the Philippines, and Java [13,20,30,38,51–53]. Two distinct subgroups of B4a can be found in Taiwanese aboriginals. The first of them, B4a1a, distinguished by a substitution at np 6719 (see Figures 2, S1), occurs frequently among the Amis and Yami, whereas the second, B4a2, is common in the southern tribes Paiwan, Puyuma, Rukai, and Yami.
Haplogroup F3b, previously labeled as R9a, has been encountered at low frequency in south and west China [30,34,37]. In Taiwanese, a high frequency of F3b was observed specifically among the three southernmost populations and is virtually absent in other tribes. This is consistent with the distribution of the respective HVS-I cluster C2 described in the study of Tajima et al. [9]. Interestingly, the HVS-I motif of all Taiwanese F3b samples was different from the motif commonly observed among the Chinese. Namely, Taiwanese F3b samples were characterized by a transversion at np 16220 (16220C) and lacked the transition at np 16335 seen in R9c. The 16220C variant of haplogroup F3b has been observed previously in two Bai samples from Yunnan Province of China and in one Japanese sample from Honshu [42,43]. In both cases, however, the difference with the closest Taiwanese HVS-I haplotype is two or more substitutions in HVS-I. Haplogroup F1a distribution is concentrated among the Tsou and Yami populations. Interestingly, among Yami, only the F1a1 subclade, defined by the HVS-I motif 16129–16162–16172–16304, was detected, although other haplogroup F subclades occur at considerably high frequency throughout other populations of Taiwan.
Finally, haplogroup D5 is spread at a moderately low frequency throughout East Asia, being most frequent in the south and absent or rare in Central Asia and Siberia [30]. Among Taiwanese aboriginal groups, distribution of D5 lineages is restricted to the three southernmost populations and the Amis. Although samples from Paiwan, Puyuma, and Rukai carry the root HVS-I haplotype of D5 or its one-step mutation descendants, the Amis harbor a derived motif with two substitutions (16148 and 16092) that has not been detected elsewhere in Asia so far.
Coalescence Times
A number of mtDNA haplogroups in Taiwan showed limited haplotype and nucleotide diversity. Consequently, according to control sequence information, sequences belonging to haplogroups B4b, D4, E2, F4b, N9a, and Y (and possibly R9c when ignoring the unique Bunun specimen) coalesced in their most recent common ancestors (MRCAs) within the last two thousand years (Table 2). These low coalescence times may be due either to drift or to bottlenecks affecting locally evolving lineages, or they may be due to founder effects following admixture with recent immigrants from outside the island. The presence or absence of matching sequence types elsewhere, as detailed above, can be considered the best possible way to evaluate these two scenarios. The nearly complete absence of haplogroup E in China makes it unlikely that the shallow time depth of E2 lineages in Taiwan could be explained by a migration from China. However, many populations from island Southeast Asia are still poorly covered, and the possibility that these lineages can derive from a migration from outside should be kept open.
Table 2 Coalescence Times of Major mtDNA Haplogroups in Taiwanese Aboriginals
More than half of Taiwanese mtDNA lineages fall into clades B4a1a, B4c1b, E1a, F1a1, F1a2, F3b, M7c1c, and M7b3 that show, with a broad range of standard errors, average coalescence times between 7.7 and 16.1 thousand years. The dates for clades M7c1c and M7b3 are similar to those of M7b1 and M7b2 previously reported in Southeast Asia [30]. It is likely that these four M7b daughter clades, together with other subclades of haplogroups B4, E, and F3b, began to diversify at the time of the rise of the sea levels after the end of the Younger Dryas cold spell approximately 11,000 years ago in distinct islands close to mainland Southeast Asia.
Taiwanese Affinities with Austronesian Populations of Oceania
PC analysis reveals that Taiwanese populations in general and the Amis specifically are more closely related to island Southeast Asian populations than to populations from mainland East Asia (see Figure 2). However, only haplogroups B4a1a and M7c1c substantiate the close affinity between Taiwanese populations and those from islands beyond the Philippines. Characteristic HVS-I motifs of haplogroups B5a, F3b, R9c, and E2 were also observed in the Philippines and Sabah from Borneo; haplogroups Y and M7b3 were seen as shared only with Philippines' populations. Finally, F4b and R9c lineages are noncharacteristic of Near and Remote Oceania [11,12,20,39].
The B4a sequences, in general, are frequent along the southeastern coast of Taiwan, but also have a wide and ancient distribution all over East Asia and island Pacific. To provide a more detailed phylogenetic resolution, we determined the complete mtDNA sequence of eight different HVS-I haplotypes observed in Taiwan that belong to haplogroup B4a. Phylogenetic analysis, including 23 published B4a complete sequences, reveals an interesting substructure of the clade. One major subclade, B4a1, can be defined now by three coding region mutations at nps 5465, 9123, and 10238 (Figure 4) that cover all Taiwanese and Oceanian B4a sequences. The B4a clade also includes samples from Korea, Japan, and China. Using a mutation rate of 1.26 ± 0.08 × 10−8 in the mtDNA coding region [54], the coalescence time of B4a1 (28.9 ± 7.4 thousand years) is consistent with its origin, likely somewhere in Southeast Asia before the last glacial maximum. Notably, besides the B4a1 subclade, which occurs at minor frequency in Han Chinese (8/332; [30,34]), at least two other subclades of B4a can be found in southern China. Similarly, among Japanese, haplogroup B4a1 is represented by three different subclades that appear as sister groups to the Taiwanese/Polynesian-specific clade B4a1a (Figure 4). On the other hand, the minor B4a2 subclade among Taiwanese has one closely related lineage among Japanese.
Figure 4 Phylogenetic Tree Relating Haplogroup B4a1 Complete Sequences
Base pair exchange is specified only for transversions. Recurrent mutations are underlined. Coalescence times are shown beside nodes.
The coalescence time of haplogroup B4a1a, based on coding region mutations, is 13.2 ± 3.8 thousand years, consistent with its HVS-I–based age estimate. To test whether B4a1a could have been imported recently to Taiwan from the mainland, we genotyped the defining markers of this clade in 47 North Vietnamese and 79 Han speakers from Fujian (unpublished data), the closest province of China to Taiwan, and we did not observe any carriers of haplogroup B4a1a. The clustering pattern within B4a1a therefore provides a unique link between Polynesian, Papuan, and Taiwanese lineages, supporting their common origin around the Younger Dryas period somewhere in island Southeast Asia, possibly in Taiwan. The Polynesian and Papuan sequences occupy a derived position in the B4a1a tree, descending from their MRCA defined by a mutation at np 14022 that was not observed in Taiwanese. The derived B4a1a1 clade in Papuans and Polynesians has a coalescence time of 9.3 ± 2.5 thousand years.
Discussion
Genetic Composition of mtDNA Lineages in Aboriginal Taiwanese
Analysis of the mtDNA haplogroup composition in aboriginal populations of Taiwan revealed highly distinctive patterns of the spread of the subgroups of common Asian specific haplogroups as compared to populations of China. On the one hand, haplogroups B, E, R9, and M7, which cover most of the lineages in aboriginal Taiwanese, have significantly lower frequencies in China. On the other hand, haplogroups A, C, G, and M8–M10, which are frequent in China, are completely absent in the nine tribes of Taiwan. In contrast to mainland China, the low haplotype diversity seen among small indigenous populations suggests that genetic drift may be responsible for generating the distinct frequency patterns reported in this study (Table 1).
Interestingly, however, haplogroups that make up the majority of lineages in Taiwanese aboriginals were also observed at high frequency (>60%; Table 1) among other island Southeast Asian populations who similarly present significant differences in their combined haplogroup frequencies with mainland East Asian populations. It is highly unlikely that the increment of the compound frequency of haplogroups B, E, R9, and M7 seen among all aboriginal populations of Taiwan could be explained by drift. It seems more plausible that the ancestral population of coastal East Asia and island Southeast Asia was already enriched by the founder lineages of these haplogroups and that drift affected the frequencies of individual haplogroups while their combined frequency throughout this region remained high.
In light of this study, the complete absence among aboriginal populations of several mtDNA haplogroups common on the Chinese mainland would suggest that the Neolithic colonizers (a) did not contribute significantly to the mtDNA pool of the pre-existing Formosan population; or (b) that the Neolithic migrants were a small group already lacking haplogroups A, C, G, D4, and M8–M10 that might have become frequent in southern China more recently.
Although possessing the same general haplogroup composition, the nine Taiwanese aboriginal populations are all significantly different from each other (in genetic distances), with southern populations showing a frequency pattern different from that of the central and northern groups (see Figure 3) and each population revealing its own specific founder haplotypes (see Figure 2). Given the low geographic distances between the sampling locations, this striking diversity between the tribes, seen also in HLA studies [18], may denote prevalence of strong inter-group cultural differences preserved through social isolation and endogamy of the tribes. Even though intermarriages may have been uncommon between the tribes, it is possible that small-scale admixture could have occurred through child adoption among the mountain tribes [55].
Fitting the mtDNA Heritage of the Taiwanese Aboriginals into the Models of Austronesian Expansion
Genetic models explaining the origins of Austronesian migration can be categorized by the three following components: the place of origin, the time scale, and the correspondence with specific archaeological evidence. Studies based on Y chromosomal and mitochondrial markers have traced in Polynesians the presence of lineages that are characteristic of Melanesians and East Indonesians but which are absent in mainland East Asia and Taiwan [8,16,25,56]. Evidence for interaction or initiation of a human settlement process, directed toward Remote Oceania, somewhere in East Indonesia or Melanesia is also supported by the phylogenetic analyses of Polynesian rats, Rattus exulans [57]. Besides the Melanesian-specific component, both human mtDNA and Y chromosomal haplogroups found in Polynesians include those that are common in populations of mainland East Asia and also in Taiwan [22]. This general share of ancestry refers, first of all, to mtDNA haplogroup B4a, which is the most frequent lineage group among Polynesians. However, the specific HVS-I motif associated with Polynesian expansion occurs only in Near and Remote Oceania whereas its immediate ancestral sequence is common throughout East Asia and has a coalescence time significantly predating the Polynesian migration [21].
Phylogenetic analysis of complete mtDNA sequences (Figure 4) in this study reveals the presence of a motif of three coding region mutations (nps 6719, 12239, and 15746) that define haplogroup B4a1a and are shared among aboriginal Taiwanese, Melanesians, and Polynesians. No mainland East Asian population has yet been found to carry lineages derived from these three positions. This suggests that the motif may have evolved in populations living in or near Taiwan at the end of the Late Pleistocene period. Considering the differences between the Late Pleistocene and present-day shorelines of Southeast Asia, the B4a1 lineages may also have evolved in regions now submerged under the sea. As long as mtDNA lineages of the Philippines and Indonesia, in particular, have not been analyzed in similar detail, the question about the precise origin of B4a1a has to be left open. The presence of an additional coding region mutation (np 14022) that defines haplogroup B4a1a1 and the HVS-I transition at np 16247 in Melanesians and Polynesians points to a maturation phase of the Austronesian migration in East Indonesia or Melanesia, during which the final Polynesian motif of mutations was established.
Austronesian is the world's most widely distributed language family, yet nine of its ten subfamilies are restricted to Taiwanese aboriginal populations [2]. The phylogeny of B4a1a mitochondrial genomes resembles the linguistic reconstruction of Austronesian languages with five of its primary branches restricted to Taiwan and the sixth branch spread all over Oceania. The 9.3 ± 2.6 thousand-year-old coalescence date we obtained using only coding region information for B4a1a1 diversification in Papuans and Polynesians predates significantly the earliest signs of Lapita culture in the region (around 3,500 BP). Nonetheless, these findings provide the first direct phylogenetic evidence for the common ancestry of Austronesian and indigenous Taiwanese maternal lineages and their maturation phase in East Indonesia or Melanesia. High frequency of a tagging mitochondrial haplotype in contrast to the absence of such in the Y chromosomes of Polynesians and Taiwanese might lend credence to the suggestion that the proto-Austronesian communities were matriarchal [58] and matrilocal (as the Amis tribe still is in Taiwan) whereby the Y chromosome pool of the initial migrants was lost after being repeatedly diluted on the way toward Polynesia.
Conclusions
Taiwanese aboriginal populations share their maternal ancestry with populations of mainland East Asia through haplogroups B, R9, and M7 as their main genetic components. At the same time the haplogroup structure at a finer phylogenetic resolution suggests relatively long-term isolation from the mainland populations. The coalescence times of B4a1a, F3b, F4b, R9c, and M7c1c lineages point to founder effects in Taiwan ranging from recent (0–2,000 years) to more ancient times (7,000–20,000 years). These results most likely reflect the drift in small endogamous populations of the island that became isolated by the rising sea levels after the last Ice Age.
The time element (13.2 ± 3.8 thousand years to the MRCA) obtained from the phylogenetic reconstruction of complete B4a1a sequences requires that we adopt a model according to which the origin of Austronesian migration can be traced back to Taiwan, and allows for the notion that it was followed by interaction periods elsewhere in Indonesia and finally in Melanesia where the complete motif specific to Polynesian B4a1a1 sequences (Polynesian motif) was developed.
Materials and Methods
In this study the sequence variation of the mtDNA D-loop HVS-I region, nps 16006–16397 were characterized in 640 samples drawn from nine Taiwan indigenous mountain tribes representative of most languages, cultures, and geographical settlements seen on the island before the last four centuries. In addition, coding region (CR) nps 9959–10917 were sequenced and the mtDNA nine-bp deletion in the mtDNA intergenic region between the COII gene and the Lysine tRNA gene (COII/tRNALYS) was screened by sequencing specific polymorphism (SSP) with primer pair L8215/H8297 [34]. Finally, significant RFLPs were used to remove unavoidable errors in haplogroup designation. The following tribes were selected: in the north of Taiwan, the Atayal (n = 109) and the Saisiat (n = 63); in the central mountain ranges, the Tsou (n = 60) and the Bunun (n = 89); in the east, the Amis (n = 98) and the Yami (n = 64); and in the south, the Rukai (n = 50), the Paiwan (n = 55), and the Puyuma (n = 52) (see Figure 1). All indigenous people had both parents belonging to the same tribe, and gave consent to participation in this study.
DNA extraction
Blood samples were collected in ACD tubes and treated at the Transfusion Medicine Research Laboratory of the Mackay Memorial Hospital in Taipei. Genomic DNA was extracted from 500 μl of buffy coat using the QIAmp DNA kit (QIAml blood kit, Qiagen inc. Chatsworth, California, United States) with minor modifications to the procedure recommended by the manufacturer.
DNA sequence analysis
Using primers L15997 and H16401 [59], a segment of 404 nucleotides from the D-loop HVS-I of the mtDNA was obtained. Primer pairs 5, 7, 8, 11–14, 19, and 24 F&R described in Rieder et al. [60] were used for typing informative RFLP sites using the following restriction enzymes: AluI (nps 5176 and 13262), BspEI (np 4710), HaeIII (np 663, 3391, and 8391), HhaI (nps 4830, 7598, and 9053), HinfI (np 9820), HpaI (np 12405), MboII (np 12704), and Tsp509I (np 5416) [34,61]. Primers 15F [60] and H10917 (5′ g
AACAg
CTAAATAggTTg 3′) were used to amplify a segment of 959 nucleotides used for sequencing both CR strands (nps 9959 to 10917). Length variation in the COII/tRNALYS (nine-bp deletion) region was assayed by PCR-SSP method, using primer pair L8215/H3274 [34]. PCR was performed in 20 μl reactions and carried out for 36 cycles using the following final conditions: 20 mM Tris-HCl (pH 8.4), 50 mM KCL, 0.2 mM deoxyribonucleotide triphosphate each, 2 mM MgCl2 , 0.25 U recombinant Taq DNA polymerase (Life Technologies, Taiwan), 5% glycerol, cresol red 0.075 mg/ml, 0.2 mM of each primer, and 30–100 ng of DNA sample. Thermal cycling conditions were 1 min. at 95 °C followed by 36 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. Following the thermal cycling, the samples were maintained at 75 °C for 10 min. Prior to sequencing, the amplified products were separated from excess dNTP and primers by first pre-treating with shrimp alkaline phosphatase (Sap) and Exo I enzymes (USB Product number US 70995 pre-sequencing Kit, Pharmacia, Taiwan) following the conditions recommended by the manufacturer (37 °C for 30 min and 85 °C for 15 min). For each individual, sequencing of nps 16006 to 16397 was performed on both strands using the Perkin-Elmer/Applied Biosystems Division (ABI Taiwan) DyeDeoxy Terminator Cycle Sequencing Kit (Foster City, California, United States) according to the recommendations of the manufacturer. Purification on a G50 sephadex column was performed before the final run on an automated DNA sequencer (ABI Model 377). In our inter-population analysis with other studies, the region including nps 16182 to 16183 was excluded because an upstream adenosine homo-polymeric region may cause poor sequencing. Consequently substitutions at nps 16182 and 16183 were not indicated in Figure 2.
Data analysis
Indices of molecular variance were calculated in ARLEQUIN package 2.0 [62]. A neighbor-joining tree was constructed with 1,000 bootstrap replicas using MEGA2 package [63]. A PC map was constructed with ADE-4 package [64–66] using HVS-I haplogroups frequencies obtained from the nine indigenous tribes of Taiwan and other regions of Asia shown in table 1. Coalescence times were calculated using the ρ statistic and HVS-I mutation rate of one transition per 20,180 years in bps 16090–16365 [67,68]. For complete coding region sequences, a mutation rate of 1.7 × 10−8 substitutions per site per million years was used assuming 5 million years for the split between human and chimpanzee lineages [69]. A more conservative rate calibration 1.26 × 10−8 [54], assuming a 6.5 million-year-old split of human and chimpanzee mtDNA lineages would imply that all the coalescence times, based on complete sequence data, that are discussed in the text would be approximately 1.3 times older.
Supporting Information
Figure S1 Ninety-Six Haplotypes Seen in 640 Taiwan Indigenous Individuals
For HVS-I, HVS II, and nps 9793−10899, sequences are numbered according to the revised reference sequence [70,71]. Only transversions are specified and the number in bracket indicates that only this region has been sequenced. Restriction enzymes have sometimes been used instead of sequencing to infer nps 9824, 10238, 10310, 10320, 10397, 10398, and 10400 (HinfI, HphI, BspMI, Tsp509I, BsrI, DdeI, and AluI, respectively). Significant diagnostic restriction enzymes are indicated at the top of each RFLP column with losses or gains of RFLP sites shown as plus or minus “9-bp del” refers to the length variation between nps 8271 and 8280 in the COII/tRNALYS region as assayed by PCR-SSP method [34] with 1 denoting presence of the nine-bp deletion and 2 denoting non-deletion. The colored columns represent lineage distribution among populations; colors are region specific and are conserved in Figure 2. The minimum evolution tree was constructed with Mega2 [63] using Tamura-Nei pairwise distances [72]. Specimens sequenced from 9982 to 10838 only are annotated with a number sign.
(2.9 MB TIF).
Click here for additional data file.
Accession Numbers
The GenBank accession numbers (http://www.ebi.ac.uk/embl/index.html) for HVS-I and HVS-II data in this article are as follows: HVS-I (AJ967036–AJ967675) and HVS-II (AJ967676–AJ968315). Complete sequence data: accession numbers AJ842744–AJ842751.
We are grateful to all Taiwan indigenous people who participated in this project. We would like to acknowledge Richard Villems (Tartu University), Victor Mair, Peter Underhill, Koji Lum, Qing-Peng Kong, and Hans-Jürgen Bandelt for corrections and comments on the manuscript. The project was supported by grant No 90–2320-B-195–002 from the National Science Council of Taiwan and grant NHRI-EX93-9218BI from the National Health Research Institute. TK received support from Estonian Science Foundation grant 5574.
Competing interests. The authors have declared that no competing interests exist.
Author contributions. JAT and ML conceived and designed the study. ML contributed DNA samples. JHL, CJH, CLL, CLH, and ZYL performed the sequence analysis. JAT and TK performed population genetic analyses. JAT, TK, and ML wrote the paper.
¤ Current address: Transfusion Medicine Laboratory, Mackay Memorial Hospital, Taipei, Taiwan
Citation: Trejaut JA, Kivisild T, Loo JH, Lee CL, He CL, et al. (2005) Traces of archaic mitochondrial lineages persist in Austronesian-speaking Formosan populations. PLoS Biol 3(8): e247.
Abbreviations
COII/tRNALYSmtDNA intergenic region between the COII gene and the Lysine tRNA gene
D-loopdisplacement loop
HLAhuman leukocyte antigen
HVS-Ihyper-variable segment I
MRCAmost recent common ancestor
mtDNAmitochondrial DNA
npnucleotide position
PCprincipal components
RFLPrestriction fragment length polymorphism
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| 15984912 | PMC1166350 | CC BY | 2021-01-05 08:28:15 | no | PLoS Biol. 2005 Aug 5; 3(8):e247 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030247 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1598491310.1371/journal.pbio.0030248Research ArticleBioinformatics/Computational BiologyGenetics/Genomics/Gene TherapyInfectious DiseasesMicrobiologyEubacteriaHomo (Human)The Genome Sequence of Rickettsia felis Identifies the First Putative Conjugative Plasmid in an Obligate Intracellular Parasite First Putative Conjugative Plasmid IdentifiedOgata Hiroyuki [email protected]
1
Renesto Patricia
2
Audic Stéphane
1
Robert Catherine
2
Blanc Guillaume
1
Fournier Pierre-Edouard
1
2
Parinello Hugues
2
Claverie Jean-Michel
1
Raoult Didier [email protected]
2
1 Structural and Genomic Information Laboratory, UPR 2589, IBSM, CNRS, Marseille Cedex, France,2 Unité des Rickettsies, UMR 6020, IFR 48, CNRS, Faculté de Médecine, Marseille Cedex, FranceMoran Nancy Academic EditorUniversity of ArizonaUnited States of America8 2005 5 7 2005 5 7 2005 3 8 e2488 3 2005 11 5 2005 Copyright: © 2005 Ogata et al.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Using the Genomic Shortcut to Predict Bacterial Behavior
We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular α-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp) and a long (62,829 bp) form. R.
felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin–antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as β-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria.
Rickettsia felis is an obligate intracellular bacterium that lives in fleas and causes spotted fever in humans. Its genome sequence provides the first evidence that such bacteria can undergo conjugation.
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Introduction
Rickettsiae are obligate intracellular small gram-negative bacteria associated with different arthropod hosts. Many Rickettsia species infect human beings and are responsible for mild to severe diseases. Rickettsia felis, the agent of the flea-borne spotted fever rickettsiosis, exhibits several specificities among the currently recognized Rickettsia species. After being identified in fleas in 1990 [1], R. felis has been found worldwide in flea species such as Ctenocephalides felis, parasitizing cats and dogs, and Pulex irritans. R. felis is transovarially transmitted in these insects [2]. Several cases of human infection caused by R. felis have been reported [3,4]. Rickettsia species are phylogenetically classified into two groups: the typhus group and the spotted-fever group (SFG). R. felis belongs to the SFG, together with tick-associated Rickettsia species such as R. conorii, R. sibirica, and R. rickettsii. However its lifestyle resembles that of R. typhi (typhus group), which is also hosted and transovarially transmitted by fleas. Furthermore, R. felis is known to coinfect fleas with Bartonella henselae, B. quintana, and Wolbachia pipientis [5]. The culture conditions of R. felis were established in 2001 using Xenopus laevis tissue culture (XTC) cells at relatively low temperatures (optimally at 28 °C) [3]. Besides these features, little is known about this pathogen. To date, six Rickettsia genome sequences are available. These are from two typhus group species (R. prowazekii [6] and R. typhi [7]) and four SFG species (R. conorii [8], R. sibirica [9], R. rickettsii, and R. akari). To further identify the specificities of R. felis, we determined its genome sequence.
Results
General Genome Features
The genome of R. felis comprises three replicons: a 1,485,148 bp circular chromosome and two circular plasmids identified for the first time in the genus Rickettsia (Figure 1). The predicted total complement of 1,512 protein-coding genes (open reading frames [ORFs]) is the largest among currently sequenced Rickettsia genomes (Table 1). Of these, 1,402 (92.7%) exhibited homologs in the nonredundant database and 1,080 (71.4%) were assigned putative functions.
Figure 1 Circular Representation of R. felis, R. conorii, and R. prowazekii Genomes
The three outer circles represent the chromosomes of R. felis, R. conorii, and R. prowazekii, respectively, with specific ORFs colored in red and nonspecific ORFs colored in black. Colinear genome fragments are highlighted by a shared background color, with their relative orientations indicated by arrows. The two inner circles represent two R. felis plasmids (pRF and pRFδ), with ORFs in the region unique to pRF colored in red.
Table 1 Comparison of R. felis and Other Published Rickettsia Genomes
The R. felis chromosome exhibits a long-range (24−277 kbp) colinearity relative to other Rickettsia genomes, although it is more frequently interrupted by inversions/translocations than is observed between other Rickettsia genomes (Figure 2A). This colinearity allowed the precise assessment of orthologous relationships between ORFs of five Rickettsia species (R. felis, R. conorii, R. sibirica, R. prowazekii, and R. typhi). On this basis, we identified 530 R. felis–specific ORFs, that were either absent or degraded (split or fragmented) in the other four Rickettsia genomes (Tables 2 and 3). Consistently, the R. felis genome exhibited a much higher number of gene families than other Rickettsia species (see Table 1). The R. felis–specific ORFs included a remarkably high number of paralogs for transposases, surface cell antigens (sca), global metabolism regulators (spoT), and proteins containing protein–protein interaction motifs such as ankyrin repeats and tetratricopeptide repeats (TPRs). Furthermore, we identified many other ORFs putatively associated with the adaptations of R. felis to its host environment or with its pathogenesis.
Figure 2 Genome Comparisons of R. felis and R. conorii
Red dots represent homologous genomic segments greater than 150 bp identified by BLASTN (E-value < 10−3).
(A) Comparison between R. felis and R. conorii chromosomes. Vertical and horizontal green lines indicate the positions of transposase ORFs in R. felis and in R. conorii, respectively.
(B) Self-comparison of R. felis chromosome.
Table 2
R. felis–Specific Genes Encoded in the Chromosome
Table 2 Continued
Table 3
R. felis ORFs Encoded in pRF Plasmid
Plasmids
The two R. felis plasmids, named pRF and pRFδ, are 62,829 bp and 39,263 bp long, respectively. Their topologies and sizes were confirmed experimentally (Figures S1 and S2). The pRF plasmid contains 68 ORFs, of which 53 (77.9%) exhibited homologs in public databases and 44 (64.7%) were associated with functional attributes. The nucleotide sequences of pRFδ and pRF are identical, except for an additional 23,566-bp segment that contains 24 ORFs (pRF15–pRF38) in pRF (see Table 3). These plasmids are likely to be R. felis specific since all attempts to detect specific plasmid sequences by polymerase chain reaction (PCR) from DNA of available reference rickettsial species were unsuccessful. In contrast, the same assays against 30 fleas naturally infected by R. felis resulted in amplification of the plasmid sequences in all cases.
Plasmids are referred to as conjugative or nonconjugative. The former are disseminated by conjugation from cell to cell, while the latter are only vertically transmitted. The pRF plasmid encodes several homologs of proteins involved in the different conjugative steps (see Table 3; Figure S3). First, it exhibits a split gene (pRF38/pRF39) homologous to the traA
Ti of the Agrobacterium tumefaciens tumor-inducing plasmid [10]. TraATi is thought to be a DNA-processing machinery with nickase and helicase activities to generate the transfer strand from the origin of transfer (oriT) [10]. Second, the pRF encodes another split gene (pRF43/pRF44) homologous to the traD
F in the Escherichia coli F plasmid. TraDF is a “coupling protein” that connects the DNA-processing machinery (and transfer strand) to the mating pair formation (Mpf) apparatus, a type IV secretion system (T4SS) [11]. Finally, pRF exhibits an ORF (pRF47) similar to TraGF, a protein involved in the F-pilus assembly and aggregate stabilization [12].
Despite the presence of these ORFs linked to the initiation of plasmid transfer, the pRF sequence lacks clear homologs for the proteins involved in the Mpf apparatus found in other bacteria. Nevertheless, the R. felis chromosome (as well as other Rickettsia genomes) encodes most of the components of T4SS, which are highly similar to the vir genes of A. tumefaciens. Since the R. felis T4SS components (virB2 [RF1075], virB3 [RF0087], virB4 [RF0088], virB6 [RF0089, RF0090, RF0091, RF0092, and RF0093], virB8 [RF0463 and RF0465], virB9 [RF0462 and RF0466], virB10 [RF0467], virB11 [RF0468], and virD4 [RF0469]) are conserved in all sequenced Rickettsia genomes that lack plasmids, the primary suspected role of the R. felis T4SS is to translocate virulence factors to hosts. However, the T4SS of A. tumefaciens (vir) and Legionella pneumophila (dot/icm) have been shown to function both as DNA-transfer machineries and as effector translocators [13]. Thus, the R. felis T4SS may also promote the transfer of DNA as in A. tumefaciens. We also noticed that the R. felis chromosome exhibits a DNA primase gene (RF0786) similar to TraC found in the E. coli IncP plasmid. TraC initiates the replication of transferred DNA strands in the recipient cells. Finally, the R. felis chromosome encodes a protein (RF0020) similar to competence protein ComE3, a protein (RF0964) similar to the F-pilin acetylation protein TraX, and a split gene (RF0705/RF0706) homologous to the P-pilus assembly protein FimD. In conclusion, the presence of those putative conjugative transfer genes suggests that the R. felis plasmids have been acquired by conjugation and that R. felis may still retain the capacity of transferring plasmids.
Genome Plasticity
We identified 333 repeated DNA sequences (50 to 2,645 bp long) in the R. felis genome, accounting for 4.3% of the sequence, a proportion markedly higher than in other sequenced Rickettsia genomes (see Table 1; Figure 2B). The major source of those repeats is the proliferation of transposase genes, for which we identified 82 copies (or inactivated derivatives). Among other obligate intracellular bacteria, only W. pipientis
wMel [14] and Parachlamydia sp. UWE25 [15] exhibit such a high number of large mobile genetic elements. The occurrence of highly similar transposase sequences appears to play a major role in the plasticity of the R. felis genome (see Figure 2A). Transposase ORFs were identified at most extremities of the R. felis genomic segments colinear with the R. conorii genome, suggesting that the R. felis chromosome has been rearranged many times through recombination mediated by these mobile sequences. With the use of the GRAPPA software inferring the most parsimonious genome-rearrangement scenario, we estimated at least 11 inversion events between R. felis and R. conorii. In contrast, only four inversions are required to associate more distantly related R. conorii and R. prowazekii genomes. In addition to transposases, we identified eight phage-related ORFs (see Table 2). The R. felis genome thus appears to have been invaded more frequently by such foreign DNAs than other Rickettsia species. Besides long repeats, Rickettsia genomes are known to contain a number of small palindromic repeats (Rickettsia palindromic elements [RPEs]) capable of invading both coding and noncoding regions [16]. We identified 728 RPEs in the R. felis genome. Of these RPEs, 85 were found within ORFs and three were found in RNA-coding genes.
The R. felis chromosome and plasmids share several homologs, suggesting gene exchanges between these replicons. Of 68 ORFs in pRF, 11 have a close homolog (>50% amino acid sequence identity) in the chromosome; these are seven transposases, patatin-like phospholipase (pRF11), thymidylate kinase (pRF13), and two small heat-shock proteins (pRF51 and pRF52). Among these, patatin-like proteins exhibit the most intriguing phylogeny (Figure S4). The genomes of five Rickettsia species (R. prowazekii, R. typhi, R. conorii, R. sibirica, and R. felis) exhibit chromosomal patatin-like phospholipase gene (pat1). Gene organization around pat1 is similar between these Rickettsia. Interestingly, a phylogenetic analysis for these Pat1 and the plasmid-encoded Pat2 indicates a close relationship between Pat1 (RF0360) and Pat2 of R. felis, together being an outgroup of Pat1 sequences of other Rickettsia, suggesting a gene replacement of the chromosomally encoded pat1 by the plasmid-encoded pat2 in the lineage leading to R. felis.
Most R. felis genes with orthologs in other Rickettsia have probably been inherited vertically from a common ancestor. On the other hand, genes without orthologs in other Rickettsia may have been acquired by lateral gene transfer. To test this hypothesis, we analyzed the taxonomic distribution of BLASTP best hits of R. felis ORFs against the nonredundant database (excluding rickettsial sequences) (Figure S5). R. felis ORFs with orthologs in other Rickettsia matched preferentially (64%) with sequences from the same taxonomic group as R. felis (i.e., α-proteobacteria). In contrast, the BLAST best hits for the chromosomal ORFs lacking orthologs in other Rickettsia were found preferentially in γ-proteobacteria (31%; 58 ORFs) and cyanobacteria (18%; 33 ORFs). The taxonomic distributions of the best matches for these two ORF sets were significantly different (p < 0.001; χ2 test). This result suggests that many R. felis-specific genes may originate from distantly related organisms by lateral transfer. However, methods based on nucleotide composition bias failed to identify unambiguous candidates for lateral gene acquisition in R. felis.
Surface Antigens
The sca family is one of the largest paralogous gene families in Rickettsia [8]. Five sca members have been identified in the previously published Rickettsia genomes. Several Sca proteins are known to account for major antigenic differences between Rickettsia species [17] and may play important roles in adhesion to host cells [18]. Sca proteins are characterized by highly variable N-terminal sequences and a conserved C-terminal autotransporter β-domain, which translocates the N-terminal part outside the outer membrane. The R. felis genome exhibits the highest number of sca genes among currently available Rickettsia genomes. We identified nine intact sca paralogs (sca1, sca2, sca3, sca4, sca5/ompB, sca8, sca9, sca12, and sca13) as well as four fragmented or split paralogs (sca0/ompA, sca7, sca10, and sca11). Reverse transcriptase–polymerase chain reaction (RT-PCR) experiments demonstrated that, under mild log growth phase, all R. felis
sca paralogs were transcribed, including split ones. Phylogenetic analyses suggest that ancient duplication events gave rise to these paralogs before the divergence of Rickettsia species. We noticed that sca genes exhibit highly different patterns of presence/absence across different Rickettsia species (Table S1). Only ompB and sca4 are conserved in all available Rickettsia genomes [19], remaining members being degraded or absent in one or more species. Together with the accelerated amino acid changes, differential gene degradation of sca paralogs probably contributes to the intra-species variation of those cell-surface proteins and might be linked with their adaptation to different host environments.
R. felis is genetically and serologically classified into the SFG of Rickettsia [20]. However, cross-reactivities caused by both proteins and lipopolysaccharides have been found with R. typhi using mouse sera [2] and human sera (Figure S6). R. conorii rarely cross-reacts with R. typhi. We therefore suspected that genes found in both R. felis and R. typhi, but missing in R. conorii, might be responsible for the cross-reactivities of R. felis and R. typhi. A list of such genes includes a sca family gene (sca3), encoding a protein with a predicted molecular weight of 319 kDa, and rfaJ for the lipopolysaccharide 1,2-glucosyltransferase (Table 4).
Table 4
R. felis ORFs Present in R. typhi but Absent or Degraded in R. conorii and R. sibirica
Adaptation to Environment
Transcriptional regulation may be of critical importance in R. felis, as the numbers of spoT, the gene regulating “alarmone,” and chromosomal toxin–antitoxin modules are higher in the R. felis genome than in any other sequenced bacterial genome.
SpoT and RelA are two hallmark enzymes regulating global cellular metabolism of E. coli in response to starvation [21]. These enzymes control the concentration of alarmone, (p)ppGpp (guanosine tetra- and pentaphosphates), which in turn acts as an effector of transcription. Remarkably, R. felis exhibits 14 spoT, (spoT1–13 and 15) paralogs (Figure S7). Using RT-PCR, we examined the transcription status of 14 R. felis
spoT genes. All the spoT ORFs were transcribed. We classified these ORFs into two groups, based on their alignment against the sequence of the Streptococcus dysgalactiae Relseq that possesses both (p)ppGpp hydrolase and synthetase activities [22]. The first group (SpoT1–10, 14, and 15) was aligned with the sequence of the hydrolase domain, and the second group (SpoT11, 12, and 13) with the sequence of the synthetase domain. Being consistent with the previous observation [23], our phylogenetic analyses suggest that each paralogous gene group originated in early duplication events before the divergence of Rickettsia species. Notably, every sequenced Rickettsia genome encodes at least one ORF exhibiting hydrolase catalytic residues and one ORF exhibiting synthetase catalytic residues, suggesting that both hydrolase and synthetase functions are required for Rickettsia. We also found that seven spoT
(spoT1–4 and 7–9) genes were located in the R. felis chromosome next to a gene encoding a transporter of the major facilitator superfamily (MFS) including proline/betaine transporters. MFS is also a large paralogous gene family composed of at least 23 ORF members in R. felis.
Toxin–antitoxin systems are composed of tightly linked toxin and antitoxin gene pairs and ensure stable plasmid inheritance when they are encoded in plasmids. In these systems, the toxic effect of a long-lived toxin is continuously inhibited by a short-lived antitoxin only when whole systems are maintained. The toxin–antitoxin modules have also been found on the chromosomes of many free-living prokaryotes, but have rarely been found in obligate intracellular bacteria [24,25]. In the R. felis chromosome, we identified 16 toxin genes (RF0016, RF0095, RF0271, RF0456, RF0490, RF0602, RF0701, RF0732, RF0787, RF0792, RF0898, RF0911, RF0956, RF1272, RF1286, and RF1368) and 14 antitoxin genes (RF0015, RF0094, RF0272, RF0457, RF0489, RF0601, RF0702, RF0731, RF0779, RF0788, RF0899, RF0910, RF0957, and RF1369), comprising at least 13 modules in operon structures. It is suggested that toxin–antitoxin systems, when encoded on the bacterial chromosome, might be involved in selective killing (a primitive form of bacterial apoptosis) or reversible stasis of bacterial subpopulations during periods of starvation or other stress [26,27]. It is also tempting to speculate that the toxin–antitoxin system could be targeted to the eukaryotic host cells. In this case, this system may help to maintain the presence of bacteria in the host. Notably, in the chromosomally encoded mazEF system of E. coli, the toxin action is regulated by (p)ppGpp. The large number of toxin–antitoxin modules in R. felis, as well as a number of spoT paralogs, might thus be linked to the synchronization of its multiplication within eukaryotic hosts.
It is probable that five R. felis–specific ORFs are related to its capacity of antibiotic resistance. We identified a streptomycin resistance protein homolog (RF0774), a class C β-lactamase, AmpC (RF1367), a class D β-lactamase (RF1275), a penicillin acylase homolog with conserved catalytic residues (RF1137), and an ABC-type multidrug transport-system protein, MdlB (RF0981). AmpC β-lactamase is known to be induced by AmpG of the MFS, which was also identified in the R. felis genome (RF0265, RF0608, RF0834, and RF1247). In vivo β-lactamase activity of R. felis was measured using high-performance liquid chromatography (see below).
Adaptation to Eukaryotic Hosts
R. felis may have developed a specific mechanism to cross-talk with its eukaryotic hosts. It exhibits 22 ankyrin-repeat-containing proteins and 11 TPR-containing proteins. These two protein motifs are frequently found in eukaryotic proteins, but their distributions are rather limited in viruses and bacteria, in both of which they appear to be linked with pathogenicity.
The ankyrin repeat is a protein–protein interaction motif, involved in transcription initiation, cell cycle regulation, cytoskeletal integrity, and cell-to-cell signaling [28]. Anaplasma phagocytophilum, a closely related intracellular α-proteobacterium, exhibits a protein containing ankyrin repeats (AnkA), which was detected in the cytoplasm and the nucleus of infected eukaryotic cells (human leukemia-60) [29]. According to the Superfamily database [30], only 15 bacterial species possess more than three ankyrin-repeat-containing proteins, and two species exhibiting the highest number of ankyrin repeats are obligate intracellular bacteria, W. pipientis (21 proteins) and Coxiella burnetii (20 proteins), although Wu et al. [14] reported slightly different numbers of ankyrin-repeat-containing proteins for these species. A recent genome analysis of a facultative intracellular bacterium, L. pneumophila, revealed 20 proteins with ankyrin repeats [31]. Ankyrin repeats were also found in more than 30 ORFs of the giant virus Acanthamoeba polyphaga Mimivirus [32].
TPR, composed of a motif of 34 amino acids organized in tandem, is also recruited by different proteins and facilitates protein–protein interactions [33]. Its role in the adaptation of parasites to their hosts has been suggested. The R. felis genome exhibits 11 TPR-containing ORFs (seven in the chromosome and four in the pRF plasmid). Only Leptospira interrogans (the agent of leptospirosis), Treponema species (including the agent of syphilis), and L. pneumophila [31] exhibit a high number of both TPR and ankyrin repeats. These organisms are eukaryotic parasites. The cryptococcal crooked neck 1 gene of Cryptococcus neoformans (a yeast), containing 16 copies of TPR, appears associated with its virulence [34].
Host Invasion/Pathogenesis
Plasmids often carry out functions that benefit bacteria in their survival or expression of virulence. pRF exhibits two ORFs that are possibly associated with the pathogenesis of R. felis: a hyaluronidase and a patatin-like protein. The hyaluronidase homolog (pRF56) exhibits a significant homology to hyaluronidase NagI (1,297 aa) of Clostridium perfringens. Hyaluronidases, which depolymerize hyaluronic acid—an unbranched polysaccharide ubiquitously present in the extracellular matrix of animal tissues—are known as “spreading factors” [35]. Another ORF (pat2) exhibits a significant homology to patatin-like phospholipases. Its paralog (pat1) was also identified in the chromosome, as already mentioned. Patatin is the major storage glycoprotein found in potato tubers, but also exhibits phospholipase A2 activity for protection from infection. Proteins containing patatin-like domains are more frequently found in pathogenic than in nonpathogenic bacteria. McLeod et al. [7] suggested that patatin-like proteins might be responsible for the phospholipase A2 activity identified some years ago in rickettsiae [36].
Potential host-invasion capacity is also provided by R. felis–specific ORFs found on the chromosome, for instance, a chitinase homolog (RF0413) and a chitin-binding protein homolog (RF0710). Chitin is a homopolymer of N-acetylglucosamine and a major component of the exoskeleton of arthropods and of the peritrophic envelope of insects, a lining layer of the midgut. These genes may facilitate the access of bacteria to the insects' gut epithelial cells. R. felis may also use chitin as a nutrient source, as does Vibrio cholerae [37]. We identified a homolog (RF0268) for ecotin, an E. coli periplasmic protein inhibiting activities of a variety of proteases. Two R. felis–specific ORFs (RF0449 and RF0855) exhibit the complete NACHT NTPase domain. In eukaryotes, this NTPase domain has been found in proteins implicated in apoptosis as well as in immune/inflammatory responses [38]. The presence of this domain in other bacterial ORFs is limited to several lineages, such as cyanobacteria and Streptomyces, and their functions are unknown.
Higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) is a recently identified domain detected in a few prokaryotes. We found four genes (two were split) exhibiting HEPN at the C-terminus, and a nucleotidyl transferase domain at the N-terminus. Among other bacteria, only A. tumefasciens, Thermotoga maritima, and Sinorhizobium melitoti were found to exhibit HEPN-containing genes [39]. The nucleotidyl transferase domain has been associated with several classes of bacterial enzymes responsible for resistance to aminoglycosides. HEPN was also found in the human sacsin protein, a chaperonin implicated in a neurodegenerative disease. Finally, R. felis exhibits an ortholog (RF0371) for R. conorii RickA, which induces its actin-based motility [40].
Phenotypic Post-Genomics Analysis
The obligate intracellular nature of R. felis hindered progress in the detailed characterization of its phenotypic diversity. Here, we envisaged post-genomics as a way of associating in vivo phenotypes of these bacteria to genomic features. The presence of pili-associated genes prompted us to investigate, by electron microscopy, the presence of such appendages on the cell surface. This approach led to the first characterization of pili on the surface of a Rickettsia; we observed two forms of pili at the surfaces of R. felis (Figure 3). One form of pili establishes direct contact between bacteria, providing a very typical figure of Mpf apparatus; these pili are probably specialized in conjugation. The other form of pili forms small hair-like projections emerging out from the cell surface; these pili are probably involved in the attachment of the bacteria to other cells. Without pili, many disease-causing bacteria lose their invasion capability. The latter type of pili might be considered as virulence factors, as described for Francisella tularensis [41,42].
Figure 3 Visualization of R. felis Pili by Transmission Electron Microscopy
Bacteria collected from the supernatant of R. felis–infected XTC cells were negatively stained.
(A) Sexual pilus observed between two bacteria.
(B) R. felis also possesses small appendages likely to be fimbriae pili.
As previously mentioned, we also found a RickA homolog in the R. felis genome [40]. Based on this finding, we performed immunofluorescence assays. The orientations of actin filaments beside bacteria are distinct from the stress fibers of the host. This further suggests that R. felis is probably capable of using the actin cytoskeleton to disseminate through eukaryotic cells, a method exploited by other SFG rickettsiae [40] (Figure S8). Another R. felis phenotypic character suggested from genomic analyses (three ORFs for patatin-like proteins) was its hemolytic capacity. We confirmed experimentally that R. felis lyses erythrocytes, this effect being inhibited by dithiothreitol. Another genome-guided discovery was β-lactam inhibition, which reached 57% and 53% of the concentration and the minimal inhibitory concentration, respectively, following 2 h incubation of R. felis with amoxicillin. Despite being preliminary results, these findings illustrate the fact that whole-genome sequencing offers opportunities to rapidly gain a better understanding of the phenotypic characters of a fastidious microorganism.
Discussion
R. felis is the first obligate intracellular bacterium exhibiting a possible conjugative plasmid. Of the nine previously published studies of members of the Order Rickettsiales (six in Rickettsiaceae, three in Anaplasmateceae), none exhibited a plasmid. Several other obligate intracellular bacteria, such as Chlamydia muridarum, Chlamydophila caviae, C. burnetii, Wigglesworthia glossinidia, and Buchnera aphidicola, are known to possess plasmids. Recently, the reannotation of the genome of Parachlamydia, an obligate intracellular bacteria living in amoeba, predicted an F-like DNA conjugative system encoded in a genomic island [43]. However, no conjugation has yet been observed for those plasmids and genomic island. Transformation of obligate intracellular bacteria remains an elusive goal, although preliminary work on several obligate intracellular bacteria has been reported with limited results [44]. The possible conjugative plasmid identified in R. felis may provide a molecular basis for the future development of new genetic transformation tools in rickettsiae.
R. felis is hosted by fleas, as are R. typhi, B. henselae, W. pipientis, and Yersinia pestis. There are surprisingly few common genomic features between R. typhi and R. felis. R. typhi genetically resembles R. prowazekii despite having a lifestyle similar to that of R. felis (Table S2). The comparison with W.
pipientis is interesting. This intracellular bacterium also multiplies in arthropods (including fleas) and is transmitted transovarially. The most relevant finding in its genome was the detection of repetitive mobile DNA elements. Many ankyrin repeats and several TPRs were also found. It appears that R. felis and W.
pipientis share common genomic features, possibly because of their similar niches (we found two Ct. felis fleas in France coinfected with W. pipientis and R. felis). They both differ significantly from their immediate neighbors [45,46]. Moreover, the phylogenetic relationship and hosts of R. felis and R. prowazekii (transmitted by lice) are comparable with those for B. quintana (transmitted by lice) and B. henselae (transmitted by fleas) [47]. B. henselae exhibits a larger genome with more repeats and integrases than B. quintana. Y. pestis, transmitted by fleas, also exhibits many more insertion sequences than its close relative, Y. pseudotuberculosis [48]. Altogether, flea-infecting bacteria appear to exhibit a specific evolution (i.e., more repeats, transposases, and/or integrases) compared with their non-flea-infecting neighbors.
For obligate intracellular bacteria such as rickettsiae, few phenotypic characters have been observed. To date, four intracellular bacterial genomes have been entirely sequenced, the procedure being completed in 7 y or less after their first identification or culture, including R. felis [14,15,49,50]. In the present study, the genome sequencing of R. felis provided evidence of the presence of conjugative plasmids, two types of pili, hemolytic activity, β-lactamase activity, and intracellular motility. We believe that for such recently identified/cultured fastidious organisms, complete genome sequencing is a very potent and timesaving strategy to identify unrecognized phenotypic properties.
Materials and Methods
Bacterial purification and DNA extraction
R. felis (strain California 2) was cultivated on XTC cells growing on RPMI with 5% fetal bovine serum, supplemented with 5 mM L-glutamine. The purification of the bacteria was performed by different steps. First, the bacteria were treated in the presence of 1% trypsine in K36 buffer for 1 h at 37 °C, then centrifuged and digested by DNAseI for 1 h at 37 °C to reduce the eukaryotic DNA contamination. The sample was loaded on a renograffin gradient and the bands of the purified bacteria were washed in K36, treated again by DNAseI. After inactivation with EDTA (50 mM), the bacteria were resuspended in TE, dispatched in 150-μl tubes and stored at −80 °C. Depending on this initial concentration, one or two tubes were diluted in 1 ml of TNE (10 mM Tris [pH 7.5], 150 mM NaCl, 2 mM EDTA) and incubated for 5 h at 37 °C in the presence of lysozyme (2 mg/ml). Lysis was performed for 2 h at 37 °C by adding 1% SDS and RNAseI (25 μg/ml). Overnight treatment with 1 mg/ml of proteinase K followed at 37 °C. After three phenol–chloroform extractions and alcoholic precipitation, the DNA was resuspended in 30 μl of TE and its concentration was estimated by agarose gel electrophoresis.
Pulsed-field agarose gel electrophoresis
The concentrated bacterial suspension was included in 1% (vol/vol) Incert agarose gel blocks (BMA, Rockland, Maryland, United States). The agarose blocks were digested by Proteinase K (1 mg/ml) (Eurobio Laboratories, Paris, France) in 1% lauroylsarcosine and 0.5 M EDTA (pH 8) (Sigma-Aldrich, St. Louis, Missouri, United States) for 24 h at 50 °C. Fresh Proteinase K was then added and the incubation was continued for 24 h. The blocks were then washed twice in TE (pH 7.6) for 30 min at room temperature. Proteinase K inactivation was performed through incubation in a 4% phenylmethylsulfonyl fluoride (MBI Fermentas, Burlington, Canada) solution for 1 h at 50 °C. This inactivation step was carried out twice. The blocks were then washed two to three times in TE and stored in 0.5 M EDTA (pH 8) at 4 °C. Before restriction enzyme digestion, the agarose blocks were equilibrated twice with TE for 15 min. Digestion was carried out for 4 h, then fresh enzyme was added and the incubation was continued overnight. The digested agarose blocks and molecular-weight markers (Low Range PFG Marker, Lambda Ladder PFG Marker [New England Biolabs, Beverly, Massachusetts, United States]) were equilibrated in 0.5× TBE (50 mM Tris, 50 mM boric acid, 1 mM EDTA).
Each agarose block was laid in a 1% PFEG agarose (Sigma-Aldrich) solution in 0.5× TBE. Pulsed-field gel electrophoresis was carried out on a CHEF-DR II device (Bio-Rad, Hercules, California, United States) under different electrophoresis conditions. The 1% agarose gel was run at 200 V using ramped pulse times from 1 to 5 s for 10 h to observe the pattern of small DNA fragments (2–48 kb). The migration was taking place under the following two consecutive conditions: (i) a ramping time from 3 to 10 s at 200 V for 12 h, with the pattern representative for 48- to 242-kb fragments, then (ii) a ramping time from 20 to 40 s at 180 V for 15 h, with the pattern representative for 145- to 610-kb fragments.
Shotgun of R. felis genome and sequencing strategy
Three shotgun genomic libraries were constructed by mechanical shearing of the genomic DNA using a Hydroshear device (GeneMachine, http://genome.nhgri.nih.gov/genemachine/). DNA fragments were blunt-ended using T4 DNA polymerase (New England Biolabs) and ligated to the BstXI adapter. Fragments of 3, 4.5, and 7 kb were separated on a preparative agarose gel (FMC BioProducts, Rockland, Maryland, United States), extracted with Qiaquick kit (Qiagen, Valencia, California, United States), and ligated into pCDNA2.1 (Invitrogen, Carlsbad, California, United States) for the two smaller inserts and into pCNS (a low copy number vector; C. R., unpublished data) for the largest one. DNA cloning was performed using electrocompetent E. coli DH10B Electromax cells (Invitrogen). Plasmid DNAs were purified and pools of 96 clones were analyzed by gel electrophoresis to validate the libraries. DNA sequencing of insert ends was carried out using Big Dye 3.1 terminator chemistry on an automated capillary ABI3700 sequencer (Applied Biosystems, Foster City, California, United States).
Sequences were analyzed and assembled into contigs using Phred, Phrap, and Consed software [51] taking all sequences into account. Sequences were considered valid when at least 75% of the nucleotides had a Phred score of more than 20. The finishing of the genome sequencing included only additional directed reactions that were performed on an ABI3100 sequencer. Two circular plasmid molecules of 63 and 38 kbp, respectively, were identified from the assembled sequences. On the chromosome, three small regions of 41, 155, and 64 bp failed by dropping of sequence. A number of parameters (DMSO, glycerol, hybridization, and elongation temperature) were tested one by one or were combined to sequence over these gaps. We finally succeeded with the association of another type of chemistry, D-rhodamine with 2 M betaine. We designed and used 420 primers (i) to close the sequencing gaps by walking either on shotgun subclones or on the chromosome and (ii) to improve sequence regions of low quality.
The integrity of the assembly was validated by comparing the restriction patterns obtained by pulsed-field gel electrophoresis with those deduced from the electronic consensus sequence. The selection of restriction enzymes was based on rare sites. We analyzed single digests of R. felis DNA. The main restriction enzymes used for these studies were ApaI, AfeI, FspI, and SbfI. This comparative study confirmed the predicted length of the R. felis DNA fragments.
The structures for pRF and pRFδ plasmids were controlled by specific primer amplifications (see Figure S1). Three PCRs were performed and the amplification results were in agreement with the expected hypothesis. These PCR results validate the two distinct plasmid forms (62.8 and 39 kbp, respectively). Meanwhile, a Southern blot was performed through a pulsed-field electrophoresis gel. Uncut genomic R. felis DNA and R. felis DNA digested by the restriction enzyme PvuI (corresponding to a unique site in the pRF-specific region) were analyzed. These blocks of DNA were loaded twice onto the gel with the molecular-weight markers: Lambda Marker (Bio-Rad) and Low Range PFG Marker (New England Biolabs) as described above, with a pulse time from 1 to 5 s for 12 h at 180 V. The gel was treated and transferred onto Hybond N+ (Amersham Biosciences, Little Chalfont, United Kingdom) with a vacuum blot. The DNA was fixed by heating for 2 h at 80 °C, and the membrane was cut into two pieces. Two probes were derived from two PCR products. The first, pRFh–pRFi (726 bp), was designed within the pRF-specific insert, and the second, pRFa–pRFg (251 bp), was designed to encompass the deletion site of the pRFδ. These two probes were labeled with dCTP32 and hybridized at 65 °C for 17 h on each membrane. Membranes were washed three times in 1× SSC and 0.1% SDS at 65 °C. The exposure time ranged from 6 h to overnight at −80 °C on ECL film. The hybridizations were clearly established on R. felis digested by PvuI and led to one signal with the pRFh–pRFi probe and two signals for the two plasmid structures with the pRFa–pRFg probe at a predicted molecular weight compatible with our prediction (see Figure S2).
We tested 30 samples of fleas naturally infected by R. felis obtained from different geographic areas (Algeria [11 fleas], France [15 fleas], and New Zealand [four fleas]) with three pairs of primers: (i) primers designed in the traD gene (pRF37F1/R1), (ii) primers in the pRF plasmid (pRFe–pRFf), and (iii) primers in the pRFδ plasmid (pRFa–pRFg). We confirmed positive PCR products of (i) 196 bp, (ii) 208 bp, and (iii) 251 bp for all the 30 cases.
Annotation
We predicted protein-coding genes (ORFs) using SelfID [52] as previously described [8]. tRNA genes were identified using tRNAscan-SE [53]. Database searches were performed using BLAST programs [54] against Swiss-Prot/TrEMBL [55], the NCBI CDD database [56], and SMART [57]. The number of transposases, ankyrin/TPR-containing genes, autotransporter domains, and integrases were computed using PSI-BLAST with NCBI/CDD entries related to those domains with an E-value threshold of 10−5. Repeated DNA sequences were identified with the use of RepeatFinder [58], by ignoring the sequence similarity between pRF and pRFδ. To identify Rickettsia palindromic elements, we used hidden Markov models [59] based on the previously identified RPE sequences [60].
By taking advantage of genome colinearity, we identified orthologous relationships of genes in R. felis, R. conorii, R. sibirica, R. prowazekii, and R. typhi with the use of Genomeview (S. Audic, unpublished software). Based on the gene orthology, we defined R. felis–specific ORFs, which were of one of the following three classes: Class I ORFs exhibiting no homologous ORFs in the other four Rickettsia genomes; Class II ORFs exhibiting homologous ORFs but no orthologous ORFs in the other four Rickettsia genomes; and Class III ORFs exhibiting orthologous ORFs in some or all of the other four Rickettsia, all of which exhibit degraded (split or fragmented) genes relative to the R. felis ORF. Plasmid-encoded ORFs were by definition classified into Class I or II. A gene composed of more than one ORF was defined as “split gene.” A gene composed of a single ORF whose length is shorter than 50% of the longest ortholog was defined as a “fragmented” ORF. We used T-Coffee [61] and MEGA [62] for multiple sequence alignment and phylogenetic tree analyses, respectively. The analyses of horizontal gene transfer were performed by BLAST search against the Swiss-Prot/TrEMBL nonredundant database, excluding rickettsial sequences, as well as by methods based on nucleotide composition bias [63,64]. We obtained the minimum number of inversions to associate a pair of Rickettsia genomes using GRAPPA release 2.0 [65].
Ultrastructural characterization of pili by electronic microscopy
R. felis cells were carefully collected from the supernatant of XTC cells infected for 5 d and grown at 28 °C. Following centrifugation (400 g, 10 min), bacteria were fixed for 1 h at 4 °C in glutaraldehyde (2.5% in phosphate-buffered saline [PBS]). Cells were then washed in PBS and placed on a carbon–formvar-coated 400-mesh copper grid (Electron Microscopy Sciences, Hatfield, Pennsylvania, United States) for 15 min then negatively stained with 2% phosphotungstic acid for 10 s, before analysis by electron microscopy (Philips Morgagni 268D, Philips Electronics, Eindhoven, the Netherlands).
Estimation of β-lactamase activity
To evaluate the level of β-lactamase activity, 104
R. felis cells grown on XTC cells and then sonicated were mixed with amoxicillin to a final concentration of 20 μg/ml, and incubated for 2 h at 28 °C. The concentration of amoxicillin was measured in the R. felis + amoxicillin suspension as well as in a suspension of XTC cells without bacteria + amoxicillin, before and after incubation, using high-performance liquid chromatography. In addition, the minimum inhibitory concentrations of these four suspensions were estimated by growth inhibition of a Micrococcus luteus strain.
RNA extraction and RT-PCR
Approximately 6.5 × 105 bacteria were used to infect one 25-cm3 flask of confluent XTC cells maintained at 28 °C. Infected cells were harvested 48 h later, centrifuged (12,000 g, 10 min), and pellets were immediately frozen in liquid nitrogen before being stored at −80 °C. Total RNA was isolated by using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. At the end of the extraction procedure, all samples were treated with RNase-Free DNase Set (Qiagen) for 30 min. The concentration and quality of isolated RNA were determined with the Agilent 2100 bioanalyzer (Agilent Technologies, Englewood, New Jersey, United States). Aliquots of the DNase-treated total RNA samples were stored at −80 °C until use. RT-PCR was performed from 2 μl of RNA (25 μl final reaction volume) with the Superscript One-Step RT-PCR with Platinum Taq (Invitrogen). Possible DNA contamination was assessed with the Expand high-fidelity polymerase (Roche, Basel, Switzerland). Cycling conditions were 30 min at 50 °C, 5 min at 95 °C, and 40 cycles at 30 s at 95 °C, 30s at 50 °C, and 1 min at 72 °C, followed by a final extension cycle of 7 min at 72 °C. The RT-PCRs were conducted on the PTC-100 thermocycler (Bio-Rad). Amplification products were run on 2% (wt/vol) agarose gels, and the DNA was stained with ethidium bromide. The size of the PCR product was determined by comparison with DNA molecular-weight marker VI (Boehringer Ingelheim, Ingelheim, Germany).
Detection of F-actin and immunofluorescence staining
Vero cells grown to semiconfluence on glass coverslips were infected with R. felis for 24–48 h at 28 °C in a humidified CO2 incubator (5% CO2). Infected cells were then fixed for 1 h at 4 °C with formaldehyde (3% wt/vol in PBS supplemented with 1 mM MgCl2 and 1 mM CaCl2), washed three times in PBS, and then made permeable with 0.2% Triton X-100 in PBS for 1 min. After three washings in PBS, the coverslips were incubated for 1 h with a monoclonal anti–R. felis antibody. Bacteria were visualized by staining with anti-mouse-Alexa 594 antibody (1:300) and F-actin with FITC-phalloidin (1:250). The coverslips were mounted using Fluoprep (BioMérieux, Marcy-l'Etoile, France) and were examined with a confocal laser scanning microscope using a 100× oil immersion objective lens.
Hemolysis experiments
Human blood (10 ml) was centrifuged (1,500 g, 10 min), and after three PBS washings, erythrocytes were resuspended in 20 ml of PBS. This suspension (100 μl) was mixed with 800 μl of PBS and 100 μl of rickettsial suspension (106, 105, and 104 bacteria, respectively). In some experiments, rickettsiae were incubated for 1 h at 35 °C in the presence of 2 mM DTT. Complete hemolysis was determined by adding 900 μl of H2O to erythrocytes, and spontaneous hemolysis corresponded to control without bacteria. Following 3 h of incubation at 35 °C, the samples were fixed using paraformaldehyde (0.3% final concentration) and centrifuged. Hemoglobin release was estimated by measurement of the optical density of the supernatant at 545 nm. This experiment was performed in duplicate.
Primers
The sequences of the primers for PCR and RT-PCR are provided in Table S3.
Supporting Information
Figure S1 Confirmation of Plasmid Topologies for pRF and pRFδ by PCR
(A) The locations of the three primer sets (pRFa–pRFb, pRFc–pRFd, and pRFa–pRFd) used to validate the presence of the two distinct plasmid forms are indicated.
(B) The result of the PCR assay with these primers. Two pairs of primers (pRFa–pRFg and pRfh–pRFi) used to obtain the probes for the Southern blot (see Figure S2), as well as another pair of primers (pRF37F1/R1) used in plasmid detection in fleas infected by R. felis, are also indicated in (A).
(1.5 MB TIF).
Click here for additional data file.
Figure S2 Characterization of R. felis Plasmids by Southern Blot
The two membranes (A and B) loaded with R. felis genomic DNA (#1/4) and R. felis DNA digested by PvuI (#2/3) were hybridized either by the probe pRFa–pRFg or by pRFh–pRFi.
(39 KB PDF).
Click here for additional data file.
Figure S3 A Model for the Conjugative Plasmid Transfer of R. felis
This model is based on gram-negative bacterial conjugation systems involving T4SS. Homologs responsible for different steps of conjugation were identified in the R. felis genome. DNA-processing machinery, plasmid-encoded pRF38/39 (TraATi); the coupling protein, plasmid-encoded pRF43/44 (TraDF) or chromosomally encoded virD4 (RF0469); Mpf apparatus, chromosomal genes for virB2 (RF1075), virB3 (RF0087), virB4 (RF0088), virB6 (RF0089, RF0090, RF0091, RF0092, and RF0093), virB8 (RF0463 and RF0465), virB9 (RF0462 and RF0466), virB10 (RF0467), and virB11 (RF0468); priming for DNA replication in the recipient cell, chromosomally encoded RF0786 (TraC).
(57 KB PDF).
Click here for additional data file.
Figure S4 Phylogenetic Trees for the Patatin-Like Proteins, Thymidylate Kinases, and Small Heat-Shock Proteins
Phylogenetic trees were constructed using the neighbor-joining method with Jones-Taylor-Thornton model.
(16 KB PDF).
Click here for additional data file.
Figure S5 Taxonomic Distribution of BLAST Best Hits of R. felis ORFs
R. felis ORFs were searched against the nonredundant database (excluding rickettsial sequences). The distribution difference between ORFs with rickettsial orthologs and ORFs lacking rickettsial orthologs remained significant even after the removal of transposase ORFs.
(16 KB PDF).
Click here for additional data file.
Figure S6 Cross-Reactivity of R. felis
Western immunoblot showing the preferential cross-reactivity of antibodies with R. felis and R. typhi in a patient with murine typhus (lanes a–c), and with R. felis and R. conorii in a patient with Mediterranean spotted fever (lanes d–f). Lanes a and d, R. conorii antigen; lanes b and e, R. typhi antigen; lanes c and f, R. felis antigen; MM, molecular mass.
(30 KB PDF).
Click here for additional data file.
Figure S7 Domain Structures and the Presence/Absence Patterns of spoT Genes in Different Rickettsia
With reference to the S. dysgalactiae Relseq, four (H: 53H, 77H, 78D, and 144D) and five (241R, 243K, 251K, 264D, and 323E) catalytic residues were examined for the (p)ppGpp hydrolase and synthetase domains, respectively. ORF sizes were those for R. felis genes, except SpoT14, for which the R. prowazekii ORF size is indicated. a, absent; Ch, conserved hydrolase catalytic residues; Cs, conserved synthetase catalytic residues; s, split or fragmented genes.
(17 KB PDF).
Click here for additional data file.
Figure S8 Confocal Laser Analysis of R. felis–Infected Vero Cells
Bacteria were stained by indirect immunofluorescence using a monoclonal anti–R. felis antibody followed by an anti-mouse-Alexa 594 antibody (red). F-actin was stained with FITC-phalloidin (green). Arrows indicate R. felis with actin tail.
(29 KB PDF).
Click here for additional data file.
Table S1 Distribution of sca Genes among Rickettsia Genomes
(30 KB DOC).
Click here for additional data file.
Table S2 Comparison of Different Features of Bacteria Infecting Fleas with Their Close Relatives
(32 KB DOC).
Click here for additional data file.
Table S3 Nucleotide Sequences of the Primers Used in the Present Study
(28 KB DOC).
Click here for additional data file.
Accession Numbers
The genome sequence of R. felis is accessible via GenBank (http://www.ncbi.nlm.nih.gov/Genbank) under the accession numbers: CP000053, CP000054, and CP000055. The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl) accession number for Cl. perfringens NagI is Q8XM09. The Pfam (http://www.sanger.ac.uk/Software/Pfam/) accession number for ComE3 is PF03772. The Protein Data Bank (http://www.rcsb.org/pdb/) accession number for S. dysgalactiae Relseq is 1VJ7.
We wish to thank Jean Weissenbach and his team at Genoscope for the shotgun sequencing, Bernadette Giumelli and Thi Tien N. Guyen for technical assistance in sequencing of the R. felis genome, Bernard Campagna for electronic microscopy analysis, Sylvianne Robineau for RT-PCR experiments, Claude Nappez for production of monoclonal antibodies against R. felis, and Deborah Byrne for carefully reading the manuscript. We gladly acknowledge the financial support of Marseille-Nice Genopole.
Competing interests. The authors have declared that no competing interests exist.
Author contributions. HO performed genome annotation and coordinated the bioinformatics analyses. PR performed electron microscopic analysis of pili, immunofluorescence assay of actin-based motility, and hemolytic activity assay. SA and CR assembled genome sequences, performed sequence finishing, and carried out experiments to characterize the plasmids. CR constructed genomic libraries. GB performed most phylogenetic analyses. PEF performed β-lactamase activity assay and contributed to the bioinformatics analyses. HP contributed to the sequencing. JMC provided laboratory (computing) support and supplied ideas. DR provided laboratory (experimental) support, supplied ideas, and coordinated the experimental aspects of the work. All authors contributed in drafting the manuscript.
Citation: Ogata H, Renesto P, Audic S, Robert C, Blanc G, et al. (2005) The genome sequence of Rickettsia felis identifies the first putative conjugative plasmid in an obligate intracellular parasite. PLoS Biol 3(8): e248.
Abbreviations
HEPNhigher eukaryotes and prokaryotes nucleotide-binding domain
MFSmajor facilitator superfamily
Mpfmating pair formation
ORFopen reading frame
PBSphosphate-buffered saline
PCRpolymerase chain reaction
(p)ppGppguanosine tetra- and pentaphosphates
RPE
Rickettsia palindromic element
RT-PCRreverse transcriptase–polymerase chain reaction
SFGspotted-fever group
TPRtetratricopeptide repeat
T4SStype IV secretion system
XTC cell
Xenopus laevis tissue culture cell
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| 15984913 | PMC1166351 | CC BY | 2021-01-05 08:28:15 | no | PLoS Biol. 2005 Aug 5; 3(8):e248 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030248 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1608950210.1371/journal.pbio.0030254Research ArticleCell BiologyDevelopmentGenetics/Genomics/Gene TherapyHematologyDanio (Zebrafish)Homo (Human)Functional Analysis of Human Hematopoietic Stem Cell Gene Expression Using Zebrafish Functional Analysis of HSC Gene ExpressionEckfeldt Craig E
1
Mendenhall Eric M
1
Flynn Catherine M
1
Wang Tzu-Fei
1
Pickart Michael A
2
Grindle Suzanne M
3
Ekker Stephen C
2
Verfaillie Catherine M [email protected]
1
1 Department of Medicine, Division of Hematology, Oncology, and Transplantation, and Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, United States of America,2 Genetics, Cell Biology, and Development and Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota, Minneapolis, Minnesota, United States of America,3 Cancer Center Bioinformatics Division, University of Minnesota, Minneapolis, Minnesota, United States of AmericaZon Leonard I. Academic EditorHarvard Medical School and Children's HospitalUnited States of America8 2005 5 7 2005 5 7 2005 3 8 e25412 11 2004 14 5 2005 Copyright: © 2005 Eckfeldt et al.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Fish and Chips: A Fast Track to Understanding Blood Development
Although several reports have characterized the hematopoietic stem cell (HSC) transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow CD34+CD33−CD38−Rholoc-kit+ cells, enriched for hematopoietic stem/progenitor cells with CD34+CD33−CD38−Rhohi cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO)-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23%) of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global gene profiling of HSCs.
Microarray studies are combined with a functional screen in zebrafish to identify new genes that are involved in the function and differentiation of hematopoietic stem cells.
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Introduction
Hematopoiesis is the process by which hematopoietic stem cells (HSCs) give rise to all hematopoietic lineages during the lifetime of an individual. To sustain lifelong hematopoiesis, HSCs must self-renew to maintain or expand the HSC pool [1], and they must differentiate to form committed hematopoietic progenitor cells (HPCs) that progressively lose self-renewal potential and become increasingly restricted in their lineage potential. A combination of extrinsic and intrinsic signals are thought to converge to regulate HSC differentiation versus self-renewal decisions, but the molecular mechanisms that regulate these processes are poorly understood [2].
A multitude of cytokines have been cloned that affect HSCs and HPCs; however, to date none of these, alone or in combination, can induce the symmetrical, self-renewing HSC cell division in vitro that is required for HSC expansion. Recently, several novel regulators of HSC fate decisions have been identified. For instance, overexpression of Hoxb4 results in expansion of murine and human HSCs with an increased competitive repopulation potential [3–5]; novel extrinsic regulators implicated in self-renewal of HSCs include Notch [6], Wnt [7,8], and the morphogens, SHH (sonic hedgehog) [9] and BMP4 (bone morphogenetic protein 4) [9]. While the discovery of these novel regulators provides credence to the hypothesis that extrinsic and intrinsic signals can influence HSC fate, a more global gene and/or protein expression analysis of human HSC should provide additional insight into pathways that support HSC self-renewal.
Our current understanding of the expressed gene profile of HSCs comes primarily from murine HSCs that can be purified to near homogeneity [10–14]. The inability to purify human HSCs to similar degrees of homogeneity makes study of the transcriptome of human HSCs more difficult. Human HSCs and HPCs are CD34 positive, while cells that engraft in severe combined immunodeficiency (SCID) mice are enriched in the CD34+Lineage(Lin)−CD38− fraction [15]. As fewer than 1/500 CD34+Lin−CD38− cells can repopulate SCID mice [15], the expressed gene profile of CD34+Lin−CD38− cells is likely only partially enriched for HSC-specific genes [12,16]. We previously demonstrated that the rhodamine (Rho) 123− and c-kit+ subpopulation of CD34+Lin−CD38− cells (Rholo) cells are highly enriched for primitive HPCs with myeloid–lymphoid initiating cell (ML–IC) capacity relative to CD34+CD38−CD33−Rhohi (Rhohi) cells [17]. Thus, such selection separates CD34+Lin−CD38− cells into HSC-enriched and HSC-depleted populations.
We hypothesized that comparison of the transcriptome of Rholo and Rhohi cells from umbilical cord blood (UCB) and bone marrow (BM) should identify conserved genes and gene pathways that define the human HSC. Because of the inherent limitations of using gene expression data to infer biological gene function, we also assessed the hematopoietic role of these genes in a high-throughput in vivo functional genomics screen in the zebrafish. Using this strategy we have not only identified a series of genes that may represent novel regulators of human HSC fate decisions, but this work also represents, to our knowledge, the first example of a functional genetic screening strategy that is a critical step toward obtaining biologically relevant functional data from global gene-profiling studies.
Results/Discussion
ML–ICs Are Highly Enriched in Rholo Compared to Rhohi Cells
The study of human HSCs has been limited since the CD34+Lin−CD38− fraction of hematopoietic cells, commonly used as an HSC-enriched population, contains fewer than 0.2% SCID-repopulating cells [15], suggesting considerable heterogeneity. We have shown that ML–IC s, single hematopoietic cells that can generate several daughter cells capable of reinitiating long-term myeloid and long-term lymphoid cultures, are highly enriched by selecting the Rholo fraction of CD34+Lin−CD38− cells. While the Rholo population still only contains 15%–25% ML–ICs and therefore remains heterogeneous, the enrichment factor is 5- to 10-fold greater than CD34+Lin−CD38− cells [17]. Similar to our previous studies, the ML–IC frequency was greater than or equal to 10-fold higher in UCB Rholo compared to Rhohi cells (Figure S1).
Genes Differentially Expressed between Rholo and Rhohi Cells from Both UCB and BM
We hypothesized that comparing genes differentially expressed between Rholo and Rhohi cells from ontogenically distinct sources would identify conserved genes and gene pathways that govern self-renewal and differentiation of human HSCs. The experimental design used is illustrated in Figure 1. We defined differentially expressed probe sets as those with p < 0.05, using a paired Student's t-test. By taking into account the variability present in primary cell populations, this provides a more accurate analysis of differential gene expression compared with the commonly used fold change cutoff.
Figure 1 Fluorescence-Activated Cell Sorting and Gene Expression Analysis
UCB or adult BM CD34+CD33−CD38−Rholoc-kit+ (Rholo; stem cell enriched) and CD34+CD33−CD38−Rhohi (Rhohi; stem cell depleted) cell populations were sorted for subsequent global gene expression profiling. Total RNA was isolated from Rholo and Rhohi cell populations prior to linear amplification and labeling for hybridization to the Affymetrix HG-U133 GeneChip set (approximately 45,000 probe sets) and subsequent data analysis.
We identified 2,707 and 4,667 probe sets differentially expressed between Rholo and Rhohi cells from UCB and BM, respectively (see Tables S1 and S2). The fidelity of our microarray results was confirmed using quantitative RT-PCR (Q-RT-PCR) (see Figure S2). We focused our further analysis on 277 unique transcripts, represented by 304 probe sets that were differentially expressed between Rholo and Rhohi cells from both UCB and BM with a fold change greater than 1.5 in either UCB or BM (Table S3).
Among the conserved genes enriched in Rholo cells, many have been implicated in early hematopoiesis, including CDKN1A, a cell cycle regulator required for maintenance of murine HSCs [18], and ABCB1, the ABC-transporter family member responsible for the Rholo phenotype [17]. Several transcription factors (TFs) known to play a role in early hematopoiesis or leukemogenesis were also identified, including HLF, involved in leukemogenic chromosomal translocations [19] and EVI1, a TF associated with myeloid leukemias [20]. Other TFs without a known role in hematopoiesis were also more highly expressed in Rholo cells, including HMGA2, a high-mobility group gene, and the zinc finger TFs ZNF165, ZNF331, and KLF5. All Rholo-enriched genes are listed in Table S3. As demonstrated in previous HSC gene-profiling studies [12–14], more than 40% of genes enriched in Rholo cells lack a functional annotation, are hypothetical proteins, or are expressed sequence tags, and thus may represent currently uncharacterized regulators of HSC fate decisions (Figure S3).
Some genes with well-established roles in HSC self-renewal and early differentiation are not present in the Rholo enriched gene list. However, most of these were differentially expressed in both datasets, but differences did not reach statistical significance. For instance, LMO2 [21] and GATA
2 [22], known to be involved in HSC development and self-renewal, were expressed significantly higher in BM Rholo than in Rhohi cells. Although similar trends were seen in UCB Rholo cells, these differences were not statistically significant (Table S4). Conversely, HOXB4 [4] expression was significantly higher in UCB Rholo than in Rhohi cells, but this difference was not statistically significant in BM. Although our stringent criteria for differential expression likely contribute to the omission of some genes that might be differentially expressed, another explanation might be that expression of these genes is maintained when cells differentiate from a Rholo to a Rhohi stage. The latter is consistent with the fact that most known HSC-associated genes were expressed at much higher levels than the normalized average microarray expression level in Rholo and in Rhohi cells from both UCB and BM (Table S4).
Conserved genes enriched in the Rhohi cells included LEF1, an effector of Wnt signaling expressed in pre-B and T cells [23], and NOTCH2, involved in hematopoietic differentiation cell-fate decision [24]. Several TFs known to play a role in hematopoietic cell differentiation were more highly expressed in Rhohi compared to Rholo cells, including HELLS and MAFB [25,26]. Additional TFs with no known role in hematopoietic development were also enriched in Rhohi cells, such as the zinc finger homeobox gene, ZFHX1B and the polycomb genes, EZH2 and SUZ12, the latter required for germ cell development [27]. Globin (Hb) gene family members were also more highly expressed in UCB and BM Rhohi than in Rholo cells. Consistent with the ontogenic expression patterns of fetal versus adult Hb genes, Hbγ genes were more highly expressed in perinatal UCB Rhohi cells, while Hbβ genes were more highly expressed in adult BM Rhohi cells. Additional genes enriched in Rhohi cells are listed in Table S3.
Because of functional redundancy among gene families, we examined the data for common differentially expressed gene family members. The Id family of transcriptional repressors [28] was enriched in the Rholo fraction, but was represented by different family members in UCB (ID4) and BM (ID1, ID2, and ID3). Similarly, various H1 and H2 histone genes were enriched in the Rholo fraction in both datasets, but were represented by distinct family members.
We also evaluated whether common differentially expressed genes were concentrated on specific chromosomes. We found that genes were not only concentrated on certain chromosomes, but at specific g-band addresses. Of the genes enriched in Rholo cells, 9% reside at 6p21, a region involved in recurrent chromosomal translocations in myeloid [29] and lymphoid [30] leukemias, and home to the PIM1 oncogene [31]. Six members of the H2B and one member of the H1 histone family, as well as CDKN1A, more highly expressed in Rholo than Rhohi cells, reside at 6p21. The remaining Rholo-enriched genes at 6p21 consist of six class II major histocompatibility complex (MHC) family members and a putative testis-specific zinc finger TF, ZNF165. H1 and H2 histone gene family members [11,13], class II MHC antigens [12,13], and CDKN1A [13] were also found among the genes identified in studies characterizing the transcriptome of murine HSC. The differential expression of such a large number of genes located at this chromosomal address suggests that, like CDKN1A, other genes located at 6p21 with as yet unknown hematopoietic function may play a role in HSC proliferation or differentiation.
We also compared the genes expressed more highly in Rholo
versus Rhohi cells with published gene expression data. Comparison with the study by Ivanova et al. [12] that compared human CD34+Lin−CD38− with CD34+Lin−CD38+ cells, yielded only seven genes in common: ARMCX2, CRYGD, HLF, KIAA1102, RBPMS, SLCO3A1, and SSBP2. The lack of overlap may not be that surprising as Rholo and Rhohi cells are subpopulations of the CD34+Lin−CD38− population used by Ivanova et al. Comparison of genes expressed more highly in Rholo
versus Rhohi cells with genes expressed more highly in murine side population/KLS/CD34− compared to total BM cells published by Ramalho-Santos et al. [13] identified 16 likely orthologs and 38 common gene family members (Table S5), suggesting that HSC-specific genes are conserved across species.
In vivo functional genomics screen in zebrafish
Because gene profiling per se does not prove functional importance, we developed an in vivo functional genomics screen in zebrafish (Figure 2). The zebrafish, Danio rerio, is an ideal organism for high-throughput genetic screens [32] as organogenesis is highly conserved from zebrafish to man [33]. There is abundant evidence that hematopoiesis in zebrafish occurs via a highly conserved genetic program. As in mammals, hematopoiesis in zebrafish occurs via specification of mesoderm to a hemangioblast stage that subsequently commits to either HSC or angioblasts [34], and genes and signals involved in specification (BMP signaling) and commitment (vascular endothelial growth factor signaling; flk1, also known as kdr; lmo2; scl, also known as tal1; gata2;
gata1) are conserved from fish to man [35]. This high degree of homology in the genetic control of zebrafish and human hematopoietic development makes genetic screens in zebrafish a powerful tool to elucidate the role of genes in hematopoiesis. Additionally, rapid reverse genetic screens can be accomplished using morpholino antisense oligonucleotides (MOs) to knock down gene expression in the developing zebrafish embryo [36].
Figure 2 Functional Genomics Screen in Zebrafish
The hematopoietic function of differentially expressed candidate genes was determined by injecting MOs into one- to two-cell embryos from gata1:DsRed Tg zebrafish to disrupt gene expression. Injected embryos were scored at 30–48 hpf for the presence of DsRed+ blood cells by fluorescence microscopy. Subsequently, MO-targeted embryos with gross hematopoietic defects were analyzed for the expression of the early hematopoietic markers gata1 and scl by whole-mount in situ hybridization, and the late hematopoietic markers hbae1 and lcp1 by Q-RT-PCR.
From the 277 unique transcripts that were differentially expressed between Rholo and Rhohi cells of both UCB and BM (Table S3), we eliminated genes with known function in hematopoiesis, MHC genes, histones, and genes that are known to play a role in glucose and protein metabolism and RNA and DNA synthesis, resulting in a final list of 158 genes. Of these, we identified a putative zebrafish ortholog for 86, and designed MOs against 61 (Table S6). The 61 MOs were injected in one- to two-cell zebrafish embryos and assayed for effects on blood development. Initial dosing studies identified 16/61 MOs that reduce blood cell production without confounding toxicities (Table 1). The 16 MOs induced a blood defect in more than 70% of embryos in two or more independent injections. Blood defects identified by gata1:DsRed transgenic (Tg) fluorescence microscopy were confirmed by Q-RT-PCR of the erythroid-specific hbae1 and myeloid-specific lcp1 transcripts in MO-targeted embryos compared to uninjected controls in three or more independent experiments of n = 5 embryos per experiment. A greater than 2-fold reduction in both erythroid and myeloid gene expression levels were seen for five of seven MOs analyzed (see Table 1; Figures 3 and 4). Thus, the addition of Q-RT-PCR to the screening process provides an independent confirmation and quantitation of the observed phenotypes, thereby limiting the false-positive rate, while maintaining the high-throughput nature of the screen. Additionally, the observed reduction of both erythroid and myeloid gene expression following knockdown of candidate genes is consistent with their presumed roles in HSC fate decisions prior to specification of the common myeloid progenitor. The validity of the Q-RT-PCR analysis was corroborated by analysis of hbae1 and lcp1 transcript levels in gata1 MO-targeted embryos, in which there was a virtually complete loss of hbae1 expression and an almost 2-fold increase in myeloid-specific lcp1 transcripts (Figure 4) consistent with the published expression patterns for these genes following loss of gata1 expression [37]. Analysis of vascular development by injecting MOs into fli1:enhanced green fluorescent protein (EGFP) Tg embryos, which express EGFP in endothelial cells, demonstrated no major abnormalities in vascular morphogenesis or remodeling that precludes the circulation of the remaining blood cells, indicating that the hematopoietic defect is not secondary to a vascular phenotype (Figure 4; Table S7).
Figure 3 Representative Hematopoietic Phenotypes Observed in MO-Targeted Zebrafish Fluorescence microscopic images of gata1:DsRed Tg zebrafish embryos display the hematopoietic phenotypes observed for six MO-targeted zebrafish embryos compared to an uninjected control
Phenotypes shown are representative of greater than 70% of injected embryos at 48 hpf (greater than or equal to three experiments of n > 40 embryos). Hematopoietic defects were quantified by Q-RT-PCR for the expression of erythroid-specific hbae1 and myeloid-specific lcp1 transcripts in MO-targeted embryos relative to uninjected clutchmate controls (greater than or equal to three experiments of n = 5 embryos; * p < 0.05)
Figure 4 spry4 Is Required for Normal Hematopoietic Development in Zebrafish Embryos
(A) The observed frequency of hematopoietic defects in gata1:DsRed Tg zebrafish embryos are indicated for two independent spry4-targeted MOs (MO1 and MO2; black bars), a four-base mismatched control MO (MM MO; no bar indicates a 0% frequency), a low-dose injection of MO1 and MO2 individually and in combination (gray bars), and MO2 coinjected with a human SPRY1 DNA expression vector or a GFP control (white bars) (error bars = standard deviation of the mean, *p < 0.05, **p ≤ 0.01).
(B) Representative pictures of the phenotypes seen with spry4 MO1 at 48 h using fli1:EGFP/gata1:DsRed double Tg zebrafish embryos that have EGFP+ vascular endothelial cells and DsRed+ erythroid cells. The embryos display a more drastic reduction in DsRed+ blood cells with some blood pooling and pericardial edema at higher MO doses (++MO) without major vasculature defects. The gata1:DsRed and fli1:EGFP Tg images are representative of three experiments of n > 40 embryos each.
(C) Q-RT-PCR analysis of hbae1 and lcp1 transcripts in spry4 MO1, control mismatch MO and a gata1 MO-injected zebrafish at 48 hpf (*p < 0.05).
(D) Injection of 30 pg of human SPRY1 DNA expression vector resulted in an expansion of DsRed+ hematopoietic cells in the posterior ICM in greater than 50% of successfully injected embryos at 32 hpf (three experiments of n = 30), and a representative bright-field image is pictured with the fluorescence micrograph overlaid (left). Q-RT-PCR analysis of hbae1 and lcp1 transcripts in SPRY1 overexpressing embryos relative to uninjected clutchmate controls (three experiments of n = 5 embryos) (right).
Table 1 Genes Differentially Expressed between Human Rholo and Rhohi Cell Populations That Have a Functional Role in Zebrafish Hematopoietic Development
The greater than 20% frequency of blood defects seen in our screen compares very favorably with the 0.5%–1% frequency of hematopoietic phenotypes seen by ethylnitrosourea mutagenesis screens that mutate genes in a near random fashion [38] and the 4% of hematopoietic phenotypes seen in a morpholino-based functional screen of the zebrafish secretome [39] (S. C. Ekker, unpublished data). The high incidence of blood defects also demonstrates that the candidate genes identified by comparing the transcriptome of Rholo and Rhohi cells represent genes with important roles in HSC biology. We were unable to identify a candidate ortholog in zebrafish for 72/158 of the differentially expressed human genes. This may be because currently only one quarter of the zebrafish genome is high-quality finished sequence. Hence, some genes with important roles in hematopoiesis may have been untested. However, from a sample of ten genes that lacked a zebrafish match, only one has a likely ortholog in the Fugu or Medaka sequencing projects, and thus incomplete genome coverage provides only a partial explanation. Alternative possibilities include a reduced level of primary sequence conservation between functional orthologs that would have been missed using our simple comparative genomics criteria, or that a number of genes are not conserved between fish and man, and thus might be less important for the conserved processes of hematopoietic self-renewal and differentiation. Therefore, the relatively high incidence of blood defects in conserved genes may in part reflect “evolutionary filtering” in our screen. Consistent with this hypothesis, all zebrafish genes whose mutation resulted in a visible embryonic phenotype identified using a retroviral insertion strategy have a likely human ortholog [40].
Although the frequency of blood defects is high, the screen is not as well suited for the identification of knockdown phenotypes that result in increased HSC proliferation and/or differentiation. We and others have successfully shown that dramatically increased hematopoietic development can be modeled in zebrafish, as is the case for knockdown of the BMP-antagonist chd (chordin) and the corresponding dino mutant [41,42]; however, more modest increases in hematopoietic cell production or skewing of lineage differentiation may be undetectable. Although these caveats may lead to underestimation of the true frequency of genes with a role in early hematopoietic development and differentiation, the screening procedure used has proven effective for extracting functional information from a global gene expression profiling dataset in a high-throughput manner.
Of note, viable knockout mice exist for 4/14 genes identified in our functional screen (Sparc, Irak3, Ccr7, and Prkch) [43–46]. One could argue that if a viable knockout mouse exists, the gene of interest may not be important in hematopoiesis. However, lack of an overt hematopoietic phenotype does not preclude a role of a gene in HSC self-renewal and differentiation, as this may only be detectable under conditions where the hematopoietic system is stressed or in transplantation experiments. For example, Hoxb4
−/− mice develop normally and present with only subtle differences in spleen and BM cellularity [47]. However, the proliferative response of HSC in vitro and in vivo is decreased, consistent with the observation that overexpression of Hoxb4 supports expansion of competitive repopulating units and SCID-repopulating cells [4,5].
Characterization of Sprouty Family Members in Zebrafish Hematopoiesis
To further verify the hematopoietic role of genes identified by gene array, we performed a more extensive evaluation of the zebrafish targeted with an MO against spry4 (spry4 morphant or spry4MO). Although human SPRY1 was differentially expressed, an MO against zebrafish spry4 was used, as it is expressed in the region of the lateral plate mesoderm, the first site of zebrafish hematopoiesis [48], and it was the full-length zebrafish Sprouty gene with the greatest protein homology to human SPRY1. Recently the partial sequence of a potential zebrafish spry1 ortholog was predicted by Ensembl's gene prediction software based on genomic sequence information. However, the single exon that was predicted does not contain an ATG start codon, or a conserved splice donor or acceptor site, and the putative zebrafish spry1 sequence only partially covers the human SPRY1 gene. Moreover, the genomic location of Ensembl's putative zebrafish spry1 is currently not known, and therefore it is not possible to use syntenic relationships to determine the most likely zebrafish ortholog for human SPRY1. At present, there is not sufficient sequence data available to design gain- or loss-of-function experiments for the putative zebrafish spry1, thus precluding an analysis of hematopoietic function in the zebrafish model. Therefore, spry4 is currently the best full-length, MO-targetable candidate ortholog for human SPRY1, and based on our results, at the very least zebrafish spry4 and SPRY1 share a conserved function in hematopoiesis.
To confirm the specificity of MO targeting in the spry4MO, a second spry4 MO of independent sequence and a four-base mismatched spry4 MO were injected into zebrafish embryos. Injection of the independent spry4 MOs induced a hematopoietic phenotype in more than 65% of injected embryos, while the four-base mismatched MO did not induce any phenotypic changes (Figure 4). Q-RT-PCR for hbae1 and lcp1 transcripts also did not show changes in expression in four-base mismatched MO embryos. Furthermore, the two independent spry4 MOs acted synergistically when coinjected (Figure 4). In addition to the blood phenotype, a slight facial outgrowth was seen at a low frequency. Also, a weak dorsalization phenotype was seen at 2-fold higher MO doses based on a decreased somite size.
To rule out the possibility that the hematopoietic defect observed in the spry4MO was secondary to a vascular defect, we injected spry4 MO into fli1:EGFP Tg zebrafish. While the resulting embryos exhibited minor defects in cardinal vein remodeling and morphogenesis of intersegmental vessels in the posterior tail, there were no major defects in vascular development (Figure 4). The integrity of the vascular network in spry4MO fish was further demonstrated by the unimpeded circulation of the remaining DsRed+ blood cells in gata1:DsRed Tg spry4MO (data not shown).
Thisse et al. [35] have shown that overexpression of zebrafish spry4 mRNA leads to an expansion of the posterior intermediate cell mass (ICM) [48]. We overexpressed human SPRY1 in gata1:DsRed Tg zebrafish embryos, and observed a similar dose-dependent expansion of DsRed+ blood cells in the posterior ICM (Figure 4). Q-RT-PCR analysis of embryos overexpressing SPRY1 revealed a 1.7- and 1.4-fold increase hbae1 and lcp1 mRNA levels, respectively, confirming the observed expansion of blood cells in the ICM. Coinjection of human SPRY1 cDNA with spry4 MOs ameliorated the spry4MO phenotype, yet another confirmation of the specificity of MO targeting (Figure 4). The similar hematopoietic phenotypes observed following the overexpression of either human SPRY1 or zebrafish spry4 indicate that these genes encode proteins with a similar functional potential in blood development. In addition to the blood phenotype, the overexpression often caused a reduction and/or curve in the posterior tail (Figure 4) not seen in the overexpression of zebrafish spry4 [48].
Characterization of hematopoietic gene expression in the spry4MO by whole-mount in situ hybridization revealed a reduction in scl expression at four somites (8/15), and virtually no scl (12/20) or gata1 (18/25) expression at 20 somites (Figure 5), consistent with a defect in mesodermal commitment to HSCs and/or HSC proliferation and differentiation. The few hematopoietic cells that are present in the morphant are hemoglobinized based on o-dianisidine staining (data not shown). To determine whether the hematopoietic defects observed in spry4MO were the result of a defect in mesoderm specification during development, we performed whole-mount in situ hybridization for the vasculature-specific flk1 and muscle-specific myod transcripts. At 10 h postfertilization (hpf) there was a slight defect in myod expression (5/10), while myod expression at 26 hpf was comparable to wild type (21/21) (Figure 5). This suggests that there were no major defects in mesodermal commitment in the spry4 morphants. Considering the nearly absent expression of the early hemangioblast and HSC cell marker scl, these results may suggest that the defect induced in the spry4MO occurs prior to HSC specification. However, the normal expression pattern of other mesodermal genes, such as flk1 and myod at 26 hpf in the spry4MO (Figure 5), indicate that the hematopoietic phenotype is not merely a consequence of defective specification of mesoderm. Finally, the hematopoietic gene cmyb, a presumed marker of definitive HSC in zebrafish [49], was absent at 38 hpf (10/12) (data not shown), suggesting that the spry4MO embryos are devoid of definitive HSC.
Figure 5 spry4 Is Required for Early Hematopoietic Development, but Not Mesodermal Commitment, in Developing Zebrafish Embryos
(A) Expression of the early hematopoietic marker scl was reduced at the four-somite stage (arrow) and virtually absent at the 20-somite stage in spry4MO embryos compared to uninjected controls.
(B) The more mature hematopoietic marker gata1 was also reduced in spry4MO compared to uninjected zebrafish embryos at 20 somites.
(C and D) In contrast to hematopoietic genes, the mesodermal-specific flk1 (vasculature) transcripts were expressed at similar levels to uninjected controls at 26 hpf, and myod (muscle) had a slight defect (arrowhead) at 10 hpf, while expression was similar to controls at 26 hpf. All images are representative of greater than or equal to two experiments of n ≥ 8 embryos.
In vertebrates, Sprouty family members act as antagonists for fibroblast growth factor (FGF), vascular endothelial growth factor, and epidermal growth factor signaling, and they may be involved in feedback regulation, as Sprouty gene expression is induced by activation of these signaling pathways [50]. Sprouty genes antagonize receptor tyrosine kinase signaling at the level of the Ras/Raf/mitogen-activated protein kinase pathway; however, they also can serve as positive regulators of these pathways in some cell types [50]. Therefore, our current hypothesis is that SPRY1 may affect HSC by modulating FGF-mediated, perhaps in combination with other receptor tyrosine kinase-mediated, signaling. In fact, three of the 14 genes that induce a hematopoietic defect in the zebrafish screen, SPRY1,MAFB and SPARC, are all involved in FGF signaling [50–52], thus suggesting a role for FGF in hematopoiesis. Studies are ongoing to confirm a role of SPRY1 in mammalian hematopoiesis, by testing the effect of overexpression of SPRY1 on the repopulating ability of human and murine HSCs. Similar definitive studies in zebrafish and subsequent confirmation in mammalian HSC models are underway for genes that were functionally validated in our zebrafish screen. We believe that the sequential genetic screen in zebrafish followed by confirmation in mammalian models as described here will establish a hematopoietic function for genes identified by gene array analysis in a high-throughput and efficient manner.
Materials and Methods
Isolation of Rholo and Rhohi cell populations from UCB and BM
Human UCB from full-term delivered infants and BM from healthy donors were obtained after informed consent in accordance with guidelines approved by the University of Minnesota Committee on the Use of Human Subjects in Research. Each biologically distinct replicate was comprised of one to four donors for UCB and individual donors for BM samples. CD34+CD38−CD33−Rholoc-kit+ and CD34+CD38−CD33−Rhohi fractions were selected by sequential Ficoll-Hypaque separation, MACS column depletion, and fluorescence-activated cell sorting as previously described [17]. Postsort analysis demonstrated that sorted populations contained fewer than 1%–2% contaminating cells from the opposing population (see Figure 1).
Determination of ML–IC frequencies
ML–IC frequencies for UCB samples (n = 3) were determined as previously described [17]. An ML–IC was defined as a single cell that gave rise to at least one LTC–IC and one NK–IC. Results are presented as ML–IC frequency ± standard deviation of the mean.
Processing of RNA samples and oligonucleotide microarray analysis
Total cellular RNA was isolated from UCB (n = 5) and BM (n = 4) Rholo and Rhohi cells using the PicoPure RNA Isolation Kit (Arcturus, Mountain View, California, United States) per the manufacturer's instructions. Seven to 10,000 Rholo and Rhohi cells were sorted directly into 100 μl extraction buffer (XB) provided with the PicoPure RNA Isolation Kit prior to RNA isolation. Labeled cRNA was generated by one round of IVT-based, linear amplification using the RiboAmp OA RNA Amplification Kit (Arcturus) followed by labeling with the Enzo Bioarray HighYield RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, New York, United States) according to manufacturer's instructions. Samples were hybridized to Affymetrix HG-U133 A and B chips (Affymetrix Inc., Santa Clara, California, United States) were washed, and scanned at the University of Minnesota Affymetrix Microarray Core Facility as described in the Affymetrix GeneChip Expression Analysis Technical Manual.
Oligonucleotide microarray data analysis
Affymetrix HG-U133 GeneChips were processed using GeneData Refiner software (GeneData, San Francisco, California, United States) to assess overall quality. Feature intensities for each chip were condensed into a single intensity value per gene using the Affymetrix Statistical Algorithm (MAS 5.0) with τ = 0.015, α1 = 0.04, α2 = 0.06, and a scaling factor of 500. Expression data was analyzed using GeneData's Expressionist and Microsoft Excel (Microsoft, Redmond, Washington, United States). Differential gene expression for comparison of Rholo versus Rhohi cells was defined by using a paired Student's t-test with a threshold of p< 0.05, and a paired fold change was used to rank gene lists. Differentially expressed genes were classified according to their respective gene pathways and gene ontologies when available by using the Web-based Affymetrix NetAffx analysis tool (http://www.affymetrix.com) and the National Institutes of Allergy and Infectious Disease Database for Annotation, Visualization and Integrated Discovery analysis tool (DAVID) (http://apps1.niaid.nih.gov/david).
Microarray Q-RT-PCR confirmation
Labeled cRNA was reverse transcribed to generate cDNA using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, California, United States) according to manufacturer's instructions. Q-RT-PCR was performed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, California, United States). Briefly, 3 ng of cDNA was amplified by 40 cycles of a two-step PCR reaction (95 °C for 15 s denaturation and 60 °C for 1 min. annealing/elongation) containing 100 nM gene-specific primers (Table S8) and the SYBR Green PCR Master Mix (Applied Biosystems). Gene expression was normalized using human ACTB (also β-actin) expression levels. Results are presented as percent expression relative to uninjected clutchmate control embryos ± standard deviation of the mean.
High-throughput loss-of-function genetic screen in zebrafish
The likely zebrafish orthologs of differentially expressed human genes were identified using Ensembl's gene homology prediction program (http://www.ensembl.org, build Zv3) in combination with comparison of the human protein sequence to the Institute for Genomic Research zebrafish EST database (http://www.tigr.org, release 13–15). The criteria for a likely ortholog was ≥ 40% amino acid identity over the entire length of the protein or ≥ 50% if the fish protein sequence was only partial. MO (Gene Tools, Philomath, Oregon, United States) sequences were designed complimentary to the region of translational initiation of the zebrafish orthologs in order to inhibit protein translation (Table S6) and injected into one- to two-cell zebrafish embryos as previously described [36]. MOs were injected at 1.5, 3, 4.5, and 6 ng into 50–70 embryos derived from mating Tg zebrafish containing a fluorescent DsRed reporter cassette driven by the gata1 promoter (gata1:DsRed Tg) or a fluorescent EGFP reporter cassette driven by the fli1 promoter (fli1:EGFP Tg). MO-targeted (morphant) zebrafish were evaluated for defects in hematopoietic and vascular development compared to uninjected controls from the same embryo clutch at 30 and 48 hpf, using fluorescence microscopy for visualization of DsRed+ blood cells and EGFP+ vasculature.
Whole zebrafish Q-RT-PCR confirmation
Total RNA was isolated from five MO-injected zebrafish embryos at 48 hpf with hematopoietic defects, determined based on gata1:DsRed+ hematopoietic cell production, or five uninjected clutchmate controls using the RNeasy Mini Kit (Qiagen, Valencia, California, United States) according to the manufacturer's instructions. Total RNA was incubated with DNaseI (Invitrogen) to digest contaminating genomic DNA, and reverse transcribed to generate cDNA using SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol. Q-RT-PCR was performed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Briefly, 1/20 of the total cDNA from five zebrafish embryos was amplified by 40 cycles of a two-step PCR reaction (95 °C for 15 s denaturation and 60 °C for 1 min. annealing/elongation) containing 100 nM gene-specific primers (Table S8) and the SYBR Green PCR Master Mix (Applied Biosystems). Gene expression was normalized using a zebrafish gapd expression level. Results are presented as percentage expression relative to uninjected clutchmate control embryos ± standard deviation of the mean.
Whole-mount in situ hybridization
The scl, myod, flk1, gata1, and cmyb riboprobes were generated and whole-mount in situ hybridization of zebrafish embryos was conducted as previously described [41].
Overexpression of human and zebrafish Sprouty gene family members
The XhoI and KpnI fragment of the SPRY1 open reading frame (ORF) (Open Biosystems, Huntsville, Alabama, United States) was cloned into pENTR1A (Invitrogen) and subsequently transferred into a modified pFRM2.1 zebrafish expression vector using the Gateway cloning system (Invitrogen) to create the pFRM2.1–SPRY1 vector. pFRM2.1–SPRY1 was coinjected with pFRM2.1–EGFP at a 5:1 ratio into the yolk/cell interface of one-cell gata1:DsRed Tg zebrafish embryos as described for MO injections. Defects in hematopoietic development of EGFP+ embryos were analyzed by comparison to embryos from the same clutch injected with pFRM2.1–EGFP alone, using fluorescence microscopy to visualize DsRed+ blood cells.
Supporting Information
Figure S1 Frequency of ML–IC in UCB Rholo and Rhohi Cell Populations
ML–ICs are highly enriched in Rholo (white bar) compared to Rhohi (black bar) cells from UCB (n = 3, *p < 0.05).
(3.6 MB TIF).
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Figure S2 Confirmation of Differential Gene Expression Using Q-RT-PCR
The Rholo to Rhohi fold change for DLK1, ABCB1, BMP6, HELLS, CDC25A, MAFB, and S100A8 was determined using Affymetrix GeneChip analysis (gray bars) and Q-RT-PCR (black bars) to confirm the fidelity of the microarray results for (A) adult BM (n ≥ 2) and (B) UCB (n ≥ 3).
(7.2 MB TIF).
Click here for additional data file.
Figure S3 Gene Ontology Classifications of Conserved Differentially Expressed Genes
Percentages of Gene Ontology classifications of the genes differentially expressed between Rholo and Rhohi cells from both UCB and adult BM (p < 0.05 and fold change > 1.5 in either UCB or BM).
(2.7 MB TIF).
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Table S1 Probe Sets Differentially Expressed between UCB-Derived Rholo and Rhohi Cells (p < 0.05)
(437 KB XLS).
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Table S2 Probe Sets Differentially Expressed between Adult BM-Derived Rholo and Rhohi Cells (p < 0.05)
(738 KB XLS).
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Table S3 Conserved Probe Sets Differentially Expressed between Rholo and Rhohi Cells from Both UCB and Adult BM (p < 0.05 and FC > 1.5 in Either UCB or BM)
(75 KB XLS).
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Table S4 Fold Changes, p-Values, and Average Expression Levels for Genes with Well-Established Roles in HSC Proliferation and Differentiation
(17 KB XLS).
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Table S5 Comparison of Conserved Rholo-Enriched Genes with Ramalho-Santos et al. [13] Dataset
(22 KB XLS).
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Table S6 Morpholino Sequences for Targeted Genes
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Table S7 Complete Phenotypic Descriptions for the 14 Zebrafish Morphants with Confirmed Blood Defects
(19 KB XLS).
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Table S8 Q-RT-PCR Primer Sequences
(16 KB XLS).
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Accession Numbers
The National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) Entrez Gene accession numbers for the following genes are: ABCB1 (5243), ARMCX2 (9823), BMP4 (652), C12orf2 (11228), CCR7 (1236), Ccr7 (12775), CDKN1A (1026), chd (30161), cmyb (30519), CRYGD (1421), EVI1 (2122), EZH2 (2146), FLJ14917 (84947), flk1 (also known as kdr) (58106), FOXM1 (2305), gata1 (30481),
GATA2 (2624), gata2 (30480), hbae1 (30597), HDHD2 (84064), HELLS (3070), HLF (3131), HMGA2 (8091), Hoxb4 (15412), HOXB4 (3214), HSPC039 (51124), IRAK3 (11213), Irak3 (73914), KIAA1102 (22998), KLF5 (688), lcp1 (30583), LEF1 (51176), LMO2 (4005), lmo2 (30332), MAFB (9935), MGC15875 (85007), MRPS6 (64968), myod (30513), NOTCH2 (4853), PIM1 (5292), PRKCH (5583), Prkch (18755), RBPMS (11030), scl (also known as tal1) (30766), SHH (6469), SLC40A1 (30061), SLCO3A1 (28232), SNX5 (27131), SPARC (6678), Sparc (20692), spry4 (114437), SPRY1 (10252), SSBP2 (23635), SUZ12 (23512), ZFHX1B (9839), ZNF165 (7718), and ZNF331 (55422).
The microarray data in this manuscript have been deposited in the GEO database (http://www.ncbi.nlm.nih.gov/geo/, and have been assigned the accession number GSE2666.
We thank Brent Bill, Aubrey Nielsen, and Shridhar Sivasubbu for zebrafish injections, Greg Veltri and Joel Sederstrom for cell sorting at the University of Minnesota Flow Cytometry Facility, Dr. Leonard Zon for the gata1:DsRed Tg zebrafish line, and Drs. Stephanie Salesse and David Largaespada for critical review of the manuscript. This research was supported by Medical Scientist Training Program (GM008244) and National Cancer Institute (NCI) (CA009138) training grants to CEE, a MinnCResT training fellowship to MAP sponsored by the National Institute of Dental and Craniofacial Research (DE07288), NCI Program Project Grant (CA065493) to CMV, and National Institute of General Medical Sciences R01 (GM63904) to SCE.
Competing interests. University of Minnesota and Stephen C. Ekker are cofounders and shareholders of a small biotechnology company called Discovery Genomics Inc. that has the exclusive license for the commercial use of morpholinos in zebrafish. Dr. Ekker is a consultant to this company.
Author contributions. CEE, EMM, SCE, and CMV conceived and designed the experiments. CEE, EMM, CMF, and TFW performed the experiments. CEE, EMM, CMF, TFW, SMG, SCE, and CMV analyzed the data. MAP and SMG contributed reagents/materials/analysis tools. CEE, EMM, SCE, and CMV wrote the paper.
Citation: Eckfeldt CE, Mendenhall EM, Flynn CM, Wang TF, Pickart MA, et al. (2005) Functional analysis of human hematopoietic stem cell gene expression using zebrafish. PLoS Biol 3(8): e254.
Abbreviations
BMbone marrow
EGFPenhanced green fluorescent protein
FGFfibroblast growth factor
HPChematopoietic progenitor cell
hpfhours postfertilization
HSChematopoietic stem cell
ICMintermediate cell mass
MHCmajor histocompatibility complex
ML–ICmyeloid–lymphoid initiating cell
MOmorpholino antisense oligonucleotide
Q-RT-PCRquantitative RT-PCR
Rhorhodamine
SCIDsevere combined immunodeficiency
TFtranscription factor
Tgtransgenic
UCBumbilical cord blood
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| 16089502 | PMC1166352 | CC BY | 2021-01-05 08:21:25 | no | PLoS Biol. 2005 Aug 5; 3(8):e254 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030254 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030278SynopsisBioinformatics/Computational BiologyGenetics/Genomics/Gene TherapyInfectious DiseasesMicrobiologyEubacteriaHomo (Human)Using the Genomic Shortcut to Predict Bacterial Behavior Synopsis8 2005 5 7 2005 5 7 2005 3 8 e278Copyright: © 2005 Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
The Genome Sequence of Rickettsia felis Identifies the First Putative Conjugative Plasmid in an Obligate Intracellular Parasite
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While many infectious bacteria remain outside human cells as they do their damage, others, including the various species of Rickettsia, take up residence inside. This makes them especially hard to study in vivo. In this issue, Hiroyuki Ogata and colleagues show that important clues to bacterial phenotype and pathogenicity can be learned about such an intracellular pathogen by sequencing and analyzing its genome.
The bacterium they studied was R. felis, which is carried by fleas and infects cats, dogs, and even humans. Its close relatives include species that cause the deadly human diseases of typhus and Rocky Mountain spotted fever. The researchers found that the R. felis genome includes not only the expected large circular chromosome, but also two small circular plasmids, bits of DNA carrying relatively few genes that are often transferred from bacterium to bacterium. This is the first species of Rickettsia in which plasmids have been found. While Ogata et al. have not yet proved that R. felis plasmids are exchanged between bacterial cells, the larger of the two plasmids contains several genes known to facilitate this type of exchange, called conjugation.
The bacterial chromosome itself appears to code for about 1,500 proteins, the largest so far of the sequenced Rickettsia genomes. More than 500 of these appear to be unique to R. felis. Among these are a large number of transposases, enzymes that cut and paste chromosomal DNA, whose existence correlates with the large amount of repeated DNA in the genome (about 5% of the total), and the finding that the R. felis chromosome has been rearranged many times. Indeed, the chromosome bears traces of multiple types of mobile gene elements, along with gene transfers back and forth between the chromosome and the plasmids, and acquisition of genes from other, non-rickettsial, bacteria.
The authors also scanned the genome for clues to the behavior of the bacterial cell. They found genes that in other bacteria code for pili, hair-like protrusions from the cell membrane. This clue prompted them to look for pili with electron microscopy, and they found them—two types, in fact, which appear to play roles in conjugation and cell adhesion. They also found a gene that induces polymerization of actin filaments in the host cell, suggesting that R. felis uses the host cytoskeleton to get around inside the cell, as do other Rickettsia species.
The common flea can carry Rickettsia felis bacteria in its cells
The genomic shortcut to predicting bacterial behavior may have applications in other intracellular species both in Rickettsia and beyond. Perhaps even more importantly, the discovery of plasmids in R. felis may provide a key tool for study of other Rickettsia species. Plasmids can be modified to carry other genes, and as such may offer a route for examining the biology of the more pathogenic species, including those that cause typhus, a widespread and often deadly disease.
| 0 | PMC1166353 | CC BY | 2021-01-05 08:21:24 | no | PLoS Biol. 2005 Aug 5; 3(8):e278 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030278 | oa_comm |
==== Front
PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030279SynopsisCell BiologyDevelopmentGenetics/Genomics/Gene TherapyHematologyDanio (Zebrafish)Homo (Human)Fish and Chips: A Fast Track to Understanding Blood Development Synopsis8 2005 5 7 2005 5 7 2005 3 8 e279Copyright: © 2005 Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Functional Analysis of Human Hematopoietic Stem Cell Gene Expression Using Zebrafish
==== Body
Each day, the bone marrow of an adult makes upwards of 200 billion new red blood cells, along with lesser numbers of white blood cells and platelets. This process, called hematopoiesis, depends on hematopoietic stem cells (HSCs), which divide to make both more stem cells and progenitor cells that differentiate into all the cell types of the blood. The genetic controls on this process are poorly understood. In this issue, Catherine Verfaillie and colleagues show how a two-stage analysis, generation of transcript microarrays followed by functional validation in zebrafish, can identify key regulators of the hematopoietic process.
The study of human hematopoiesis has been hampered in part because it's not possible to use surface markers to identify and isolate HSCs, a technique used to purify other cell types. The authors used a strategy they had previously developed to isolate HSCs from bone marrow and umbilical cord blood that produces a yield up to 10-fold greater than standard protocols for purifying human HSC. Gene expression in this HSC-enriched cell population was then compared with that in an HSC-depleted population using transcript microarrays (“RNA chips”) to identify those genes whose expression was most different between the two groups of cells. They identified 277 genes whose expression in both marrow and cord blood significantly different between the HSC-enriched and -depleted populations.
Using a zebrafish microarray, researchers discovered which human genes are active during hematopoiesis, the formation of blood cells
Of these 277 genes, Verfaillie and colleagues identified 61 whose functions were not already known and which had close matches in the zebrafish, a small fish in which hematopoiesis follows essentially the same path as in humans. To prevent expression of these 61genes, they designed complementary antisense molecules against them, and injected them into zebrafish embryos. In 14 of the 61 genes, knocking down expression led to observable defects in hematopoiesis.
The authors note that three of these 14 genes are involved in signaling of fibroblast growth factor, a powerful regulator of development, suggesting that fibroblast growth factor may play a central role in hematopoiesis. More generally, they believe that the combination of using gene transcript microarrays to identify candidates and producing antisense molecules in zebrafish for functional screening of these candidates offers a way to quickly identify genes with central roles in vertebrate development.
| 0 | PMC1166354 | CC BY | 2021-01-05 08:21:24 | no | PLoS Biol. 2005 Aug 5; 3(8):e279 | utf-8 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030279 | oa_comm |
==== Front
PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0030279SynopsisCell BiologyDevelopmentGenetics/Genomics/Gene TherapyHematologyDanio (Zebrafish)Homo (Human)Fish and Chips: A Fast Track to Understanding Blood Development Synopsis8 2005 5 7 2005 5 7 2005 3 8 e279Copyright: © 2005 Public Library of Science.2005This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Functional Analysis of Human Hematopoietic Stem Cell Gene Expression Using Zebrafish
==== Body
Each day, the bone marrow of an adult makes upwards of 200 billion new red blood cells, along with lesser numbers of white blood cells and platelets. This process, called hematopoiesis, depends on hematopoietic stem cells (HSCs), which divide to make both more stem cells and progenitor cells that differentiate into all the cell types of the blood. The genetic controls on this process are poorly understood. In this issue, Catherine Verfaillie and colleagues show how a two-stage analysis, generation of transcript microarrays followed by functional validation in zebrafish, can identify key regulators of the hematopoietic process.
The study of human hematopoiesis has been hampered in part because it's not possible to use surface markers to identify and isolate HSCs, a technique used to purify other cell types. The authors used a strategy they had previously developed to isolate HSCs from bone marrow and umbilical cord blood that produces a yield up to 10-fold greater than standard protocols for purifying human HSC. Gene expression in this HSC-enriched cell population was then compared with that in an HSC-depleted population using transcript microarrays (“RNA chips”) to identify those genes whose expression was most different between the two groups of cells. They identified 277 genes whose expression in both marrow and cord blood significantly different between the HSC-enriched and -depleted populations.
Using a zebrafish microarray, researchers discovered which human genes are active during hematopoiesis, the formation of blood cells
Of these 277 genes, Verfaillie and colleagues identified 61 whose functions were not already known and which had close matches in the zebrafish, a small fish in which hematopoiesis follows essentially the same path as in humans. To prevent expression of these 61genes, they designed complementary antisense molecules against them, and injected them into zebrafish embryos. In 14 of the 61 genes, knocking down expression led to observable defects in hematopoiesis.
The authors note that three of these 14 genes are involved in signaling of fibroblast growth factor, a powerful regulator of development, suggesting that fibroblast growth factor may play a central role in hematopoiesis. More generally, they believe that the combination of using gene transcript microarrays to identify candidates and producing antisense molecules in zebrafish for functional screening of these candidates offers a way to quickly identify genes with central roles in vertebrate development.
| 0 | PMC1166355 | CC BY | 2021-01-05 08:21:24 | no | PLoS Biol. 2005 Aug 5; 3(8):e281 | latin-1 | PLoS Biol | 2,005 | 10.1371/journal.pbio.0030281 | oa_comm |
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BMC BiochemBMC Biochemistry1471-2091BioMed Central London 1471-2091-6-111592978810.1186/1471-2091-6-11Research ArticleDetermination of thermodynamic parameters of Xerocomus chrysenteron lectin interactions with N-acetylgalactosamine and Thomsen-Friedenreich antigen by isothermal titration calorimetry Damian Luminita [email protected] Didier [email protected] Mathias [email protected] Laurent [email protected] IPBS UMR 5089, Biotechnologie des Protéines, 205 route de Narbonne, 31077 Toulouse, France2 IUB, School of Engineering and Science, D-28727 Bremen, Germany2005 1 6 2005 6 11 11 20 10 2004 1 6 2005 Copyright © 2005 Damian et al; licensee BioMed Central Ltd.2005Damian et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Lectins are carbohydrate-binding proteins which potentially bind to cell surface glycoconjugates. They are found in various organisms including fungi. A lectin from the mushroom Xerocomus chrysenteron (XCL) has been isolated recently. It shows insecticidal activity and has antiproliferative properties.
Results
As the monosaccharide binding specificity is an important determinant of lectin function, we determined the affinity of XCL for the galactose moiety. Isothermal titration calorimetry studies revealed a dissociation constant Kd of 5.2 μM for the XCL:N-acetylgalactosamine interaction at 27degreesC. Higher affinities were observed at lower temperatures and higher osmotic pressures. The dissociation constant was five hundred times higher for the disaccharide beta-D-Gal(1–3)-D-GalNAc, Thomsen-Friedenreich (TF) antigen (Kd of 0.94 μM). By using fetuin and asialofetuin in interaction with the XCL, we revealed its ability to recognize the Thomsen-Friedenreich motif on glycoproteins.
Conclusion
The XCL antiproliferative effect and the TF antigen specificity presented in this work suggest that XCL and ABL may have similar binding mechanisms. The recent structure determination of these two proteins lead us to analyse these interactions in the light of our thermodynamic data. The understanding of this type of interaction may be a useful tool for the regulation of cell proliferation.
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Background
Lectins are carbohydrate-binding proteins found in various organisms including fungi [1,2]. Despite the large amount of informations available on lectin sequence and specificity, relatively little is known about their biological significances. The abundance and the variety of carbohydrate specificities of lectins raised the interest to use them for isolation and analysis of complex carbohydrates, cell separation and studies of cell surface architecture [3]. For a long period, legume lectins were the model system of choice to study the molecular basis of carbohydrate-lectin recognition. They are easy to purify in large quantities, and they exhibit a wide variety of carbohydrate specificities despite strong sequence conservation [4].
Mushroom lectins have captured the attention of investigators on account of their antiproliferative, immunomodulatory, antitumor and cytotoxic activities, and more than 50 mushroom lectins have been reported [5]. We recently isolated a lectin from Xerocomus chrysenteron (XCL) [6]. The X ray crystal structure resolution of XCL revealed a tetrameric assembly and an unexpectedly similarity with actinoporins [7]. XCL was reported as an insecticidal protein [6] and shares antiproliferative properties against two mammalian cell lines [8]. We can also mention Agaricus bisporus lectin (ABL), another mushroom lectin well known for its reversible antiproliferative effects [9].
ABL is a member of a group of proteins, which bind the Thomsen-Friedenreich (TF) antigen selectively and with high affinity. TF antigen is represented by galactosyl β-1, 3-N-acetylgalactosamine and is common in malignant and pre-malignant epithelia [10,11]. There are three other well known dietary TF-binding lectins: jacalin from the seeds of jackfruit Artocarpus integrifolia, the peanut lectin from peanut Arachis hypogaea, and amaranth lectin from Amaranthus caudatus. These four lectins have been used in histochemistry for identification of the TF antigen in tissues [12,13].
As previously reported by Rosen and al. [14], ABL belongs to a lectin fungi family based on sequence homology and N-acetylgalactosamine and galactose affinity. At present this family contains: Agaricus bisporus lectin (ABL), Arthrobotrys oligospora lectin, Xerocomus chrysenteron lectin (XCL), Pleurotus cornucopiae lectin, Gibberella zeae lectin, Paxilus involutus lectin [15]. The sequence homology between XCL and its family members varies from 65% to 35% suggesting that all these lectins could recognize TF antigen.
Here we focus on XCL binding constants for specific sugars and quantify the underlying thermodynamic parameters of the carbohydrate-XCL lectin interactions by direct measurement of the enthalpy using isothermal titration calorimetry method. We found that XCL recognizes TF antigen with high affinity (Kd: 1 μM).
Results
Sugar – XCL interaction
Red blood cell agglutination by XCL was inhibited when galactose, lactose and N-acetylgalactosamine was added to the system but no effect was seen with glucose, fucose, fructose, sorbitol, mannose and sucrose [6]. We first performed a titration of lactose and galactose to the protein, however the low binding affinity of both sugars was below the detection limit of the method (data not shown). Subsequently, we investigated the N-acetylgalactosamine/XCL interaction. With N-acetylgalactosamine, the acetamide group on the galactose ring can bring one more hydrogen bond, which can contribute to the enthalpy of the reaction and affinity values, and then titration was possible [16].
Figure 1A shows a titration of N-acetylgalactosamine into XCL protein at 27°C, together with a least squares fit. An apparent monotonic decrease in the heat release evolves when increasing amount of ligand is added, suggesting that XCL displays only one type of binding site and the absence of allostery between the four sites present on the tetramer. The fit of the data based on the one type site model reveals a binding constant of 192 M-1 and a reaction enthalpy of – 6.27 kcal/mol when the monomer concentration was considered for the calculations. As this affinity is very small, the enthalpy was estimated separately in another experiment where small quantities of lectin were injected into a N-acetylgalactosamine containing solution. We obtained a ΔH value of – 25.20 kcal/mole corresponding to four independent binding sites (data not shown).
Figure 1 ITC profile and treatment of data of XCLtag and N-acetylgalactosamine interaction using 25 mM Na2HPO4/NaH2PO4 as buffer. A: Top: raw data obtained from 98 automatic injections (3 μl each), by titration of 50 mM N-acetylgalactosamine into 0,394 mM XCLtag solution. Bottom: the integrated curve showing the experimental points (■) and the best fit (-) B: Comparison of titrations realized at two different temperatures. Red scatter (): 10°C data titration; black scatter (■): 27°C data titration.
Binding of carbohydrates to a number of proteins is characterised by small enthalpy and heat capacity changes. Hydrogen bonding interactions are essentially enthalpically driven with little change in the heat capacity, while hydrophobic interactions are essentially entropically driven [17]. Measurements performed at 10°C using the same titration conditions indicate that the enthalpy of binding of N-acetylgalactosamine does not vary significantly with temperature and small changes in the heat capacity are observed. The fit of the data with one set of site model (figure 1B, red spectra) gave an affinity value of 362 M-1 and no important change of the reaction enthalpy was observed (- 6.25 kcal/mole). In many cases, binding of saccharides to lectins is coupled to changes in solvent accessibility that result in negative, albeit small, ΔCp values [16]. This is also the case of XCL – N-acetylgalactosamine interaction.
Variation of osmotic stress allows to measure the energetic contribution of the solvatation effect on the enthalpy of the reaction [18]. The water activity was reduced by adding 10 % (w/v) PEG 8000 to the system. An increase in the binding constant value (280 M-1) and a reduction of the binding enthalpy (- 5.82 kcal/mole) were observed. Thermodynamics parameters, which characterize the XCL – N-acetylgalactosamine interaction are summarised in Table 1. The ΔG values and the deduced binding constants are higher at low temperature or under osmotic stress.
Table 1 Thermodynamic values, which characterize the interaction of XCL with N-acetylgalactosamine
Ka, M-1 -ΔH, kcal/mole -ΔG, kcal/mole -ΔS, cal/moleK Nr exp.
27°C 192 ± 5 6.27 ± 0.10 3.122 10.5 2
27°C, 10% PEG 280 ± 4 5.82 ± 0.05 3.347 8.2 4
10°C 362 ± 3 6.25 ± 0.03 3.301 10.4 2
The N-acetyllactosamine was also used as a ligand and the affinity constant at 27°C is in the range of 50 M-1 but with significant errors. These errors are due to an uncertainty in fitting the data at Ka values of smaller than 100 M-1.
We especially checked the XCL interaction for TF antigen, β-D-Gal(1–3)-D-GalNAc, since ABL was previously shown to bind this disaccharide with a high affinity constant. A ΔH value of -9.13 kcal/mole and an affinity constant of 1.06 105 M-1 were obtained (fig. 2 and table 2). This value is 500 fold higher than the affinity constant determined for N-acetylgalactosamine interaction.
Figure 2 Binding isotherms acquired by titration of 0.95 mM β-D-Gal(1→3)-D-GalNAc into 0,14 mM XCLtag solution at 27°C, using 25 mM Na2HPO4/NaH2PO4 as buffer.
Table 2 Thermodynamic values of binding with Thomsen-Friedenreich antigen (TF), fetuin and asialofetuin
Ka, 105 M-1 -ΔH, kcal/mole -ΔG, kcal/mole -ΔS, cal/moleK Nr exp.
TF 1.06 ± 0.44 9.13 ± 0.29 6.81 ± 0.27 7.61 2
Fetuin 5.9 ± 1.4 21.5 ± 0.5 7.89 45.35 3
Asialofetuin 25.9 ± 0.6 16.8 ± 2.5 8.84 26.51 2
Glycoproteins – XCL interaction
Fetuin and asialofetuin, which bear the TF antigen motif, were used to test XCL interactions with glycoproteins. Fetuin contains six oligosaccharides chains, namely three carbohydrate units O-linked to Thr or Ser residues and three complex glycans, N-linked to Asn residues [19]. A fourth O-linked residue may exist in the fetuin structure [20]. In fetuin, the exposed Gal residues of both O-linked and N-linked saccharides are linked to sialic acid residues, which are absent in asialofetuin.
Several titrations of fetuin and asialofetuin to a XCL containing solution (see concentrations in material and methods) were performed at 27°C. The binding isotherms for the titration of fetuin and asialofetuin into a XCL solution are presented in figure 3A and 3B respectively and the thermodynamic data are presented in Table 2. Affinity constant for asialofetuin (2.59 106 M-1) was found 4 times higher than for fetuin (5.9 105 M-1). This suggests that XCL binds asialofetuin more avidly than the native fetuin, and therefore that the presence of sialic acid reduces the affinity of XCL towards such glycans. The binding stoichiometry is of 0.23, which could correspond to 4 similar binding sites either on fetuin or asialofetuin. The binding enthalpy of XCL – fetuin/asialofetuin is of -21.5 kcal/mole and -16.8 kcal/mole respectively. This significant difference in the binding enthalpies of almost 5 kcal/mole leads us to conclude that in fetuin the sialic acids do contribute to the energy of binding.
Figure 3 Binding isotherms corresponding to the titration of 38 automatic injections (5 μl each) of 2 mM fetuin (A) or 2 mM asialofetuin (B) into 0,25 mM XCLtag solution at 27°C, using 25 mM Na2HPO4/NaH2PO4 as buffer.
Discussion
As we mentioned in the introduction, there are several TF-binding lectins. Althought they recognize the same motif, they have different actions on the proliferation phenomenon [21]. For example, PNA stimulates the proliferation of human intestinal epithelial cells [22] while ABL is a potent inhibitor of proliferation [9]. The fact that XCL shows a dose-dependent inhibition of proliferation [8] suggests that its effects could be mediated by glycoproteins bearing TF antigen. We first check the binding of XCL with free TF antigen. Our results lead us to suggest that water molecules involved in the sugar-lectin binding may contribute to the energy of the reaction. This is in agreement with the Chevernak and Toone work since the amount of heat liberated on the binding of ligands with a variety of proteins was significantly smaller (0.4 – 1.8 kcal/mol) when heavy water was used like solvent [18]. In the case of XCL, the affinity enhancement observed when the galactose is linked to the N-acetylgalactosamine suggests the existence of an extended binding site [23]. An increase in the binding enthalpy is also observed when disaccharides replace monosaccharides in XCL-sugar complexes. This increase correlates with the addition of direct hydrogen bonds and more extensive van der Waals [24] interactions between the protein and the ligand. Sugar binding site determined on ABL by RX crystallography shows that water molecules are involved in this interaction as we hypothesised [25].
On cell-surface glycoproteins, the epitope structure of TF antigen is α-linked to either serines or threonines [26]. The affinity constants of XCL obtained for fetuin and asialofetuin are higher than for free TF antigen. This difference could be explained by an implication of several residues of the glycoprotein in the interaction with the lectin. Nevertheless, residues potentially involved in this interaction are not serine or threonine linking TF antigen [25]. Then it would be interesting to investigate the implication of the spatially surrounding residues in this interaction.
Conclusion
At present, only limited informations on the thermodynamics datas of the lectin-sugar recognition are available and much work remains to be done to understand the underlying forces that govern these interactions. In this study, we investigate the specificity of XCL for carbohydrates and especially for Thomsen-Friedenreich antigen and glycoproteins bearing this disaccharide. Kinetic studies using a resonant mirror biosensor reported a binding affinity value of 3.3 106 M-1 for the asialofetuin-ABL interaction [27] which is very close to that of the asialofetuin-XCL interaction (2.59 106M-1). The XCL antiproliferative effect [8] and the TF antigen specificity presented in this work suggest that XCL and ABL may have similar binding mechanisms. The recent structure determination of XCL and ABL lead us to currently analyse these interactions in the light of our thermodynamic data.
Methods
Materials
All products, mono- and di-saccharides, fetuin and asialofetuin from fetal bovine serum were purchased from sigma.
XCL expression and purification
A fusion protein containing histidine tag, TEV site and XCL was expressed in E. coli BL21-DE3 strain. The histidine tag was added to facilitate the purification of the recombinant protein on an affinity column using nickel as ligand [28] and the TEV site was added to eliminate the tag by incubation with TEV protease [29]. Freshly transformed BL21(DE3) cells were grown overnight in a NZY/agar – kanamycin medium at 37°C. Colonies of bacteria were grown in an NZY medium at 37°C. When an O.D.600 nm of 1 was reached, the induction of T7 RNA polymerase with IPTG (final concentration 0.4 mM) was realized. Then the culture medium was allowed to grow overnight, at 16°C. Cells were harvested by centrifugation, washed and then lysed by sonication. Isolated XCL was purified by affinity chromatography on Ni-NTA column and dialyse methods. Protein purity was assessed using overloaded SDS-PAGE gels with Coomassie blue staining. XCL concentrations were determined spectrophotometrically from molar extinction coefficients at λ = 280 nm, ε = 31150.
Isothermal titration calorimetry, ITC
Isothermal titration calorimetry was performed using a VP-ITC microcalorimeter from Microcal Inc. (Northampton, MA). Several experiments were performed to determine the binding constant values. In individual titrations, injections of 3 to 10 μl of carbohydrate/glycoprotein were added by computer-controlled 296 μl microsyringe at an interval of 200 seconds into the XCL solution (cell volume = 1.437 ml). The experiments were realized at 27°/10°C and a stirring speed of 300 rmp. 10% (w/v) PEG 8000 was used for some of the experiments. As the lectin affinity for sugars is relatively small, high sugar and protein concentrations were required. The XCL concentration varied between 0.14-0.4 mM, the monosaccharide concentrations between 30–50 mM, 0.95–3 mM for TF antigen and glycoproteins concentrations between 0.66–2 mM. The carbohydrates were dissolved in the buffer solution (25 mM Na2HPO4/NaH2PO4, pH = 7) from the last protein dialysis.
Several blind titrations were performed to determine and correct for unspecific heat contributions (heat of dilution).
The experimental data were fitted to a theoretical titration curve using software supplied by Microcal, with ΔH (enthalpy change in kcal/mole), Ka (association constant in M-1) and n (number of binding sites), as adjustable parameters. The monomer concentration was used throughout the analysis. The instrument was calibrated using the built in mode of electric field heat pulses. Thermodynamic parameters were calculated from the relation:
ΔG = ΔH - TΔS = - RT ln Ka
where the ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding; T is the absolute temperature and R = 1.98 cal mol-1K-1.
Authors' contributions
LD carried out the protein preparation and microcalorimetry work and drafted the manuscript. DF participated in the design of the study, MW participated in the microcalorimetry experiment and interpretations. LP conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by the EU project Nanocapsules with functionalized surfaces and walls HPRN-CT-2000-00159.
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| 15929788 | PMC1166539 | CC BY | 2021-01-04 16:36:49 | no | BMC Biochem. 2005 Jun 1; 6:11 | utf-8 | BMC Biochem | 2,005 | 10.1186/1471-2091-6-11 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-1231591068410.1186/1471-2105-6-123Research ArticleGenome comparison without alignment using shortest unique substrings Haubold Bernhard [email protected] Nora [email protected]öller Friedrich [email protected] Thomas [email protected] Department of Biotechnology & Bioinformatics, University of Applied Sciences, Weihenstephan, Germany2 Institute of Genetics, Universität zu Köln, Zülpicher Straße 47, 50674 Köln, Germany3 Berlin Center for Genome Based Bioinformatics and Freie Universität, Berlin, Germany2005 23 5 2005 6 123 123 9 11 2004 23 5 2005 Copyright © 2005 Haubold et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Sequence comparison by alignment is a fundamental tool of molecular biology. In this paper we show how a number of sequence comparison tasks, including the detection of unique genomic regions, can be accomplished efficiently without an alignment step. Our procedure for nucleotide sequence comparison is based on shortest unique substrings. These are substrings which occur only once within the sequence or set of sequences analysed and which cannot be further reduced in length without losing the property of uniqueness. Such substrings can be detected using generalized suffix trees.
Results
We find that the shortest unique substrings in Caenorhabditis elegans, human and mouse are no longer than 11 bp in the autosomes of these organisms. In mouse and human these unique substrings are significantly clustered in upstream regions of known genes. Moreover, the probability of finding such short unique substrings in the genomes of human or mouse by chance is extremely small. We derive an analytical expression for the null distribution of shortest unique substrings, given the GC-content of the query sequences. Furthermore, we apply our method to rapidly detect unique genomic regions in the genome of Staphylococcus aureus strain MSSA476 compared to four other staphylococcal genomes.
Conclusion
We combine a method to rapidly search for shortest unique substrings in DNA sequences and a derivation of their null distribution. We show that unique regions in an arbitrary sample of genomes can be efficiently detected with this method. The corresponding programs shustring (SHortest Unique subSTRING) and shulen are written in C and available at .
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Background
Sequence comparison is traditionally carried out using alignments. The alignment procedure ensures that only homologous positions are compared and corresponding algorithms form the classical core of bioinformatics [1-3]. Once a sequence alignment has been computed, it can be used to determine, for example, signature oligonucleotides or unique genomic regions among a group of closely related organisms.
Perhaps surprisingly, the applications of alignments just mentioned – signature oligos and detection of unique genomic regions – do not necessarily involve an alignment step. Since the computation of alignments tends to take time proportional to the product of the lengths of the sampled sequences, elimination of this step often leads to dramatic increases in the speed of sequence analysis algorithms [4].
Our method of alignment-free sequence comparison is based on the idea of "shortest unique substrings", that is, the shortest substrings of a sequence which are not found elsewhere. Consider for example the sequence S = ACCG. It contains substrings, of which the following eight are unique: {A, AC, ACC, ACCG, CC, CCG, CG, G}. Two of these are shortest unique substrings: {A, G}. Such global shortest unique substrings can occur anywhere in S. In contrast, we define local shortest unique substrings to be tied to a specific position in S. More formally, we determine for every position i in S the length x of the substring S[i..i + x - 1] such that it is unique while S[i..i + x - 2] is not. In the case of our example string, the result is x = 1 for the first position, x = 2 for the second, x = 2 for the third, and x = 1 for the last. Figure 1A gives a graphical representation of these local shortest unique substrings.
So far we have only considered the forward strand of a given DNA sequence. In the presence of the reverse strand, the set of x-values changes to x = 1 for the first and x = 2 for the second position. No well-defined unique substrings start at the third or the last position of the sequence. Figure 1B illustrates the shortest unique substrings found on the forward and reverse strands of our example sequence. It is important to realize that when dealing with double-stranded DNA, the set of shortest unique substrings is different from that found in single-stranded DNA. However, complementarity of DNA ensures that the complement of a local shortest unique substring is again a unique substring, though not necessarily a shortest unique substring (cf. Figure 1B). In contrast, the complement of a global shortest unique substring is also a global shortest unique substring.
Application of shortest unique substrings to biological problems requires both their efficient detection and knowledge of their probability distribution. The latter is derived in this paper. As to the detection of shortest unique substrings, a data structure known as the suffix tree can readily be used for this purpose [4]. We demonstrate the utility of shortest unique substrings for sequence analysis by applying them to three tasks: (i) identification of signature oligo nucleotide sequences, (ii) detection of unique as well as repeat regions in the genome of Mycoplasma genitalium, and (iii) detection of unique genomic regions in strain MSSA476 of the human pathogen Staphylococcus aureus when compared to four other staphylococcal genomes.
Results
Global shortest unique substrings in Caenorhabditis elegans, human, and mouse
The genome of C. elegans is one of the smallest metazoan genomes sequenced to date. It consists of five autosomes and one sex chromosome, amounting to 100.29 Mb of sequence information [5]. When searching for global shortest unique substrings in this genome, we found a single complementary pair of unique motifs of length 10 located on chromosome 1. Considering the next shortest unique substrings (length 11) we found a total of 10,509 such motif pairs distributed among the five chromosomes. Note that the search for these globally unique substrings was done with respect to the forward and backward strands of the complete genome.
We repeated this analysis for the human genome, which is the largest genome sequenced to date. It consists of 22 autosomes and two sex chromosomes totalling 2.84 Gb published sequence data [6]. We found 215 pairs of global shortest unique substrings of length 11 distributed on the autosomes and the X-chromosome. The Y chromosome contained no unique sequences of length 11 but 135 globally unique sequence pairs of length 12.
We were puzzled by the fact that – with the exception of the single instance of a unique substring pair of length 10 on chromosome 1 of C. elegans – the shortest unique substrings in humans had the same length (11) as those found in C. elegans, even though the human genome is 28 times larger than that of the nematode. When repeating the search for global shortest unique substrings in the mouse genome, whose size is similar to that of human (2.49 Gb), we found a matching result: there were 255 shortest unique substring pairs of length 11 distributed among the autosomes and the X-chromosome. On the Y-chromosome there were 38 unique substring pairs of length 12. The fact that the highly repetitive Y chromosome contained global unique substrings of length 12 as compared to length 11 on autosomes suggested that the length of shortest unique substrings is inversely related to genome information content. In order to further explore whether particularly short unique substrings are associated with functional regions of the genome, we investigated the distribution of globally shortest unique substrings among 1 kb upstream regions of annotated genes.
A total of 29 out of 2 × 350 shortest unique substrings were located among a non-redundant set of 16,286 human 1 kb upstream regions. The probability of observing a single hit in an upstream region with one shortest unique substring is equal to the fraction of the published genome (considering again forward and backward strands) covered by upstream regions. This is
fh = 16286 × 1000/(2 × 2.84 × 109) ≈ 0.003.
The probability of observing 29 or more hits to upstream regions under the null hypothesis of equal distribution is
A similar result was obtained for the mouse genome. Here a total of 13,985 non-redundant upstream regions contained 22 of the 2 × 293 shortest unique substrings. The probability of finding a single hit with one shortest unique substring is again equal to the fraction of the published genome covered by the upstream regions:
fm = 13985 × 1000/(2 × 2.49 × 109) ≈ 0.003.
The probability of observing 22 or more hits to upstream regions by chance is
In other words, both in the human as well as in the mouse genome shortest unique substrings are clustered close to genes. A complete list of the shortest unique substrings with hits to upstream regions is available as supplementary material (see Additional file 1).
So far we have concentrated on the overall, i.e. global, shortest unique substrings. In the following sections we extend this analysis to include all local shortest unique substrings.
Empirical distribution of local shortest unique substring lengths
The pathogenic bacterium Mycoplasma genitalium has one of the smallest genomes known for any free-living organism [11]. The 580,074 bp of its genome encode 480 open reading frames and would be expected to be void of redundant, that is repetitive, sequences. However, when plotting the length of all local shortest unique substrings contained in its genome, we detected 26 non-overlapping shortest unique sequences longer than 100 bp, the longest of which spanned 244 bp (Figure 2A). In other words, the genome of M. genitalium contains a perfectly conserved repeat sequence that is 244 - 1 = 243 bp long. The statistical significance of these repeats is illustrated in Figure 2B, which displays the lengths of the shortest unique substrings in a shuffled version of M. genitalium's genome. In such a scrambled sequence devoid of biological meaning no shortest unique substring is longer than 21 bp. Having found surprisingly short, as well as surprisingly long shortest unique substrings, we proceed by deriving the null distribution of shortest unique substring lengths.
Distribution of local shortest unique substring lengths
As explained further in the Methods section, the probability of finding shortest unique substrings of length x, , is the number of unique substrings of length x, , minus the number of unique substrings of length x - 1, , divided by the genome length l:
where
and 2p represents the GC-content of the genome (p ∈ [0, 1/2]). Figure 3 demonstrates that the fit between equation (1) and the empirical distribution of local shortest unique substrings (cf. Figure 2B) is excellent. Equation (1) provides an efficient method for establishing the statistical significance of any given length of a local shortest unique substring. Using equation (1) and knowing that the GC-content of the genome of C. elegans is 0.3544, one expects to find by chance alone 1.6 unique substrings of length and 10 and 20,441 unique substrings of length 11. These values agree with what we have found in the actual genome of C. elegans (one pair of unique substrings of length 10, and 10,509 pairs of length 11). However, again based on equation (1), the probability of finding in the human genome (GC-content = 0.4088) a unique substring of length 11 is less than 10-100. This is equivalent to an expected number of only 2.4 × 10-94 of such unique substrings and in sharp contrast to the observed value of 215 pairs of unique substrings of this length. The same holds for mouse. Clearly, the lengths of the shortest unique substrings found in mouse and human cannot be explained by chance.
In addition to quantifying the probability of finding shortest unique substrings, equation (1) also allows us to estimate the lengths of unique oligos for arbitrary genomes. In the case of the human genome the length distribution is shown in Figure 4. Notice that this distribution is strongly skewed to the right with 30 being the highest length with an expected occupancy of ≥ 1.
Since equation (1) describes the length distribution of shortest unique substrings in random sequences, it is also the null-distribution for multiple, but phylogenetically unrelated, sequences. This fact can be exploited for comparative genome analysis.
Comparing five strains of Staphylococcus aureus
Staphylococcus aureus is a human pathogen notorious for its resistance to multiple antibiotics [7]. Five genomes of this bacterium are publicly available, which makes it one of the best characterized bacterial species. Our aim was to take strain MSSA476 [7] and identify the regions unique to its genome when compared to the four other strains available. Given the GC-content of Staphylococcus aureus and the combined length of the five genomes analyzed, we calculated the expected distribution of shortest unique substring lengths using equation (1). Figure 5 shows that for unrelated S. aureus sequences of the same combined length (14.21 × 106) and composition (GC-content 0.33) as the strains investigated, the lengths of shortest unique substrings are expected to range from 9 to 27. We decided to analyze the local shortest unique substrings of length ≤ 10 in strain MSSA476 in the presence of the genomes of the four other strains. Figure 6 displays the cumulative distribution of the local shortest unique substrings of length ≤ 10 along the genome of strain MSSA476. Regions unique to MSSA476 contain a high density of such substrings and hence stand out as jumps in the cumulative plot. Figure 6 shows two such jumps. These correspond exactly to the two unique regions ΦSa4 and SCC476 recently annotated as the only two unique regions in MSSA476 [7].
Discussion
The search for unique substrings has a long tradition in molecular biology. It is fundamental for many sequence-based identification techniques, and affects PCR primer design as well as the development of specific antibodies. A DNA or protein sequence of length n contains substrings, which is also an upper limit for the number of unique substrings. This means that in most real world situations there is in an excess of unique substrings to choose from. Since a given unique substring remains unique upon extension, we decided to concentrate on the shortest unique substrings. In their global version they have minimal length with respect to the entire sample of sequences investigated. In contrast, their local version is defined for substrings starting from a specific position in the genome. There are of the order of n such local shortest unique substrings from which all the remaining unique substrings can be generated. Shortest unique substrings therefore lead to considerable space reduction when dealing with unique substrings.
Our technique for detecting such unique substrings is applicable to protein as well as to DNA sequence data. Antibodies are widely used in basic biomedical research; in addition, there is growing interest in applying them as therapeutics. A major design goal in generating antibodies to a given protein in all of these contexts is to maximize their specificity. Since the entire proteome of important biomedical model organisms, including human, is known, epitope selection might be guided not only by considerations of antigenicity, but also of uniqueness. In a preliminary study of the human proteome we found that 88% of the 27,175 human proteins we looked at contain at least one unique hexapeptide. Given that a typical epitope consists of 6 to 12 amino acids, this suggests that our method of detecting shortest unique substrings coupled with epitope prediction programs might also be useful for antibody development.
However, in this paper we have concentrated on shortest unique substrings in genomes. The fact that the length of global shortest unique substrings does not exceed 11 in autosomes of both C. elegans and humans is intriguing given the widely differing sizes of the two genomes and the extremely small probability of observing unique sequences of length 11 by chance in the human genome. Since the length of global shortest unique substrings remained constant after we had removed repetitive elements from the genomes, we take this as an indication that genomes contain a core of high-complexity sequences which determine the length of global shortest unique substrings. The size of this high-complexity core is apparently much less variable across metazoan genomes than raw genome size, hence the observed constancy of global shortest unique substring lengths.
These global shortest unique substrings can be used as starting points for developing signature oligos. Such oligos are widely used in biotechnology and taxonomy. A typical application in biotechnology is PCR-primers that should be unique to the target sequence. In taxonomy a recent initiative for the "Barcoding of Life" attempts to "label" all extant species by assigning a short unique DNA-sequence to them.
The probability of finding a shortest unique substring of some length can be readily computed using equation (1). However, this equation is highly sensitive to the value of the parameter p, which describes the sequence composition. Hence, local variation in sequence composition will strongly affect the expected length of both local and global shortest unique substrings. This fact may also have contributed to our observation that shortest unique substrings cluster in upstream regions of genes in both the mouse and the human genomes. The euchromatic part of the human genome has an average GC-content of 0.41 (, Human genome assembly hg16), which is similar to the value of 0.42 for the mouse (, Mouse genome assembly mm4). In contrast, the global shortest unique substrings we found in humans have a GC-content of 0.59 and those found in mouse have a GC-content of 0.61. The upstream regions of genes tend to be GC-rich in human (GC-content = 0.53) as well as mouse (GC-content = 0.50), which might account for the clustering of global shortest unique substrings in these regions.
Detection of unique genomic regions is traditionally done by alignment-based approaches. However, the run-time of these algorithms depends non-linearly on the number and lengths of the input sequences and also on the degree of relatedness of the input sequences. In contrast, the scheme for detecting unique genomic regions proposed in this paper has a run time that is strictly linear in the combined lengths of the input sequences.
Conclusion
There is currently a lot of interest in comparative genomics [8]. In many of these projects detection of regions unique to a genome is one of the first steps towards functional annotation (e. g. [7]). Given equation (1), the size distribution of shortest unique substrings in random, i.e. unrelated, sequences can be predicted. This leads to our method of detecting unique genomic regions from an arbitrary set of input sequences without the need for alignment. Its usefulness for comparative genomics is clearly demonstrated in the case of the genomes of S. aureus, where we could rapidly detect the two unique regions previously annotated in one strain [7] (Figure 5).
Methods
Detection of shortest unique substrings
Two methods borrowed from computer science were used for the detection of shortest unique substrings: suffix tree construction and hashing. Suffix trees are well described by Gusfield [4] and we follow his nomenclature. To use suffix trees for detecting unique substrings, notice that the path label of any leaf is a unique substring. The set of shortest unique substrings at every position can therefore be discovered by traversing the tree once and looking up the string depth of the parent node of every leaf. This value plus one is the desired length of the shortest unique string that starts at the position indicated by the leaf.
Hashing is described, for example, by Cormen et al. [[9], ch. [11]] and we used it for detecting global shortest unique substrings in large genomes.
Unless stated otherwise, all computations presented in this paper consider both strands of the DNA sequences concerned. Note that in this case, and due to complementarity of DNA, a single parameter (p) suffices to describe nucleotide composition.
The probability distribution of local shortest unique substring lengths in nucleotide sequences
Consider a nucleotide sequence S and let 2p denote the GC-content of S (p ∈ [0, 1/2]). A shortest unique substring of length x of this nucleotide sequence is defined as a unique substring S[i..i + x - 1] where S[i..i + x - 2] is not unique. We wish to derive the probability distribution of values of x under the assumption of random sequence composition.
We start by considering a particular substring of length x consisting of k positions occupied by either G or C each. We refer to such a substring as being of type (x, k). The probability of finding a substring of type (x, k) is
Px,k = (1/2 - p)x - k pk.
Assuming that l independent trials each having a success probability of Px,k are performed, the probability of finding a particular sequence of type (x, k) exactly once is then
This expression is only approximately valid, since the nucleotide compositions of any two overlapping subtrings are not independent. Still, from now on we assume independence. The error introduced by this assumption is negligible, if the genome size, l, is large compared to the length of the considered substrings (l >> x) - which is the case we have in mind. Thus, we replace the ≈-sign in the above and following expressions by = and define
For each sequence of type (x, k), there are permutations of k "G|C" s and (x - k) "A|T" s. Some of these permutations occur zero times in S, some occur multiple times and some occur exactly once. We are interested in the latter: the number of unique substrings of type (x, k) is
In order to determine the number of unique substrings irrespective of their sequence composition, we need to sum over all possible values of k:
The number of shortest unique substrings of length x, , is then simply the number of unique substrings of length x minus the number of unique substrings of length x - 1. In order to see this, notice that all unique substrings of length x - 1 are contained in the set of unique substrings of length x. Those that are gained by adding the extra nucleotide are precisely the substrings that lose their uniqueness when reduced in length by one as required by the definition of shortest unique substring:
Finally, the probability of finding such a shortest unique substring of length x, , is the number of unique shortest substrings of length x divided by the genome length:
Implementation
The search for shortest unique substrings is implemented in our program shustring (SHortest Unique subSTRING). The distribution of shortest unique substring lengths in genomic sequences as embodied in equation (1) is implemented in our program shulen. Both pieces of software are available from .
Data
Genome sequences of the nematode (Caenorhabditis elegans) [5], mouse (Mus musculus) [10], and human (Homo sapiens) [6] as well as 1 kb upstream regions for genes in the genomes of human and mouse were obtained from the University of California Santa Cruz genome website at the following URLs:
1. nematode: (version ce2)
2. mouse: (version mm4, October 2003)
3. human: (version hg16, July 2003)
Table 1 lists the six bacterial genomes analyzed in this study.
Authors' contributions
B.H. designed and implemented the software, performed data analysis and contributed to the writing of the manuscript. N.P. was involved in data analysis, software testing and contributed to the writing of the manuscript. F.M. carried out the analysis of the upstream regions. T.W. conceived of the study of shortest unique substrings, derived their null distribution, and contributed to the writing of the manuscript. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Supplementary Material. List of Human and Mouse genes with hits to shortest unique substrings to their 1 kb upstream regions
Click here for file
Acknowledgements
We would like to thank C. Acquisti and A. Börsch-Haubold for stimulating discussions and two anonymous reviewers for comments which helped to improve the manuscript. Furthermore, we would like to thank the members of the High Performance Computing Group at the Leibniz Rechenzentrum München for advice on computational issues. F.M. is supported by a grant from the German Ministry of Education and Research (BMBF; Fkz. 0312705A). B.H. is supported financially by Dehner Gartencenter GmbH and the Stifterverband der Deutschen Wissenschaft.
Figures and Tables
Figure 1 Shustrings on forward and backward strands. Global and local shortest unique substrings ('shustrings') in the DNA-sequence ACCG. A: The global shustrings are A and G, and have length 1 (black numbers above the sequence). The numbers above the sequence indicate the length of the four local shustrings A, CC, CG and G present on the forward strand. B: In the presence of the reverse strand global as well as local shustrings may change. For some positions at the 3'end of the sequence shustrings may not be defined (here, position 3 and 4 on the forward strand). Notice that the complement of a local shortest unique substring is also unique, however not necessarily a shortest unique substring (for example the pair GT on the reverse strand and AC on the forward strand). The complement of a global shortest unique substring is again a global shortest unique substring (here the two global shustrings A and T).
Figure 2 Shustrings in Mycoplasma genitalium. A: Lengths of the local shortest unique substrings at every position along the genome of Mycoplasma genitalium. The lengths displayed minus one correspond to the lengths of substrings which are repeated at least once in the genome. B: The same as A, except that the nucleotides in the genome were shuffled (thus preserving nucleotide frequencies), which leads to the disappearance of long repeats.
Figure 3 Shustring probability distribution in the randomized genome of Mycoplasma genitalium. Observed and expected distributions of the lengths x of local shortest unique substrings. The "observed" distribution was obtained by shuffling the nucleotides of the genome of Mycoplasma genitalium (c. f. Figure 2), while the expected distribution is based on equation (1) using the genome's GC-content of 2p = 0.316 and length l = 580,074 bp.
Figure 4 Expected number of shustrings in the randomized human genome. Expected number, , of local shortest unique substrings of length x. Parameters used in equation (1) are l = 2.84·109 and 2p = 0.409 and correspond to the euchromatic part of the human genome.
Figure 5 Shustring probability distribution in five randomized strains of Staphylococcus aureus. Observed and expected distributions of the lengths x of local shortest unique substrings. Parameters l = 1.42·107 and 2p = 0.330 correspond to the combined length and average GC-content of five strains of Staphylococcus aureus.
Figure 6 Cumulative distribution of shustrings in Staphylococcus aureus. Cumulative distribution of unique substrings as a function of genome position in S. aureus MSSA476 when compared to strains MRSA252, MW2, Mu50, and N315 (c. f. Table 1). The steep jumps in the plot correspond to the two regions SCC476 (close to the origin) and ΦSa4 indicated in grey. These are known to be the sole two unique regions in the genome of MSSA476 [7]. Only local shortest unique substrings of length ≤ 10 were included in the analysis.
Table 1 Bacterial genomes analyzed in this study
organism accession number reference genome size
Mycoplasma genitalium L43967 [11] 580,074
Staphylococcus aureus MRSA252 NC 002952 [7] 2,902,619
Staphylococcus aureus MSSA476 NC 002953 [7] 2,799,802
Staphylococcus aureus MW2 NC 003923 [12] 2,820,462
Staphylococcus aureus Mu50 NC 002758 [13] 2,878,040
Staphylococcus aureus N315 NC 002745 [13] 2,814,816
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| 15910684 | PMC1166540 | CC BY | 2021-01-04 16:02:50 | no | BMC Bioinformatics. 2005 May 23; 6:123 | utf-8 | BMC Bioinformatics | 2,005 | 10.1186/1471-2105-6-123 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-1231591068410.1186/1471-2105-6-123Research ArticleGenome comparison without alignment using shortest unique substrings Haubold Bernhard [email protected] Nora [email protected]öller Friedrich [email protected] Thomas [email protected] Department of Biotechnology & Bioinformatics, University of Applied Sciences, Weihenstephan, Germany2 Institute of Genetics, Universität zu Köln, Zülpicher Straße 47, 50674 Köln, Germany3 Berlin Center for Genome Based Bioinformatics and Freie Universität, Berlin, Germany2005 23 5 2005 6 123 123 9 11 2004 23 5 2005 Copyright © 2005 Haubold et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Sequence comparison by alignment is a fundamental tool of molecular biology. In this paper we show how a number of sequence comparison tasks, including the detection of unique genomic regions, can be accomplished efficiently without an alignment step. Our procedure for nucleotide sequence comparison is based on shortest unique substrings. These are substrings which occur only once within the sequence or set of sequences analysed and which cannot be further reduced in length without losing the property of uniqueness. Such substrings can be detected using generalized suffix trees.
Results
We find that the shortest unique substrings in Caenorhabditis elegans, human and mouse are no longer than 11 bp in the autosomes of these organisms. In mouse and human these unique substrings are significantly clustered in upstream regions of known genes. Moreover, the probability of finding such short unique substrings in the genomes of human or mouse by chance is extremely small. We derive an analytical expression for the null distribution of shortest unique substrings, given the GC-content of the query sequences. Furthermore, we apply our method to rapidly detect unique genomic regions in the genome of Staphylococcus aureus strain MSSA476 compared to four other staphylococcal genomes.
Conclusion
We combine a method to rapidly search for shortest unique substrings in DNA sequences and a derivation of their null distribution. We show that unique regions in an arbitrary sample of genomes can be efficiently detected with this method. The corresponding programs shustring (SHortest Unique subSTRING) and shulen are written in C and available at .
==== Body
Background
Sequence comparison is traditionally carried out using alignments. The alignment procedure ensures that only homologous positions are compared and corresponding algorithms form the classical core of bioinformatics [1-3]. Once a sequence alignment has been computed, it can be used to determine, for example, signature oligonucleotides or unique genomic regions among a group of closely related organisms.
Perhaps surprisingly, the applications of alignments just mentioned – signature oligos and detection of unique genomic regions – do not necessarily involve an alignment step. Since the computation of alignments tends to take time proportional to the product of the lengths of the sampled sequences, elimination of this step often leads to dramatic increases in the speed of sequence analysis algorithms [4].
Our method of alignment-free sequence comparison is based on the idea of "shortest unique substrings", that is, the shortest substrings of a sequence which are not found elsewhere. Consider for example the sequence S = ACCG. It contains substrings, of which the following eight are unique: {A, AC, ACC, ACCG, CC, CCG, CG, G}. Two of these are shortest unique substrings: {A, G}. Such global shortest unique substrings can occur anywhere in S. In contrast, we define local shortest unique substrings to be tied to a specific position in S. More formally, we determine for every position i in S the length x of the substring S[i..i + x - 1] such that it is unique while S[i..i + x - 2] is not. In the case of our example string, the result is x = 1 for the first position, x = 2 for the second, x = 2 for the third, and x = 1 for the last. Figure 1A gives a graphical representation of these local shortest unique substrings.
So far we have only considered the forward strand of a given DNA sequence. In the presence of the reverse strand, the set of x-values changes to x = 1 for the first and x = 2 for the second position. No well-defined unique substrings start at the third or the last position of the sequence. Figure 1B illustrates the shortest unique substrings found on the forward and reverse strands of our example sequence. It is important to realize that when dealing with double-stranded DNA, the set of shortest unique substrings is different from that found in single-stranded DNA. However, complementarity of DNA ensures that the complement of a local shortest unique substring is again a unique substring, though not necessarily a shortest unique substring (cf. Figure 1B). In contrast, the complement of a global shortest unique substring is also a global shortest unique substring.
Application of shortest unique substrings to biological problems requires both their efficient detection and knowledge of their probability distribution. The latter is derived in this paper. As to the detection of shortest unique substrings, a data structure known as the suffix tree can readily be used for this purpose [4]. We demonstrate the utility of shortest unique substrings for sequence analysis by applying them to three tasks: (i) identification of signature oligo nucleotide sequences, (ii) detection of unique as well as repeat regions in the genome of Mycoplasma genitalium, and (iii) detection of unique genomic regions in strain MSSA476 of the human pathogen Staphylococcus aureus when compared to four other staphylococcal genomes.
Results
Global shortest unique substrings in Caenorhabditis elegans, human, and mouse
The genome of C. elegans is one of the smallest metazoan genomes sequenced to date. It consists of five autosomes and one sex chromosome, amounting to 100.29 Mb of sequence information [5]. When searching for global shortest unique substrings in this genome, we found a single complementary pair of unique motifs of length 10 located on chromosome 1. Considering the next shortest unique substrings (length 11) we found a total of 10,509 such motif pairs distributed among the five chromosomes. Note that the search for these globally unique substrings was done with respect to the forward and backward strands of the complete genome.
We repeated this analysis for the human genome, which is the largest genome sequenced to date. It consists of 22 autosomes and two sex chromosomes totalling 2.84 Gb published sequence data [6]. We found 215 pairs of global shortest unique substrings of length 11 distributed on the autosomes and the X-chromosome. The Y chromosome contained no unique sequences of length 11 but 135 globally unique sequence pairs of length 12.
We were puzzled by the fact that – with the exception of the single instance of a unique substring pair of length 10 on chromosome 1 of C. elegans – the shortest unique substrings in humans had the same length (11) as those found in C. elegans, even though the human genome is 28 times larger than that of the nematode. When repeating the search for global shortest unique substrings in the mouse genome, whose size is similar to that of human (2.49 Gb), we found a matching result: there were 255 shortest unique substring pairs of length 11 distributed among the autosomes and the X-chromosome. On the Y-chromosome there were 38 unique substring pairs of length 12. The fact that the highly repetitive Y chromosome contained global unique substrings of length 12 as compared to length 11 on autosomes suggested that the length of shortest unique substrings is inversely related to genome information content. In order to further explore whether particularly short unique substrings are associated with functional regions of the genome, we investigated the distribution of globally shortest unique substrings among 1 kb upstream regions of annotated genes.
A total of 29 out of 2 × 350 shortest unique substrings were located among a non-redundant set of 16,286 human 1 kb upstream regions. The probability of observing a single hit in an upstream region with one shortest unique substring is equal to the fraction of the published genome (considering again forward and backward strands) covered by upstream regions. This is
fh = 16286 × 1000/(2 × 2.84 × 109) ≈ 0.003.
The probability of observing 29 or more hits to upstream regions under the null hypothesis of equal distribution is
A similar result was obtained for the mouse genome. Here a total of 13,985 non-redundant upstream regions contained 22 of the 2 × 293 shortest unique substrings. The probability of finding a single hit with one shortest unique substring is again equal to the fraction of the published genome covered by the upstream regions:
fm = 13985 × 1000/(2 × 2.49 × 109) ≈ 0.003.
The probability of observing 22 or more hits to upstream regions by chance is
In other words, both in the human as well as in the mouse genome shortest unique substrings are clustered close to genes. A complete list of the shortest unique substrings with hits to upstream regions is available as supplementary material (see Additional file 1).
So far we have concentrated on the overall, i.e. global, shortest unique substrings. In the following sections we extend this analysis to include all local shortest unique substrings.
Empirical distribution of local shortest unique substring lengths
The pathogenic bacterium Mycoplasma genitalium has one of the smallest genomes known for any free-living organism [11]. The 580,074 bp of its genome encode 480 open reading frames and would be expected to be void of redundant, that is repetitive, sequences. However, when plotting the length of all local shortest unique substrings contained in its genome, we detected 26 non-overlapping shortest unique sequences longer than 100 bp, the longest of which spanned 244 bp (Figure 2A). In other words, the genome of M. genitalium contains a perfectly conserved repeat sequence that is 244 - 1 = 243 bp long. The statistical significance of these repeats is illustrated in Figure 2B, which displays the lengths of the shortest unique substrings in a shuffled version of M. genitalium's genome. In such a scrambled sequence devoid of biological meaning no shortest unique substring is longer than 21 bp. Having found surprisingly short, as well as surprisingly long shortest unique substrings, we proceed by deriving the null distribution of shortest unique substring lengths.
Distribution of local shortest unique substring lengths
As explained further in the Methods section, the probability of finding shortest unique substrings of length x, , is the number of unique substrings of length x, , minus the number of unique substrings of length x - 1, , divided by the genome length l:
where
and 2p represents the GC-content of the genome (p ∈ [0, 1/2]). Figure 3 demonstrates that the fit between equation (1) and the empirical distribution of local shortest unique substrings (cf. Figure 2B) is excellent. Equation (1) provides an efficient method for establishing the statistical significance of any given length of a local shortest unique substring. Using equation (1) and knowing that the GC-content of the genome of C. elegans is 0.3544, one expects to find by chance alone 1.6 unique substrings of length and 10 and 20,441 unique substrings of length 11. These values agree with what we have found in the actual genome of C. elegans (one pair of unique substrings of length 10, and 10,509 pairs of length 11). However, again based on equation (1), the probability of finding in the human genome (GC-content = 0.4088) a unique substring of length 11 is less than 10-100. This is equivalent to an expected number of only 2.4 × 10-94 of such unique substrings and in sharp contrast to the observed value of 215 pairs of unique substrings of this length. The same holds for mouse. Clearly, the lengths of the shortest unique substrings found in mouse and human cannot be explained by chance.
In addition to quantifying the probability of finding shortest unique substrings, equation (1) also allows us to estimate the lengths of unique oligos for arbitrary genomes. In the case of the human genome the length distribution is shown in Figure 4. Notice that this distribution is strongly skewed to the right with 30 being the highest length with an expected occupancy of ≥ 1.
Since equation (1) describes the length distribution of shortest unique substrings in random sequences, it is also the null-distribution for multiple, but phylogenetically unrelated, sequences. This fact can be exploited for comparative genome analysis.
Comparing five strains of Staphylococcus aureus
Staphylococcus aureus is a human pathogen notorious for its resistance to multiple antibiotics [7]. Five genomes of this bacterium are publicly available, which makes it one of the best characterized bacterial species. Our aim was to take strain MSSA476 [7] and identify the regions unique to its genome when compared to the four other strains available. Given the GC-content of Staphylococcus aureus and the combined length of the five genomes analyzed, we calculated the expected distribution of shortest unique substring lengths using equation (1). Figure 5 shows that for unrelated S. aureus sequences of the same combined length (14.21 × 106) and composition (GC-content 0.33) as the strains investigated, the lengths of shortest unique substrings are expected to range from 9 to 27. We decided to analyze the local shortest unique substrings of length ≤ 10 in strain MSSA476 in the presence of the genomes of the four other strains. Figure 6 displays the cumulative distribution of the local shortest unique substrings of length ≤ 10 along the genome of strain MSSA476. Regions unique to MSSA476 contain a high density of such substrings and hence stand out as jumps in the cumulative plot. Figure 6 shows two such jumps. These correspond exactly to the two unique regions ΦSa4 and SCC476 recently annotated as the only two unique regions in MSSA476 [7].
Discussion
The search for unique substrings has a long tradition in molecular biology. It is fundamental for many sequence-based identification techniques, and affects PCR primer design as well as the development of specific antibodies. A DNA or protein sequence of length n contains substrings, which is also an upper limit for the number of unique substrings. This means that in most real world situations there is in an excess of unique substrings to choose from. Since a given unique substring remains unique upon extension, we decided to concentrate on the shortest unique substrings. In their global version they have minimal length with respect to the entire sample of sequences investigated. In contrast, their local version is defined for substrings starting from a specific position in the genome. There are of the order of n such local shortest unique substrings from which all the remaining unique substrings can be generated. Shortest unique substrings therefore lead to considerable space reduction when dealing with unique substrings.
Our technique for detecting such unique substrings is applicable to protein as well as to DNA sequence data. Antibodies are widely used in basic biomedical research; in addition, there is growing interest in applying them as therapeutics. A major design goal in generating antibodies to a given protein in all of these contexts is to maximize their specificity. Since the entire proteome of important biomedical model organisms, including human, is known, epitope selection might be guided not only by considerations of antigenicity, but also of uniqueness. In a preliminary study of the human proteome we found that 88% of the 27,175 human proteins we looked at contain at least one unique hexapeptide. Given that a typical epitope consists of 6 to 12 amino acids, this suggests that our method of detecting shortest unique substrings coupled with epitope prediction programs might also be useful for antibody development.
However, in this paper we have concentrated on shortest unique substrings in genomes. The fact that the length of global shortest unique substrings does not exceed 11 in autosomes of both C. elegans and humans is intriguing given the widely differing sizes of the two genomes and the extremely small probability of observing unique sequences of length 11 by chance in the human genome. Since the length of global shortest unique substrings remained constant after we had removed repetitive elements from the genomes, we take this as an indication that genomes contain a core of high-complexity sequences which determine the length of global shortest unique substrings. The size of this high-complexity core is apparently much less variable across metazoan genomes than raw genome size, hence the observed constancy of global shortest unique substring lengths.
These global shortest unique substrings can be used as starting points for developing signature oligos. Such oligos are widely used in biotechnology and taxonomy. A typical application in biotechnology is PCR-primers that should be unique to the target sequence. In taxonomy a recent initiative for the "Barcoding of Life" attempts to "label" all extant species by assigning a short unique DNA-sequence to them.
The probability of finding a shortest unique substring of some length can be readily computed using equation (1). However, this equation is highly sensitive to the value of the parameter p, which describes the sequence composition. Hence, local variation in sequence composition will strongly affect the expected length of both local and global shortest unique substrings. This fact may also have contributed to our observation that shortest unique substrings cluster in upstream regions of genes in both the mouse and the human genomes. The euchromatic part of the human genome has an average GC-content of 0.41 (, Human genome assembly hg16), which is similar to the value of 0.42 for the mouse (, Mouse genome assembly mm4). In contrast, the global shortest unique substrings we found in humans have a GC-content of 0.59 and those found in mouse have a GC-content of 0.61. The upstream regions of genes tend to be GC-rich in human (GC-content = 0.53) as well as mouse (GC-content = 0.50), which might account for the clustering of global shortest unique substrings in these regions.
Detection of unique genomic regions is traditionally done by alignment-based approaches. However, the run-time of these algorithms depends non-linearly on the number and lengths of the input sequences and also on the degree of relatedness of the input sequences. In contrast, the scheme for detecting unique genomic regions proposed in this paper has a run time that is strictly linear in the combined lengths of the input sequences.
Conclusion
There is currently a lot of interest in comparative genomics [8]. In many of these projects detection of regions unique to a genome is one of the first steps towards functional annotation (e. g. [7]). Given equation (1), the size distribution of shortest unique substrings in random, i.e. unrelated, sequences can be predicted. This leads to our method of detecting unique genomic regions from an arbitrary set of input sequences without the need for alignment. Its usefulness for comparative genomics is clearly demonstrated in the case of the genomes of S. aureus, where we could rapidly detect the two unique regions previously annotated in one strain [7] (Figure 5).
Methods
Detection of shortest unique substrings
Two methods borrowed from computer science were used for the detection of shortest unique substrings: suffix tree construction and hashing. Suffix trees are well described by Gusfield [4] and we follow his nomenclature. To use suffix trees for detecting unique substrings, notice that the path label of any leaf is a unique substring. The set of shortest unique substrings at every position can therefore be discovered by traversing the tree once and looking up the string depth of the parent node of every leaf. This value plus one is the desired length of the shortest unique string that starts at the position indicated by the leaf.
Hashing is described, for example, by Cormen et al. [[9], ch. [11]] and we used it for detecting global shortest unique substrings in large genomes.
Unless stated otherwise, all computations presented in this paper consider both strands of the DNA sequences concerned. Note that in this case, and due to complementarity of DNA, a single parameter (p) suffices to describe nucleotide composition.
The probability distribution of local shortest unique substring lengths in nucleotide sequences
Consider a nucleotide sequence S and let 2p denote the GC-content of S (p ∈ [0, 1/2]). A shortest unique substring of length x of this nucleotide sequence is defined as a unique substring S[i..i + x - 1] where S[i..i + x - 2] is not unique. We wish to derive the probability distribution of values of x under the assumption of random sequence composition.
We start by considering a particular substring of length x consisting of k positions occupied by either G or C each. We refer to such a substring as being of type (x, k). The probability of finding a substring of type (x, k) is
Px,k = (1/2 - p)x - k pk.
Assuming that l independent trials each having a success probability of Px,k are performed, the probability of finding a particular sequence of type (x, k) exactly once is then
This expression is only approximately valid, since the nucleotide compositions of any two overlapping subtrings are not independent. Still, from now on we assume independence. The error introduced by this assumption is negligible, if the genome size, l, is large compared to the length of the considered substrings (l >> x) - which is the case we have in mind. Thus, we replace the ≈-sign in the above and following expressions by = and define
For each sequence of type (x, k), there are permutations of k "G|C" s and (x - k) "A|T" s. Some of these permutations occur zero times in S, some occur multiple times and some occur exactly once. We are interested in the latter: the number of unique substrings of type (x, k) is
In order to determine the number of unique substrings irrespective of their sequence composition, we need to sum over all possible values of k:
The number of shortest unique substrings of length x, , is then simply the number of unique substrings of length x minus the number of unique substrings of length x - 1. In order to see this, notice that all unique substrings of length x - 1 are contained in the set of unique substrings of length x. Those that are gained by adding the extra nucleotide are precisely the substrings that lose their uniqueness when reduced in length by one as required by the definition of shortest unique substring:
Finally, the probability of finding such a shortest unique substring of length x, , is the number of unique shortest substrings of length x divided by the genome length:
Implementation
The search for shortest unique substrings is implemented in our program shustring (SHortest Unique subSTRING). The distribution of shortest unique substring lengths in genomic sequences as embodied in equation (1) is implemented in our program shulen. Both pieces of software are available from .
Data
Genome sequences of the nematode (Caenorhabditis elegans) [5], mouse (Mus musculus) [10], and human (Homo sapiens) [6] as well as 1 kb upstream regions for genes in the genomes of human and mouse were obtained from the University of California Santa Cruz genome website at the following URLs:
1. nematode: (version ce2)
2. mouse: (version mm4, October 2003)
3. human: (version hg16, July 2003)
Table 1 lists the six bacterial genomes analyzed in this study.
Authors' contributions
B.H. designed and implemented the software, performed data analysis and contributed to the writing of the manuscript. N.P. was involved in data analysis, software testing and contributed to the writing of the manuscript. F.M. carried out the analysis of the upstream regions. T.W. conceived of the study of shortest unique substrings, derived their null distribution, and contributed to the writing of the manuscript. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Supplementary Material. List of Human and Mouse genes with hits to shortest unique substrings to their 1 kb upstream regions
Click here for file
Acknowledgements
We would like to thank C. Acquisti and A. Börsch-Haubold for stimulating discussions and two anonymous reviewers for comments which helped to improve the manuscript. Furthermore, we would like to thank the members of the High Performance Computing Group at the Leibniz Rechenzentrum München for advice on computational issues. F.M. is supported by a grant from the German Ministry of Education and Research (BMBF; Fkz. 0312705A). B.H. is supported financially by Dehner Gartencenter GmbH and the Stifterverband der Deutschen Wissenschaft.
Figures and Tables
Figure 1 Shustrings on forward and backward strands. Global and local shortest unique substrings ('shustrings') in the DNA-sequence ACCG. A: The global shustrings are A and G, and have length 1 (black numbers above the sequence). The numbers above the sequence indicate the length of the four local shustrings A, CC, CG and G present on the forward strand. B: In the presence of the reverse strand global as well as local shustrings may change. For some positions at the 3'end of the sequence shustrings may not be defined (here, position 3 and 4 on the forward strand). Notice that the complement of a local shortest unique substring is also unique, however not necessarily a shortest unique substring (for example the pair GT on the reverse strand and AC on the forward strand). The complement of a global shortest unique substring is again a global shortest unique substring (here the two global shustrings A and T).
Figure 2 Shustrings in Mycoplasma genitalium. A: Lengths of the local shortest unique substrings at every position along the genome of Mycoplasma genitalium. The lengths displayed minus one correspond to the lengths of substrings which are repeated at least once in the genome. B: The same as A, except that the nucleotides in the genome were shuffled (thus preserving nucleotide frequencies), which leads to the disappearance of long repeats.
Figure 3 Shustring probability distribution in the randomized genome of Mycoplasma genitalium. Observed and expected distributions of the lengths x of local shortest unique substrings. The "observed" distribution was obtained by shuffling the nucleotides of the genome of Mycoplasma genitalium (c. f. Figure 2), while the expected distribution is based on equation (1) using the genome's GC-content of 2p = 0.316 and length l = 580,074 bp.
Figure 4 Expected number of shustrings in the randomized human genome. Expected number, , of local shortest unique substrings of length x. Parameters used in equation (1) are l = 2.84·109 and 2p = 0.409 and correspond to the euchromatic part of the human genome.
Figure 5 Shustring probability distribution in five randomized strains of Staphylococcus aureus. Observed and expected distributions of the lengths x of local shortest unique substrings. Parameters l = 1.42·107 and 2p = 0.330 correspond to the combined length and average GC-content of five strains of Staphylococcus aureus.
Figure 6 Cumulative distribution of shustrings in Staphylococcus aureus. Cumulative distribution of unique substrings as a function of genome position in S. aureus MSSA476 when compared to strains MRSA252, MW2, Mu50, and N315 (c. f. Table 1). The steep jumps in the plot correspond to the two regions SCC476 (close to the origin) and ΦSa4 indicated in grey. These are known to be the sole two unique regions in the genome of MSSA476 [7]. Only local shortest unique substrings of length ≤ 10 were included in the analysis.
Table 1 Bacterial genomes analyzed in this study
organism accession number reference genome size
Mycoplasma genitalium L43967 [11] 580,074
Staphylococcus aureus MRSA252 NC 002952 [7] 2,902,619
Staphylococcus aureus MSSA476 NC 002953 [7] 2,799,802
Staphylococcus aureus MW2 NC 003923 [12] 2,820,462
Staphylococcus aureus Mu50 NC 002758 [13] 2,878,040
Staphylococcus aureus N315 NC 002745 [13] 2,814,816
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| 15921519 | PMC1166541 | CC BY | 2021-01-04 16:02:48 | no | BMC Bioinformatics. 2005 May 27; 6:127 | latin-1 | BMC Bioinformatics | 2,005 | 10.1186/1471-2105-6-127 | oa_comm |
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-5-161592706910.1186/1472-6750-5-16Methodology ArticleHigh-throughput kinase assays with protein substrates using fluorescent polymer superquenching Rininsland Frauke [email protected] Casey [email protected] Wendy [email protected] Duncan [email protected] QTL Biosystems, 2778 Agua Fria Street, Santa Fe, NM 87507, USA2005 31 5 2005 5 16 16 1 3 2005 31 5 2005 Copyright © 2005 Rininsland et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform.
Results
Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that are used in the high-throughput community to determine assay robustness (Z'-value) demonstrate the suitability of this format for high-throughput screening applications for detection of inhibitors of enzyme activity.
Conclusion
The QTL Lightspeed™ protein detection system provides a simple mix and measure "turn on" assay for the detection of kinase activity using natural protein substrates. The platform is robust and allows for identification of inhibitors of kinase activity.
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Background
Approximately 75% of the drugs in clinical use elicit their pharmacological effects by interactions with receptor or enzyme targets, such as kinases [1,2]. Methods to screen large chemical libraries for inhibitors of protein kinases include radiometric assays [3], ELISA [4], ATP consumption assays [5] and several fluorescence based assays such as time-resolved fluorescence (TRF) [6], fluorescence polarization (FP) [7,8], fluorescence energy transfer (FRET) [9] or fluorescence quench assays [10]. Assays such as FRET, FP and fluorescence quench do not require antibodies or radioactive label, and are thus attractive for HTS. However non-antibody based FP and FRET assays are restricted to the use of small, synthetic peptide substrates to monitor kinase activity. While peptide substrates are convenient for HTS purposes, those that bind with high affinity are available for only a small percentage of the >500 kinases encoded by the human genome [11]. Additionally, peptide substrates may diminish the ability to detect inhibitors that bind to docking sites of a native protein substrate or that bind to unique conformational states induced by protein substrate binding [12]. Here we report the detection of phosphorylation of the natural protein substrates Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1) by PKCα and IRAK4 using a modified version of our original assay format [13,14], which is based on superquenching of fluorescent polyelectrolytes [15,16]. The assay principle is shown in Figure 1.
Results
MBP, Histone H1 and and PHAS-1 proteins were phosphorylated with PKCα and IRAK4 as described in the Methods section. The proteins were used in their native form and were not chemically modified. An enzyme concentration-dependent increase in phosphorylation correlated with increasing fluorescence signal, demonstrating the efficacy of the QTL Lightspeed™ platform for detection of phosphorylated proteins (Figure 2). The detection of MBP phosphorylation worked equally well for protein substrates derived from either bovine or human (not shown). In order to explore the utility of the assay for screening inhibitors, the ATP competitor Staurosporine was used to inhibit enzyme activities using substrates Histone H1, PHAS-1 (Figure 3A) MBP (Figure 3B). For each protein, a concentration of Staurosporine which inhibited enzyme activity by 50% (IC50) was determined to be within the range of the reported value using a peptide substrate (9 nM) [17]: values of 3.8 nM, 1.6 nM and 0.6 nM were obtained for MBP, Histone H1 and PHAS-1, respectively (Figure 3). The IRAK4 protein assay was 3-fold more sensitive than a QTL Lightspeed™ assay performed on a peptide substrate (EC50 = 1.4 nM vs 5 nM). Using MBP as substrate, we were able to obtain IC50 values for Staurosporine of 11.5 nM, which were very similar to those obtained using a peptide substrate (19 nM, not shown).
The robustness of the assay (Z') was determined by performing 10 multiples of phosphorylation reactions using identical assay conditions. The Z' is a statistical parameter used in the drug screening community to evaluate and validate performance of assays [18]. An assay that delivers a Z' of higher than 0.5 is considered to be robust. The Z' was determined using the following equation:
For MBP, high Z' values of 0.84 and 0.8 were obtained for phosphorylation using PKCα and IRAK4, respectively. For PHAS-1 the values were 0.52 and for Histone H1 0.43. Table 1 summarizes the statistical values obtained for the three different substrates.
Discussion
Despite the existence of several different technologies to monitor phosphorylation reactions of kinases, the detection of protein substrate phosphorylation has been limited to antibody-based assays, radioactive assays or assays that measure ATP consumption rather than specific kinase activity. Antibody-based assays are restricted to available, purified antibodies, usually directed against phosphoserine and phosphothreonine residues and may have low affinity and be specific to only a single kinase. Due to safety concerns over radioisotope handling as well as cost, drug screening efforts are rapidly moving toward non-radioactive and non-antibody-based assays. FP fulfills these criteria. However, since FP signal is correlated with changes in molecular rotation rates within the fluorescence life time and since only molecules smaller than approximately 20 kDa show significant rotation within this range most protein substrates are not directly measurable by FP [19]. A prompt fluorescence quench assay relies upon the binding of a phosphate coordinating metal ion to the phosphate group on a substrate, which is labeled with a dye [10]. Upon association of the metal ion, fluorescence is quenched. Similar to FRET assays, the rapid drop in signal with increasing distance between donor and acceptor molecules makes such assays unsuitable for the detection of protein phosphorylation, even if the proteins could be labeled with appropriate dyes [9]. The QTL Lightspeed™ protein assay is a novel non-radioactive and non-antibody-based assay that follows a simple mix and measure protocol for the quantitative detection of protein phosphorylation. Unmodified proteins can be used as substrates, thus allowing for the integrity of protein structure, which may be a requirement for successful recognition and phosphorylation by kinases. Our experiments utilized 0.5 μg MBP for phosphorylation by PKCα and IRAK4, thus moving the assay into the realm of HTS with regard to cost effectiveness. In this report, we describe the use of relatively small proteins MBP and PHAS-1 (18.9 kDa and 21 kDa respectively) and show feasibility for the larger protein Histone H1 (32 kDa) as well. Since our assay does not rely on site-specific antibodies, the phosphorylation of serine, threonine and tyrosine residues is possible.
Conclusion
We demonstrate that a modified version of our QTL Lightspeed™ platform provides a simple and robust assay for monitoring protein phosphorylation. The platform does not employ radioactive labels or antibodies, which makes it specific as well as time and cost-effective. We show that the assays for PKCα are suitable for drug compound screening by obtaining IC50 values of an ATP-analog which are very close to those reported in the literature for this compound. For IRAK4, reported values were not available. Thus, it is reasonable that our assay is also well-suited for the identification of inhibitors that are not ATP-site directed competitors. Further applications of the assay could be the detection of autophosphorylated kinases or other proteins that play a role in cell signaling. Finally, the QTL Lightspeed™ platform can be used to further identify novel protein substrates for specific kinases.
Methods
Fluorescent poly-phenylene ethyneylene was synthesized and coated onto microspheres as previously described [13,14].
Bovine substrate MBP was purchased from Upstate (Charlottesville, VA). PHAS-1 was purchased from Biomol (Plymouth Meeting, PA) and Histone H1 from Upstate. The phosphopeptide tracer was a 13 amino acid peptide, which was N-terminal labeled with a rhodamine derivative. The enzyme PKCα was purchased form Biomol and IRAK4 from Upstate. The inhibitor Staurosporine was from Sigma (St Louis, MO).
Kinase assays were performed using 384-well, white Optiplates (Perkin Elmer, Wellsley, MA) in a total volume of 15 μL. Protein Kinase Cα assays were performed in assay buffer (20 mM HEPES pH 7.4; 5 mM MgCl2; 0.1 mM CaCl2, 0.1 mg/ml 1-2-Dioleoyl-sn glycerol (Avanti, Alabaster, AL) and 0.02 mg/ml phosphatidylserine (Avanti) and 0.1% w/v NaN3) using 0.5 μg MBP as substrate for 1 hour at room temperature. The concentration of ATP (IDC, Livermore, CA) was 10 μM. IRAK4 phosphorylation was performed in assay buffer containing 50 mM Tris, pH 7.4; 5 mM MgCl2; 1 mM MnCl2; 0.1% BSA and 0.09% NaN3. The concentration of ATP was 50 μM for enzyme concentration curves and 10 μM for inhibition experiments. Following the enzymatic reaction, the phosphorylated protein was mixed with 15 μL of QTL Sensor for 10 minutes. Lastly, a rhodamine-labeled phosphorylated "tracer" peptide was added with a final concentration of 0.5 μM or 125 nM for detection of MBP or Histone H1 and PHAS-1 respectively for 30 minutes at ~25°C. The fluorescence of the reaction mixture was measured using a SpectraMax Gemini XS plate reader (Molecular Devices, Inc., Sunnyvale, CA) in well scan mode using excitation wavelength of 450 nm with a 475 nm cutoff filter and emission readout at 490 nm. Curve fitting was performed using GraphPad Prism® sigmoid dose-response (variable slope) software.
List of abbreviations
EC50 = enzyme concentration at 50% of signal; ELISA = Enzyme linked immunosorbent assay; FP = Fluorescence polarization; FRET = fluorescence resonance energy transfer; IC50 = Inhibitor concentration that produces 50% of signal.
Authors' contributions
FR developed the experimental approach and wrote the manuscript; CS and WW carried out the experiments and DM made contributions to the conception of the study.
Acknowledgements
We thank Wensheng Xia, Komandoor Achyuthan and Harshini Mukundan for helpful discussions.
Figures and Tables
Figure 1 Phosphorylated protein binds to the QTL Sensor via specific phosphate binding to metal coordinating ions and inhibits the association of the dye-labeled phosphopeptide (tracer; red "starburst"). The resulting increase in fluorescence signal correlates with the extent of protein phosphorylation.
Figure 2 Enzyme concentration curve using proteins as substrates. MBP (0.5 μg/well) was phosphorylated using various amounts of PKCα and IRAK4 (2A) and Histone H1 and PHAS-1 (0.5 μg/well) were phosphorylated using various amounts of PKCα (2B) for 1 hour at room temperature (~25°C) in a 384-well white Optiplate. Following reaction, QTL Sensor was added for 10 minutes at ~25°C. Then, dye-labeled phosphopeptide tracer was added with a final concentration of 0.5 μM for detection of phosphorylation of MBP or 125 nM for Histone H1 and PHAS-1. The plate was incubated for an additional 30 minutes at ~25°C and fluorescence measured.
Figure 3 Staurosporine inhibition of enzyme activity. A concentration of 8.6 nM PKCα was used to phosphorylated Histone H1 or PHAS-1 for 60 minutes at ~25°C using various concentrations of Staurosporine (3A). Inhibition of PKCα and IRAK4 activities using MBP as a substrate were performed using decreasing concentrations of Staurosproine (3B). Following incubation, QTL Sensor was added and incubated for 10 minutes at ~25°C. Subsequently dye-labeled phosphopeptide tracer was added with a final concentration of 0.5 μM for MBP or 125 nM for Histone H1 and PHAS-1. The plate was incubated for an additional 30 minutes at ~25°C and fluorescence measured.
Table 1 QTL Lightspeed™ Protein Assay Statistical Data18. Statistics are shown for Z' Factor, signal/noise (S/N), signal/background (S/B), signal window (SW) and coefficient of variation (% CV) at maximal substrate phosphorylation. Z'Factor equal to or greater than 0.5 are an indication of a robust assay18. Ten replicate measurements were made to determine the assay statistics.
Protein Enzyme Z' Factor S/N S/B SW %CV
MBP PKCα 0.84 20.7 2.6 33.4 2.2
MBP IRAK4 0.80 23 2.6 31 4.3
PHAS-1 PKCα 0.52 9.6 0.7 9.6 2.8
Histone H1 PKCα 0.43 7.32 0.7 9.4 3.0
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| 15927069 | PMC1166542 | CC BY | 2021-01-04 16:02:57 | no | BMC Biotechnol. 2005 May 31; 5:16 | utf-8 | BMC Biotechnol | 2,005 | 10.1186/1472-6750-5-16 | oa_comm |
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BMC Cardiovasc DisordBMC Cardiovascular Disorders1471-2261BioMed Central London 1471-2261-5-131594387110.1186/1471-2261-5-13Research ArticleDeterminants of persistence in hypertensive patients treated with irbesartan: results of a postmarketing survey Burnier Michel [email protected] Bernhard [email protected] Peter [email protected] Bernard [email protected] Service de Néphrologie, CHUV, Lausanne, Switzerland2 Spital Zimmerberg, Wädenswil, Switzerland3 Medizinische Poliklinik, Universitätsspital, Zürich, Switzerland4 Division de Physiopathologie Clinique, CHUV, Lausanne, Switzerland2005 8 6 2005 5 13 13 5 12 2004 8 6 2005 Copyright © 2005 Burnier et al; licensee BioMed Central Ltd.2005Burnier et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Persistence is a key factor for long-term blood pressure control, which is of high prognostic importance for patients at increased cardiovascular risk. Here we present the results of a post-marketing survey including 4769 hypertensive patients treated with irbesartan in 886 general practices in Switzerland. The goal of this survey was to evaluate the tolerance and the blood pressure lowering effect of irbesartan as well as the factors affecting persistence in a large unselected population.
Methods
Prospective observational survey conducted in general practices in all regions of Switzerland. Previously untreated and uncontrolled pre-treated patients were started with a daily dose of 150 mg irbesartan and followed up to 6 months.
Results
After an observation time slightly exceeding 4 months, the average reduction in systolic and diastolic blood pressure was 20 (95% confidence interval (CI) -19.6 to -20.7 mmHg) and 12 mmHg (95% CI -11.4 to -12.1 mmHg), respectively. At this time, 26% of patients had a blood pressure < 140/90 mmHg and 60% had a diastolic blood pressure < 90 mmHg. The drug was well tolerated with an incidence of adverse events (dizziness, headaches,...) of 8.0%. In this survey more than 80% of patients were still on irbesartan at 4 month. The most important factors predictive of persistence were the tolerability profile and the ability to achieve a blood pressure target ≤ 140/90 mmHg before visit 2. Patients who switched from a fixed combination treatment tended to discontinue irbesartan more often whereas those who abandoned the previous treatment because of cough (a class side effect of ACE-Inhibitors) were more persistent with irbesartan.
Conclusion
The results of this survey confirm that irbesartan is effective, well tolerated and well accepted by patients, as indicated by the good persistence. This post-marketing survey also emphasizes the importance of the tolerability profile and of achieving an early control of blood pressure as positive predictors of persistence.
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Background
Angiotensin II receptor antagonists (AIIRA) have demonstrated excellent efficacy in patients with hypertension [1,2], heart failure [1,2], diabetes [3], diabetic nephropathy [4-6], and recently in post-myocardial infarction patients [7]. Such promising results allowed the US Joint National Committee on hypertension in their seventh report (JNC-7) and the European Society of Hypertension (ESH-ESC) in the 2003 guidelines to integrate this class of agents in the management of hypertension and to propose AIIRAs as an alternative treatment to ACE-inhibitors for most of the above-mentioned high-risk conditions [8,9].
However, the main message of the published guidelines remains that a normalization of arterial blood pressure in hypertensive patients is the key objective to the prevention of cardiovascular events, especially in high-risk categories, where stricter therapeutic targets and aggressive strategies to reach them have been proposed. Yet, achieving normalization of blood pressure (BP) remains a difficult task, or, in the words of the JNC-7 report [8]: "The most effective therapy prescribed by the most careful clinician will control hypertension only if the patient is motivated to take the prescribed medication and to establish and maintain a health-promoting lifestyle." In industrialized countries, the success rates in controlling blood pressure range from below 10% to 30% of the treated population, depending mainly on the definition of the therapeutic targets [10-14]. The JNC-7 report estimates that 34% of hypertensive patients in the USA manage to maintain a BP below 140/90 mmHg [8].
The inadequate persistence with therapy, i.e. the frequent switch or discontinuation of the prescribed therapy has been recognized as a frequent cause of treatment failure [15]. Persistence is a good general indicator of the satisfaction with the treatment of both patients and physicians. Among classes, a British comparative study showed ACE-inhibitors as the best and diuretics as the poorest persistence builders [15]. A large Canadian cohort study based on pharmacy prescriptions confirmed these results [16]. Recently, the first population-based studies including AIIRAs revealed that patients persist more with AIIRAs than with all other antihypertensive drugs after 6 months up to 3 years [17,18]. A cohort study with 2416 newly diagnosed hypertensive patients showed that the AIIRA irbesartan induced significantly more persistence than other drug classes and even than other AIIRAs [19]. This improved persistence has been attributed in part to the efficacy of the compounds and mainly to the low, placebo-like side effects profile [20-22]. Placebo-like tolerability has indeed been confirmed at all clinically relevant dosages of irbesartan [23].
The aim of this open prospective observational survey with 4769 hypertensive patients treated in general practices in Switzerland, was to evaluate the persistence with irbesartan in real-life settings over a period of about one year and to investigate factors affecting either positively or negatively the persistence. In addition, the tolerability profile and the effect of irbesartan on BP control were assessed.
Methods
Design of the investigation
This prospective observational survey was conducted in general practices in all regions of Switzerland from October 1997 to March 1999 i.e. shortly after the launch of irbesartan in the country. The general practitioners (GPs) were asked to document their daily routine in the treatment of hypertensive patients with irbesartan; 1390 physicians were contacted, 1045 included patients and 886 documented the treatment. They were recruited by the field forces of the sponsors of the study i.e. BMS and Sanofi-Synthelabo Switzerland. GPs filled in a baseline visit (Visit 1) form for every treated patient, and could report up to five follow up visits in the following 3–6 months (End Form). They were asked to document two arterial blood pressure measurements at every visit and to report concomitant antihypertensive medication and adverse events, as well as changes or discontinuation of treatment. At the end of the observation period, GPs reported if their patients were continuing the treatment with irbesartan, alone or in combination with other antihypertensive drugs. About 1 year after baseline, patients with ongoing treatment with irbesartan received a Compliance Form Patient, where they were asked how irbesartan was tolerated (very well, well, fairly well, poorly) and how many times weekly they took irbesartan in the preceding 3–4 weeks; a further question was how many tablets (all drugs) were taken daily. Their GPs received a Compliance Form MD, to report the last blood pressure measurement and if and when the patient had discontinued the treatment. The various forms were designed by external consultants. The data were collected by mail using self-addressed envelopes and processed by the consultants. Queries for incomplete forms were done by the sponsor representatives. No standard validated questionnaire was used. In Switzerland, this type of survey does not need to be submitted to an ethical committee.
Patient selection
All patients with newly diagnosed hypertension, or with treated hypertension requiring a change in medication according to the GP, were considered for the survey. No standardized definition was used but physicians considered a BP >140/90 mmHg as hypertension. There were neither demographic nor clinical exclusion criteria. The only condition to participate was that patients should not have been pre-treated with irbesartan. Treatment was started with irbesartan 150 qd. Thereafter, physicians were free to change the antihypertensive therapy at any time during the follow-up based on their individual therapeutic goals (usually <140/90 mmHg).
Statistical analysis
Values are presented as mean +/- sd. The statistical significance of between-group differences was computed using the 2-sided Chi-square test, the Mann-Whitney-U test or ANOVA methods as appropriate. For multivariate correlations, a logistic regression analysis with a dichotomous dependent variable was used (e.g. therapy discontinuation: yes = 1; no = 0). To support the results of the logistic regression models, Cox regression models with cumulative survival functions were further computed.
Results
Characteristics of the database
As shown in Figure 1, 5452 patients were enrolled by 1045 GPs, and 886 of those returned the therapy documentation (End Form) of 4769 patients (87.5%). This latter sample was taken to analyze the safety data and is referred to as AE-Sample (Tolerability Events). For the evaluation of the effect on BP control, all cases with at least one follow-up value were taken into account. 130 cases of the AE-Sample had no follow-up values; the remaining 4639 subjects (97.3% of the AE-Sample) are referred to as the Efficacy Sample. At the end of the treatment observation period, after an average of slightly more than 4 months (133 ± 75 days, mean ± SD), GPs reported that 3829 patients (82.5% of the Efficacy sample) continued the treatment with irbesartan. This is referred to as the Sample with Ongoing Therapy. A total of 1419 Compliance Forms MD (37.1% of the sample with ongoing therapy) and 928 Compliance Forms Patient (24.2%) were returned after on average more than 13 months from baseline (402 ± 105 days). Due to lack of completeness, some forms had to be excluded from the analysis, giving a total of 1186 valid Compliance Forms MD (31.0%) and 853 cases with both usable Compliance Form MD and Patient (22.2%).
Figure 1 Patient population and available data. Summary of the analysis patient populations of this investigation and of the data available for analysis.
Table 1 summarizes the baseline demographic and clinical data for both the previously untreated and the pre-treated patients. Almost two thirds of the patients (61,5%) entered the study receiving another therapy for high blood pressure. The most frequent reasons why GPs changed pre-treatment to introduce irbesartan were insufficient efficacy of the previous therapy (64.6%), cough (22.5%) and adverse events other than cough (16.6%). The multivariate analysis of factors correlated to pre-treatment shows that patients who switched to irbesartan from other antihypertensive drugs were older, prevalently female and from the German part of Switzerland (p < 0.001). They had significantly more risk factors, associated clinical conditions (p < 0.0001) and target organ damages than naïve patients (p = 0.0013). More pre-treated patients received a polytherapy regimen during the post-marketing surveillance (p < 0.0001).
Table 1 Baseline demographic and clinical data: Efficacy Sample.
Naïve patients Pre-treated Total
Total 1785 (38.5%) 2854 (61.5%) 4639
Gender (f/m) 882/903 1605/1249 2487/2152
Mean age ± SD 57.9 ± 12.7 63.6 ± 12.3 61.4 ± 12.8
Baseline SBP (Mean ± SD) 168.8 ± 17.8 163.5 ± 19.1 165.5 ± 18.8
Baseline DBP (Mean ± SD) 101.2 ± 8.6 96.3 ± 10.4 98.2 ± 10.0
Diabetes 165 (9.2%) 492 (17.2%) 657 (14.2%)
ISH 79 (4.4%) 408 (14.3%) 487 (10.5%)
WHO-risk
Low (<15%) 37 (2.1%) 101 (3.5%) 138 (3.0%)
Medium (15–20%) 837 (46.9%) 1190 (41.7%) 2027 (43.7%)
High (20–30%) 274 (15.4%) 526 (18.4%) 800 (17.2%)
Very high (>30%) 637 (35.7%) 1037 (36.3%) 1674 (36.1%)
For detailed explanation of WHO-risk and region categories see results. ISH = isolated systolic hypertension (defined as ≥ 140 mmHg systolic and < 90 mmHg diastolic blood pressure).
Effect on blood pressure
More that 90% of the Efficacy Sample patients received irbesartan 150 mg qd, as a monotherapy or in combination with other antihypertensives. 69% of the Efficacy Sample patients received a constant monotherapy and 5.6% a constant polytherapy. In the Efficacy Sample, the mean reduction of systolic blood pressure (SBP) from baseline to the last visit was 20.2 ± 19.5 mmHg (p < 0.001). For the diastolic blood pressure (DBP), the mean reduction was 11.7 ± 11.3 mmHg (p < 0.001). Figure 2a shows the differences between naïve and pre-treated patients. Despite previous treatment, the pre-treated group had clearly inadequate mean baseline values of SBP and DBP (163.5 and 96.3 mmHg, respectively) justifying a change in therapy. Naïve patients achieved a significantly greater reduction of both SBP and DBP than pre-treated ones. At the last visit, the pre-treated group showed higher SBP and similar DBP values in comparison to naïve patients.
Figure 2 a. Evolution of blood pressure during observation period (~4 months): Efficacy Sample. Baseline SBP (systolic blood pressure): previously untreated patients 168.8 mmHg, pre-treated 163.5 mmHg (*p < 0.0001); SBP at last visit: previously untreated 142.8 mmHg, pre-treated 146.9 mmHg (p < 0.0001). Baseline DBP (diastolic blood pressure): previously untreated patients 101.2 mmHg, pre-treated 96.3 mmHg (*p < 0.0001); DBP at last visit: previously untreated 85.9 mmHg, pre-treated 86.8 mmHg (p = 0.004). b. Reaching of therapeutic targets: Efficacy Sample. Response to treatment is defined as reaching DBP < 90 mmHg or a reduction of DBP = 10 mmHg. In real-life practice, a satisfactory objective is also the normalization of DBP (< 90 mmHg). Target = 140/90 mmHg was introduced because of digit preference of study GPs (see results).
Since GPs did not receive specific instructions about therapeutic goals in this survey, various therapeutic targets were used to evaluate the success in controlling blood pressure (Figure 2b). As shown in the figure, one third of the patients had normalized their BP (<140/90 mmHg) and two-thirds had a diastolic BP below 90 mmHg. The Sample with Ongoing Therapy, as reasonably expected, appeared slightly more successful than the Efficacy Sample in achieving the various targets (Figure 2b).
Tolerability profile
Adverse events were reported for 383 patients (8.0% of the AE-Sample), more often by older patients (>65 years: 10.2%; 55–65 years: 7.8%; = 55 years: 5.5%; p < 0.001) and by pre-treated patients (9.6% vs. 5.5% naïve; p < 0.001). Yet, in the majority of patients (90.7%), irbesartan was well tolerated according to GPs. Tolerance was reported as poor only for 131 patients (2.7%). Adverse events led to discontinuation of irbesartan in 343 cases (7.4%). The most frequent side effects are listed in Table 2, where they are compared with their occurrence listed in the Swiss prescribing information [24]. Serious adverse events, leading to death, disability, life-threatening conditions or hospitalization, were reported in 74 patients (1,3% of AE-sample), but GPs described a possible or probable connection with trial medication only in 8 cases. Very good or good tolerance was reported by 824 patients in the Compliance Form Patient (96.6% of the total), all subgroups scoring above 90%.
Table 2 Most reported adverse events in the AE-Sample (n = 4769) compared to the Swiss prescribing guidance
AE-Sample Swiss prescribing guidance
Total 383 (8.0%)
Dizziness 65 (1.4%) >1%
Nausea 53 (1.1%) >1%
Headache 43 (0.9%) >1%
Dyspepsia 24 (0.5%) 0.5–1%
Diarrhea 18 (0.4%) 0.5–1%
Palpitation 17 (0.4%) Not mentioned
Cough 15 (0.3%) 0.5–1%
Fatigue 15 (0.3%) >1%
Vomiting 11 (0.2%) >1%
Tachycardia 10 (0.2%) 0.5–1%
Persistence
3829 patients out of 4639 continued the treatment with irbesartan after the last visit (on average, more than 4 months from the start). The main reasons for discontinuation of the remaining 810 patients were the occurrence of adverse events and an insufficient efficacy (figure 3). In a logistic regression model, the factors that correlated more strongly with ongoing therapy were a reported good tolerance and reaching the a blood pressure ≤ 140/90 mmHg. Interestingly, pre-treated patients discontinued irbesartan significantly more often when the previous therapy was a fixed combination of antihypertensive agents, but not if they had stopped the pre-treatment because of cough, which on the contrary increased the probability of therapy continuation. Treatment modifications also affected persistence. Thus, if the dose of irbesartan was increased or another antihypertensive agent was added at the first follow up visit (Visit 2), patients had better chances to stay on therapy whereas, if treatment was modified after visit 2, this was associated with more discontinuations. In the former situation 9.5% of patients persisted on therapy. In contrast, if further changes in drug therapy were necessary on visit 3 because of insufficient BP control 35.9% of drug therapies were discontinued. The survival analysis (Cox regression) generally confirmed the statistically significant relationships with persistence found in the logistic regression models (Figure 4). An effect detected only by the Cox regression was the positive correlation between persistence and number of risk factors for cardiovascular diseases at baseline (RR = 0.74 for the discontinuation; p = 0.0001). Diabetes did not appear to influence persistence. In both multivariate analyses, the presence of a family history of hypertension or cardiovascular diseases reduced the chances of persistence.
Figure 3 Ongoing treatment and discontinuation reasons: Efficacy Sample. (n = 4639)Other reasons included: patient moved, blood pressure normalized, break off attempt, concurrent disease, effect too strong, lost to follow up and others. Multiple answers were possible.
Figure 4 Mean (line) and 95% confidence interval (box) for the odds ratio (OR) of the main variables correlating significantly with ongoing treatment or discontinuation in a logistic regression model; Efficacy Sample. For detailed explanations see results.
Perception of compliance
Patients with ongoing therapy at last visit were asked in the Compliance Form Patient to indicate how many irbesartan tablets they took per week during the preceding 3–4 weeks. Since after about one year only 853 patients returned the form, we have to assume a sampling bias. Nevertheless, some within-group differences are worth mentioning. 777 patients (91.1%) who returned the Compliance Form Patient reported an irbesartan intake of 6–7 times per week, i.e. more than 80% of the prescribed doses. All subgroups scored around 90% (Figure 5), but females reported a better compliance than males (92.9% vs. 89.0%; p = 0.021). Patients with isolated systolic hypertension appear to adhere better to therapy than other hypertensives (97.2% vs. 90.2%; p = 0.032), while patients with a low risk for cardiovascular events showed a lower compliance (84%, n.s.). Compliance Forms MD reported an overall ongoing treatment rate with irbesartan of 88.0% (1044 patients), and a slightly higher rate for pre-treated patients (90.4%, n = 728).
Figure 5 Self-reported compliance according to the patient for selected patient subgroups. Good compliance with treatment after 1 year; Compliance Form Patient (n = 853). Good compliance is defined as >80% adherence to the prescribed therapeutic regimen. In this case it means irbesartan intake on 6 or 7 days a week, as reported by the patients. Risk = WHO risk categories; ISH = isolated systolic hypertension; constant mono-and polytherapy; on & off = on and off treatment breaks; * = p < 0.05 compared to the rest of the patients.
Discussion
Taken together, the data of this postmarketing survey confirm that irbesartan is a well tolerated and effective antihypertensive agent when used in a real life setting at the dose of 150 mg qd. More interestingly, our data provide further insights on factors affecting either positively or negatively the persistence with antihypertensive treatment. In particular, our observations point out the importance of a good tolerability profile and of achieving a rapid control of blood pressure in enhancing persistence whereas late changes in treatment and addition of irbesartan in patients already treated with a fixed-dose combination appears to be factors promoting a lower persistence.
Prospective observational surveys are not specifically designed to evaluate the antihypertensive efficacy of a new agent since there is generally no control group and the treatment schedule is not standardized. Moreover, there may be a selection bias since the inclusion of patients was not randomised. Nevertheless, this type of survey may provide some valid information on the antihypertensive effect that may be obtained in real life conditions i.e. outside the rigorous context of a clinical trial and the rather large number of patients included certainly limit the effect of a systematic selection bias. In the present case most patients were treated with a 150 mg irbesartan tablet per day because this was the dose recommended at the time the survey was conducted, i.e. soon after the launch of irbesartan in Switzerland. All patient subgroups, but in particular naïve patients, responded positively to the treatment. In fact, in the group of patients with follow-up values and after a mean observation time of 4 months, the average reductions in blood pressure were of comparable magnitude that those obtained in clinical trials and in other post-marketing surveys [25-27]. Such a substantial reduction could be achieved in spite of the fact that irbesartan 150 mg represents the minimal recommended daily dosage nowadays.
Of note, no blood pressure target was pre-defined in our program. Moreover, one should consider that the reported blood pressure values show a clear digit preference for figures ending with a 0 or a 5. This reflects the tendency of the GPs to round blood pressure values, explainable by the wide use of sphygmomanometers. Therefore, the effect on blood pressure was assessed using different criteria. With the generally accepted targets of <140/90 mmHg or a diastolic BP below 90 mmHg, respectively 26 % and 60% of patients were controlled with 150 mg irbesartan qd. In a more recent survey, in which physicians had the opportunity to use the higher dose of 300 mg or the combinations of irbesartan 150 or 300 mg with hydrochlorothiazide 12.5 mg, the percentage of patients with a blood pressure below 140/90 mmHg increases to more than 60% [28]. Looking at sub-populations, older patients and pre-treated patients had more problems in reaching the therapeutic target chosen for analysis (≤ 140/90 mmHg). The same was true for patients with more cardiovascular risk factors, but not for people with associated clinical conditions – the highest risk factor according to the WHO 1999 guidelines, JNC-7 and ESH-ESC 2003 guidelines – who, on the contrary, reached the target more easily [8,9,29]. This observation further emphasize the known difficulty to achieve a good control of systolic blood pressure particularly in elderly patients with isolated systolic hypertension.
In post-marketing surveys in real life settings, the evaluation of safety is of great importance. More than 90% of patients tolerated the treatment well or very well according to their GPs and adverse events were reported by only 8% of them. Serious adverse events possibly or probably related to irbesartan occurred in less than ten cases. The patients with more risk factors tolerated irbesartan equally well, despite taking significantly more drugs. The most frequent adverse events were dizziness, nausea, headache, dyspepsia and diarrhea, and occurred at the rate described in the Swiss prescribing information. These data therefore confirm the excellent tolerability profile of angiotensin II receptor antagonists reported in clinical trials [30].
The main observation of the present survey is the assessment of the factors determining long-term persistence with the irbesartan treatment in our population. Out of the 4639 patients with complete follow-up data, 82.5% continued to take irbesartan for more than 4 months. When evaluating factors affecting persistence some interesting observations were made. The first and expected ones are that a good tolerability profile and the achievement of a rapid blood pressure control are positively associated with the long-term persistence with therapy. This finding would therefore encourage the use of well-tolerated antihypertensive drugs such as angiotensin II receptor antagonists at high doses in order to obtain a rapid control of blood pressure. It may also favor the use of fixed dose combinations as first line treatment since these combinations are associated with a low side-effect profile and an improved efficacy [31].
A consistent majority of the study population (61.5%) entered the trial after a failed experience with other antihypertensive drugs. Irbesartan proved to be the drug inducing more persistence in a comparative study with newly-diagnosed patients [19]; therefore, it was also interesting to appraise the persistence rate in pre-treated patients. To our surprise, patients who switched from a fixed combination treatment tended to discontinue irbesartan more often. On the contrary, patients who abandoned the previous treatment because of cough (a class side effect of ACE-Inhibitors), tended to stick more to irbesartan. Moreover, late changes in treatment schedule had a negative impact on persistence whereas early changes in treatment had a rather favorable impact on persistence. These negative influences on persistence are probably linked to the increased complexity of the treatment schedule which is known to impair compliance as well as persistence. Indeed, several previous studies have demonstrated that drug adherence decreases in proportion with the complexity of the drug regimen [32,33].
In this survey, an attempt was made to obtain information on drug adherence using simplified questionnaires addressed to the patients and physicians. Unfortunately, only a small proportion of these questionnaires were filled by the participants. There is therefore a high probability of bias towards highly compliant patients. Moreover, questionnaires are known to overestimate drug adherence. Hence it is not surprising that good compliance with the dosing schedule was reported by more than 90% of the patients after about one year treatment, meaning that this fraction of the patients reported to have taken irbesartan at least 6 times a week in the preceding 3–4 weeks. Yet, the results of more than 1000 questionnaires confirm previous observations such as the lower compliance in men and the absence of effect of age. Indeed, Degoulet at al have also reported thatmale sex is a variable significantly associated with an increased dropout rate in hypertensive patients attending a hypertension clinic [34]. Of interest is the observation that patients with a low cardiovascular risk appear to have a lower compliance whereas those presenting with an isolated systolic hypertension have a higher compliance. These findings may be related to the patients' perception of their disease and their degree of concern. Thus, patients with a low cardiovascular risk may be less motivated to take their medications whereas patients with high systolic blood pressure may be particularly concerned by their hypertension. In line with this finding, we have found recently that epileptic patients well controlled under treatment are less compliant than those experiencing repeated seizures despite treatment [35].
In conclusion, this survey confirms that irbesartan is a safe and effective antihypertensive drug in clinical practice. A high persistence with irbesartan was found in this program which is likely related to the consistent reductions in blood pressure and the good tolerability profile. This post-marketing survey has also tend to confirm in a large population of patients that achieving an early control of blood pressure may be a positive predictor of persistence. Based on this observation one could encourage physicians to start the antihypertensive therapy more aggressively using higher doses of well tolerated drugs such as angiotensin II receptor antagonists or fixed low-dose combinations in order to improve blood pressure control as well as long-term persistence.
Competing Interests
All authors had a consultant agreement with both Bristol Myers Squibb and Sanofi-Synthelabo. These companies supported the study financially and monitored the study.
Authors' contributions
MB has written the paper, BH, PG and BW have contributed to the conception of the study, to the analysis of the data and to the redaction of the paper.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This study was supported by Sanofi-Synthelabo and Bristol-Myers Squibb, Switzerland.
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| 15943871 | PMC1166543 | CC BY | 2021-01-04 16:30:07 | no | BMC Cardiovasc Disord. 2005 Jun 8; 5:13 | utf-8 | BMC Cardiovasc Disord | 2,005 | 10.1186/1471-2261-5-13 | oa_comm |
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BMC Cardiovasc DisordBMC Cardiovascular Disorders1471-2261BioMed Central London 1471-2261-5-151597210310.1186/1471-2261-5-15CommentaryPeripheral arterial disease: A high risk – but neglected – disease population Tomson Joseph [email protected] Gregory YH [email protected] University Department of Medicine, City Hospital, Birmingham B18 7QH, England, UK2005 22 6 2005 5 15 15 20 6 2005 22 6 2005 Copyright © 2005 Tomson and Lip; licensee BioMed Central Ltd.2005Tomson and Lip; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Peripheral arterial disease (PAD) is a common, progressive manifestation of atherothrombotic vascular disease, which should be managed no different to cardiac disease. Indeed, there is growing evidence that PAD patients are a high risk group, although still relatively under-detected and under treated. This is despite the fact that PAD patients are an increased mortality rate comparable to those with pre-existing or established cardiovascular disease [myocardial infarction, stroke]. With a holistic approach to atherothrombotic vascular disease, our management of PAD can only get better.
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Peripheral arterial disease (PAD) results from the narrowing of the blood vessels of the lower limbs, predominantly secondary to atherosclerotic vascular disease leading to compromised blood flow to the lower limbs. Risk factors associated with PAD include typical cardiovascular risk factors, such as older age, cigarette smoking, diabetes mellitus, hypercholesterolemia, and hypertension [1]. PAD is also very common in the western world – based on symptoms of intermittent claudication, PAD has a prevalence of up to 5%, but a higher prevalence is estimated in the general population [1,2]. As with much of cardiovascular disease, PAD can be progressive, which about a third report worsening symptoms and require surgical interventions over 5 to 10 years [3] as well as suffer a higher cardiovascular mortality risk [2-4]. One could argue that compared to the large publicity and public health initiatives on heart attacks and strokes, public recognition on the risks, morbidity and mortality associated with PAD has been relatively neglected.
Given that the pathology in PAD arises secondary to atherosclerosis (or atherothrombosis) affecting the vascular tree, PAD would be a very useful, subtle and early indicator of a high risk population. The realisation that the majority of those with PAD remain asymptomatic [4], and that they can be more effectively identified by simple non-invasive techniques warrants new hope for the primary prevention of cardiovascular risk.
In their recent article published in BMC Cardiovascular Disorders, Caro et al [5] embark upon estimating the burden of cardiovascular risk in terms of mortality, morbidity and associated risk factors in patients with PAD. Of note, they compared outcomes between the PAD cohort to those with reference populations with a first diagnosis of myocardial infarction and stroke. They report that the mortality rate amongst those with PAD was higher when compared to those who had an index myocardial infarction but lower than those who had suffered stroke. Importantly, the rates were comparable to the other high risk cohorts, with the crude five year death rate standing at 33.2% [26.6% for index myocardial infarction; 41.8% for index stroke]. Furthermore, at the end of the follow-up period, half of the patients with PAD were alive, compared to 59.6% of those in the myocardial infarction group and 51.3% of those with stroke, respectively[5]. Broadly similar findings have also been reported in previous studies involving patients with PAD symptoms [6,7].
As expected, risk factor analyses revealed that men with PAD were more likely to have atherothrombotic complications, angina, myocardial infarction, stroke, and death [5]. Also, the risk of myocardial infarction was significantly increased with in those aged over 65 years, especially with concomitant angina, diabetes mellitus, heart failure, and hypertension – the increase in cardiovascular risk in those with PAD was dependent on the number of additional risk factors at the time of diagnosis in comparison to those who did not have any other risk factors at baseline, illustrating how risk factors are additive to each other, resulting in a 'cumulative' cardiovascular risk[5]. The parallels with cardiac disease are uncanny.
The study by Caro et al [5] does merit some scrutiny. As it is a database/registry-type study, and there are limitations to the data presented. For instance, temporal changes in blood pressures and laboratory values are not available for further analyses, as well as detailed information on optimization of medical therapies. Data on concomitant risk factors such as atrial fibrillation [8] could have helped in further understanding their long term impact on PAD and outcome. Case ascertainment also remains an issue, as the PAD cohort in the study by Caro et al [5] was identified based on the ICD codes used in case records. Though different methods with varying complexities are used currently [9], the ankle brachial pressure index seems a simple effective and noninvasive solution and the best mode for diagnosis. However, it would be a daunting task to ensure confirmatory evidence in a standardized detailed population examination of this magnitude. One may argue that the patients with prior stroke and myocardial infarction were at a better advantage, making them a 'more scrutinized' cohort with better provisions for active secondary risk factor modification.
The conclusion that having PAD puts you at a much higher risk which is comparable to the reference populations is therefore a justified one, and a message that needs to be emphasized. Eventhough the majority of PAD subjects remain asymptomatic, they remain a high-risk population – probably even higher than those with single cardiovascular risk factors. Of concern, risk factor reduction by lipid modifying therapy and antiplatelet therapy in those with a diagnosis of PAD is less frequently applied [10] in comparison to those with cardiac disease. Thus, PAD patients may have had a less intensive risk reduction, leading to a high cardiovascular event rate.
Should we be surprised? Cynical cardiologists would argue that these aspects are well-known for heart disease, but as PAD commonly presents to vascular surgeons (at least in the UK) the onus is on them to initiate full cardiovascular risk prevention therapies (a 'best medical therapy' strategy), such as smoking cessation and the aggressive treatment of hypertension and lipids. However, many PAD patients also have cardiac problems, and may attend blood pressure and lipid clinics. Perhaps the time has come to organize vascular clinics jointly run by vascular surgeons and vascular physicians (even cardiologists!), as one possible solution. The approach to the PAD patient should not be simply surgery (or a stent) but should include focus on aggressive risk factor modification [11,12].
However, cardiac patients are blessed with many well conducted large randomized controlled clinical trials, which PAD patients often have to depend on subgroup analyses from these studies to inform their management. Though clinical trials of lipid-lowering therapy for patients with PAD per se are not available, a systematic review by the Cochrane collaboration of seven clinical trials [13] which included 698 patients with PAD, showed a substantial but not statistically significant reduction in total mortality (odds ratio 0.21; 95% confidence interval [CI], 0.03–1.17), with lipid-lowering therapy. Furthermore, there are new studies indicating atherosclerosis regression with aggressive lipid-lowering therapy and with antihypertensive therapy, offering an effective interventional target [14,15], again emphasising the role for aggressive risk factor modification. Antiplatelet therapy with aspirin or thienopyridine drugs (ticlopidine and clopidogrel) reduces the risk of cardiovascular events in patients with established atherosclerosis [16], but in PAD patients included in the Antithrombotic Trialists Collaboration metaanalysis, there was no significant difference in cardiovascular outcomes. As in cardiac disease, blockade of the renin-angiotensin system may offer another therapeutic target. For example, in the Heart Outcomes Prevention Evaluation (HOPE) study [17], 44% of the study population had PAD and an impressive reduction in cardiovascular events was seen in the ACE inhibitor (ramipril) group, the reduction being independent of blood pressure reduction.
In conclusion, Caro et al [5] allows us to take stock of our approach to managing PAD. PAD should be managed no different to cardiac disease. Indeed, there is now growing evidence that PAD patients are a high risk group, although still relatively under-detected and under treated. This is despite the fact that PAD patients are an increased mortality rate comparable to those with pre-existing or established cardiovascular disease [myocardial infarction, stroke]. With a holistic approach to atherothrombotic vascular disease, our management of PAD can only get better.
Pre-publication history
The pre-publication history for this paper can be accessed here:
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| 15972103 | PMC1166544 | CC BY | 2021-01-04 16:30:07 | no | BMC Cardiovasc Disord. 2005 Jun 22; 5:15 | utf-8 | BMC Cardiovasc Disord | 2,005 | 10.1186/1471-2261-5-15 | oa_comm |
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BMC Endocr DisordBMC Endocrine Disorders1472-6823BioMed Central London 1472-6823-5-51592708310.1186/1472-6823-5-5Research ArticleAn IGF-I promoter polymorphism modifies the relationships between birth weight and risk factors for cardiovascular disease and diabetes at age 36 te Velde Saskia J [email protected] Rossum Elisabeth FC [email protected] Paul G [email protected] Jos WR [email protected] de Waal Henriette A Delemarre [email protected] Coen DA [email protected] Mechelen Willem [email protected] Steven WJ [email protected] Han CG [email protected] Institute for research in extramural medicine (EMGO), VU University Medical Center, Amsterdam, The Netherlands2 Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands3 Department of Pediatric Endocrinology, VU University Medical Center, Amsterdam, The Netherlands4 Department of Clinical Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands5 Institute for Cardiovascular Research and Department of Internal Medicine, VU University Medical Center, Amsterdam, and Department of Medicine, University Hospital Maastricht, Maastricht, The Netherlands6 Department of Social Medicine and Body@Work research centre for physical activity, work and health TNO-VU, VU University Medical Center, Amsterdam, The Netherlands2005 1 6 2005 5 5 5 7 2 2005 1 6 2005 Copyright © 2005 te Velde et al; licensee BioMed Central Ltd.2005te Velde et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Objective
To investigate whether IGF-I promoter polymorphism was associated with birth weight and risk factors for cardiovascular disease (CVD) and type 2 diabetes (T2DM), and whether the birth weight – risk factor relationship was the same for each genotype.
Design and participants
264 subjects (mean age 36 years) had data available on birth weight, IGF-I promoter polymorphism genotype, CVD and T2DM risk factors. Student's t-test and regression analyses were applied to analyse differences in birth weight and differences in the birth weight – risk factors relationship between the genotypes.
Results
Male variant carriers (VCs) of the IGF-I promoter polymorphism had a 0.2 kg lower birth weight than men with the wild type allele (p = 0.009). Of the risk factors for CVD and T2DM, solely LDL concentration was associated with the genotype for the polymorphism. Most birth weight – risk factor relationships were stronger in the VC subjects; among others the birth weight – systolic blood pressure relationship: 1 kg lower birth weight was related to an 8.0 mmHg higher systolic blood pressure
Conclusion
The polymorphism in the promoter region of the IGF-I gene is related to birth weight in men only, and to LDL concentration only. Furthermore, the genotype for this polymorphism modified the relationships between birth weight and the risk factors, especially for systolic and diastolic blood pressure.
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Background
Insulin-like growth factor-1 (IGF-I) is a peptide that is involved in fetal growth and cell differentiation [1,2] In addition, it has been suggested that this peptide plays a role in the regulation of glucose homeostasis and cardiovascular function [3-6] (Lower IGF-I levels are also associated with increased levels of serum low-density lipoprotein (LDL) [7]. IGF-I also plays a role in plaque development [8].
A genetic polymorphism comprising a variable length cytosine-adenine (CA) repeat sequence in the promoter region of the IGF-I gene has been identified, and is thought to influence the transcription rate of IGF-I, which in turn affects serum IGF-I levels [9]. Since IGF-levels are associated with fetal growth and adult risks for cardiovascular disease (CVD) and type 2 diabetes (T2DM), it has been suggested that the polymorphism in the promoter region of the IGF-I gene might be relevant to the fetal origins hypothesis. This hypothesis suggests that an adverse environment during the intra-uterine period negatively affects fetal growth (often estimated by birth weight), and results in adaptations that permanently change the structure and functions of the body, which leads to an increased risk for disease, such as CVD and T2DM, at adult age [10]. An alternative hypothesis is that impaired fetal growth and increased risk for CVD and T2DM share a common genetic factor [11,12] A study performed in Rotterdam in the Netherlands has recently shown that the absence of the wild type allele (192 base pair (bp)) in the promoter region IGF-I gene was related to lower birth weight [13]. In addition, the intra-uterine environment may interact with genetic polymorphisms [14]. This has already been found in other studies, two of which concerned on birth weight and genetic factors in insulin metabolism [15-17] These data raise the issue of whether or not birth weight also interacts with the IGF-I gene.
In the Amsterdam Growth and Health Longitudinal Study (AGAHLS), data on birth weight, and risk factors for CVD and T2DM have been collected. The IGF-I gene has now been analysed for the 192 bp polymorphism [9,13], in order to address the following four research questions: 1) Is the IGF-I promoter polymorphism genotype associated with birth weight? 2) Is IGF-I promoter polymorphism genotype associated with risk factors for CVD and T2DM? 3) Is birth weight associated with risk factors for CVD and T2DM and finally 4) Do intra-uterine environment and the IGF-I promoter polymorphism genotype interact? Or, in other words, is the association between birth weight and risk factors for CVD and T2DM different for each genotype of this polymorphism?
Methods
Participants
The Dutch population described in this study are all participants of the AGAHLS. The AGAHLS is an observational study with repeated measurements, which started in 1976 to monitor boys and girls with a mean age of 13 years [18]. During the most recent measurement in which 433 subjects participated, subjects had than reached a mean age of 36 years, information was obtained concerning birth weight, IGF-I promoter polymorphism genotype, glycated hemoglobin (HbA1c), carotid intima-media thickness (IMT), blood pressure, anthropometry and serum low density lipoprotein (LDL) cholesterol levels. Three-hundred-ninety-one subjects completed the birth-weight questionnaire of whom 380 had written information or information from parents. For the purpose of the present study, subjects who were born pre-term (before 37 weeks of gestation, 27 subjects), were one of a twin (11 subjects) or were of non-Caucasian ethnicity (9 subjects) were excluded. Another 69 subjects were not taken into account for the analyses because of errors or missing values in the genotyping or risk factor measurements. Finally, for 264 subjects (152 women) there were complete data sets on the IGF-I promoter polymorphism genotype, birth weight, and adult risk factors for CVD and T2DM. Subjects included for the analyses were smaller (1.76 m vs. 1.77 m, p = 0.030) and had less fat-free mass (56.5 kg vs. 57.9 kg, p = 0.056) compared to subjects not included for these analyses, but with data on the outcomes available (N = 109). On all other outcome variables the groups did not differ. (Some outcome measures had missing values, which were due to error in the specific measurement.) All subjects were apparently healthy at the time of the measurements, and none had been diagnosed with T2DM. All subjects gave written informed consent and the Medical Ethical Committee of the VU University Medical Center approved the protocol.
Birth weight
Data on birth weight were obtained by means of a questionnaire. The questions concerned birth weight, gestational age, being one of a twin, and ethnicity, and subjects were also asked about the source of the information. Only those who had received the information from their parents or had it in written documents were included, as this has been shown to be a valid method [19,20] Subjects born preterm (gestational age < 37 weeks) were excluded, this may have independent effects on adult health or influence the relationship between birth weight and adult health [21-23]. Twins were excluded because they have different fetal growth patterns, which might cause error when analysing the relationship between birth weight and adult health outcomes. Subjects who retrieved the birth weight information from their parents' memory (n = 112) had slightly higher mean birth weights (3.54 ± 0.53 kg) compared with the subjects who retrieved the requested information from written documents (n = 152; 3.44 ± 0.48 kg), but this difference was not significant (p = 0.10). Furthermore, they did not differ significantly on any of the outcome measures.
Polymorphism in the promoter region of the IGF-I gene
IGF-I promoter polymorphism genotypes were determined as described earlier [9]. In brief, DNA was isolated using standard methods. PCR was performed in a final volume of 10 μL containing 10 ng DNA, 10* Gold (Au) buffer (Perkins and Elmer), 200 M dNTP, 30 pmol of each primer, 3 mM MgCl2, 0.5 U Ampli Tag Gold polymerase (Perkins and Elmer). The PCR program concisted of 30 cycli of 30 sec 95°C, 30 sec 55°C and 30 sec 72°C and additionally 5 min of denaturation at 95°C before the first cycle and an extension of 10 min. at 72°C after the last cycle. Forward primers were labelled with FAM (Weber & May 1989) to determine the size of the PCR products by fragment analysis (ABI-Prism genetic analyser with Genescan 2.1 software). The Genescan 350/500 Tamra was used as internal size standard within the fragment analysis.
Rietveld et al[24] recently demonstrated that subjects who were homozygous for the 192 bp or the 194 bp allele had comparable IGF-I blood levels, while individuals who were homozygous for either alleles shorter than 192 bp or longer than 194 bp had significantly lower serum IGF-I levels. Therefore, we decided to regard all subjects who were homozygous for 192 bp or 194 bp, or were carrier of a 192 bp allele and a 194 bp allele as wild types (WTs). Consequently, all subjects who were carrier of a variant allele, which is either shorter than 192 bp or longer than 194 bp, were grouped as variant carriers (VCs).
Risk factors for CVD and T2DM
The following risk factors for CVD and T2DM were measured: body mass index (BMI), waist circumference, waist-to-hip ratio (WHR), total fat mass (FM), total fat-free mass (FFM), carotid intima-media thickness (IMT), systolic and diastolic blood pressure (SBP, DBP), resting heart rate, LDL cholesterol levels and HbA1c as an estimate of glucose metabolism (unfortunately no glucose measures were available). BMI was calculated as body weight (kg) divided by body height (m2). Standing height was measured with a stadiometer to the nearest 0.001 m. Body weight (kg) was measured to the nearest 0.1 kg using a spring balance scale (Van Vucht, Amsterdam, The Netherlands), with subjects dressed only in underwear. Waist (at the level of the umbilicus) and hip circumference were measured with a flexible steel tape to the nearest 0.1 cm. WHR was calculated as the ratio between waist circumference and hip circumference. Fat mass (FM) was estimated from four skinfolds (biceps, triceps, subscapular and supra iliacal) with the Durnin and Womersley equation [25]. The four skinfolds were measured according to standard procedures [26]. FFM was calculated by subtracting FM from body weight.
IMT of the right common carotid artery was obtained by an ultrasound scanner equipped with a 7.5 MHz linear array probe (Pie Medical, Maastricht, The Netherlands), as described elsewhere in more detail [27-29] SBP and DBP were assessed in the left arm at 5-minute intervals with an oscillometric device (Colin Press-Mate, model BP 8800, Komaki-City, Japan) during the entire period of ultrasound imaging when the subjects were lying in a supine position. The mean value over this entire period was calculated. Resting heart rate was measured with the same device as used for the blood pressure measurement. The mean value over this measurement period was calculated and used in the analyses.
Serum LDL and HbA1c (%) were measured from blood samples (10 ml) taken from the antecubital vein between 8.30 and 12.30 a.m. with subjects in a non-fasting state. Standard methods were used to analyse the LDL concentration and external quality control took place with target samples from a World Health Organisation reference laboratory (Lipid Standardization Laboratory, Atlanta, USA). HbA1c was determined by non-exchange high performance liquid chromatography with a modular Diabetes Monitoring System (Bio-Rad, Veenendaal, the Netherlands).
Data-analyses
A t-test was used to analyse differences in birth weight between the IGF-I promoter polymorphism genotypes. Multiple linear regression analyses were applied to study the associations between birth weight and the risk factors for CVD and T2DM. The results of the regression analyses were presented as regression coefficients (β) and the corresponding 95% confidence intervals (CI) for two different models. The first model was a crude analysis, only adjusted for gender (and for SBP and DBP in case of IMT). The second was further adjusted for adult body weight.
To investigate whether the relationship between birth weight and the risk factors for CVD and T2DM were modified by the IGF-I promoter polymorphism, multiple linear regression was performed between birth weight and all risk factors (all as continuous variables) for the two genotypes separately.
All analyses were performed with the Statistical Package of Social Science (SPSS, Chicago, USA) version 10.1. The statistical significance was set at p-value ≤ 0.05.
Results
Table 1 gives allele and genotype frequencies in this cohort. As can be seen, of all 264 subjects, 189 (111 women) were WTs for the IGF-I promoter polymorphism. The remaining 75 subjects (41 women), 4 were homozygous for alleles with variant CA repeats and 71 were heterozygous for variant alleles. The distribution of genotypes was in Hardy-Weinberg equilibrium (p = 0.17).
Table 1 Allelic and genotype frequencies for the bp repeat polymorphism at the promoter region of the IGF-I gene
Allele (bp length) Frequency N (%) Genotype Frequency N (%)
176 3 (0.5) WT 192/192 105 (39.8)
188 10 (1.9) 192/194 78 (29.5)
190 23 (4.4) 194/194 6 (2.3)
192 341 (64.6) total 189 (71.6)
194 108 (20.5) VC 192/x 53 (20.0)
196 38 (7.2) 194/x 18 (6.8)
198 5 (1.0) x/x 4 (1.5)
total 75 (28.4)
X, alleles with bp lengths other than 192 or 194; WT, Wild Type; VC, Variant Carrier
Population characteristics on all measured variables are presented in Table 2, stratified according to gender and genotype. In men, the mean birth weight was 0.2 kg lower in the VC group than in the WT group (p = 0.009). In women, no significant differences in birth weights between the genotypes were observed (p = 0.755).
Table 2 Characteristics of the adult population of the Amsterdam Growth and Health Longitudinal Study
Men Women
WT VC WT VC
Mean ± SD Mean ± SD Mean ± SD Mean ± SD
Birth weight (kg) 3.64 ± 0.47 3.40 ± 0.39 3.44 ± 0.53 3.41 ± 0.52
Height (m) 1.84 ± 0.08 1.83 ± 0.07 1.71 ± 0.06 1.72 ± 0.06
Weight (kg) 84.1 ± 10.8 85.2 ± 10.8 67.5 ± 9.3 69.2 ± 11.8
Body mass index (kg/m2) 23.5 ± 2.6 24.5 ± 2.4 22.4 ± 2.9 22.8 ± 3.8
Fat-mass (kg) 13.7 ± 5.0 15.3 ± 5.6 17.7 ± 5.3 18.4 ± 6.4
Fat-free mass (kg) 66.1 ± 6.6 66.3 ± 5.7 47.3 ± 4.5 48.7 ± 5.9
Waist circumference (cm) 85.3 ± 7.5 85.9 ± 8.8 73.3 ± 8.9 73.4 ± 8.0
Waist-to-hip ratio 0.95 ± 0.04 0.96 ± 0.05 0.83 ± 0.08 0.82 ± 0.08
Systolic blood pressure (mmHg) 121.0 ± 8.9 122.7 ± 14.4 110.7 ± 10.6 111.8 ± 12.0
Diastolic blood pressure (mmHg) 66.4 ± 6.4 67.2 ± 8.6 63.1 ± 7.2 61.6 ± 6.9
LDL concentration (mmol/l) 3.22 ± 0.80 3.52 ± 0.87 2.80 ± 0.81 2.97 ± 0.70
Carotid intima-media thickness (mm) 0.628 ± 0.099 0.636 ± 0.095 0.616 ± 0.086 0.631 ± 0.101
Resting heart rate (b/min) 73 ± 12 69 ± 11 72 ± 12 70 ± 12
Glycated hemoglobin (%) 5.3 ± 0.5 5.3 ± 0.3 5.3 ± 0.4 5.3 ± 0.3
Data is presented as means ± standard deviations (SD),
†WT, Wild type; VC, Variant carrier LDL – low lipoprotein
Subjects in the VC group had significantly higher LDL concentrations (p = 0.039). No other significant differences between the genotypes were observed.
Table 3 presents the results of the linear regression analyses for the relationship between birth weight and risk factors for CVD and T2DM. It was found that 1 kg higher birth weight was associated with 2.55 kg more FFM. However, this association decreased and lost significance after adjustment for adult body weight. In addition, birth weight was found to be associated with SBP in such a way that 1 kg lower birth weight was related to a 3.05 mmHg higher SBP. No other significant associations were observed between birth weight and risk factors for CVD and T2DM.
Table 3 Results of the linear regression analyses for the relationship between birth weight and adult risk factors for CVD and DM-2
Crude† Adjusted ‡
Outcome β 95%CI β 95%CI
Body mass index (kg/m2) 0.445 [-0.264; 1.154]
Fat-mass (kg) 0.962 [-0.361; 2.285] -0.663 [-1.605; 0.208]
Fat-free mass (kg) 2.553*** [1.224; 3.882] 0.714 [-0.085; 1.513]
Waist circumference (cm) 1.760 [-0.372; 3.892] -1.013 [-2.253; 0.227]
Waist-to-hip ratio 0.012 [-0.005; 0.028] 0.006 [-0.011; 0.022]
Systolic blood pressure (mmHg) -1.387 [-4.082; 1.309] -3.050* [-5.626; -0.475]
Diastolic blood pressure (mmHg) -0.692 [-2.445; 1.071] -1.608 [-3.329; 0.113]
Resting heart rate (b/min) 1.941 [-0.932; 4.815] 1.861 [-1.081; 4.803]
Low density lipoprotein (mmol/l) -0.079 [-0.277; 0.118] -0.154 [-0.351; 0.044]
Carotid intima-media thickness (mm) ∥ 0.011 [-0.012; 0.034] 0.011 [-0.012; 0.035]
Glycated hemoglobin (%) -0.013 [-0.109; 0.082] -0.025 [-0.123; 0.072]
Data is presented as regression coefficients and corresponding 95% confidence intervals (CI)
CVD – cardiovascular disease; DM-2 – type 2 diabetes mellitus
† Crude, only adjusted for gender; ‡, Adjusted, further adjusted for adult body weight
* p < 0.05 *** p < 0.001
∥ also adjusted for systolic and diastolic blood pressure
Table 4 shows the results of the linear regression analyses between birth weight and the risk factors for CVD and T2DM, stratified according to IGF-I promoter polymorphism genotypes. In most of the associations studied, the regression coefficient for birth weight was highest in the VC group, indicating a stronger effect of birth weight on the outcome variable. This difference was most marked in the association with SBP, in which a 1 kg lower birth weight was related to an 8.0 mmHg increase in adult SBP in the VC group, compared to a 1.4 mmHg increase in the WT group. Although, the difference between the VC group and WT group was not statistically significant (p = 0.08) for these kind of 'interactions' normally a higher significance level is used. In addition, the relationships between birth weight and DBP and FM were significant in the VC group and not in the WT group (p = 0.06 for the difference between VCs and WTs for DBP). The differences between VCs and WTs regarding all other relationships showed p-values > 0.10.
Table 4 Analyses stratified according to genotype for the relationship between birth weight and risk factors for CVD and DM-2
Wild type Variant carrier
Risk factor β 95% CI β 95% CI
BMI (kg/m2) 0.475 [-0.312; 1.262] 0.777 [-0.835; 2.338]
Fat mass (kg) -0.217 [-1.337; 0.904] -1.929* [-3.742; -0.116]
Fat free mass (kg) 0.780 [-0.212; 1.722] 0.473 [-0.897; 1.844]
Waist circumference (cm) -1.380 [-2.822; 0.061] 0.040 [-2.588; 2.668]
Waist-hip ratio 0.002 [-0.016; 0.021] 0.021 [-0.015; 0.057]
Systolic blood pressure (mmHg) -1.497 [-4.265; 1.272] -8.038* [0.014; -14.391]
Diastolic blood pressure (mmHg) -0.599 [-2.553; 1.355] -5.073** [-8.845; -1.301]
LDL cholesterol (mmol/l) -0.138 [-0.369; 0.093] -0.076 [-0.491; 0.339]
Resting heart rate (b/min) 2.540 [-0.865; 5.946] -1.995 [-8.277; 4.286]
Carotid intima-media thickness (mm)‡ 0.010 [-0.017; 0.036] 0.020 [-0.035; 0.075]
Glycated hemoglobin (%) -0.019 [-0.138; 0.101] -0.025 [-0.207; 0.157]
Data is presented as regression coefficients (β) and their corresponding 95% confidence intervals (CI)
CVD – cardiovascular disease; DM-2 – type 2 diabetes mellitus
Models were adjusted for gender and adult body weight * p < 0.05 ** p < 0.01
‡ also adjusted for systolic and diastolic blood pressure
For the associations between birth weight and adult FFM, waist circumference, LDL and resting heart rate, the regression coefficients were highest in the WT group, although none of these associations were statistically significant.
Discussion
In this study, the associations between a polymorphism in promoter region of the IGF-I gene, birth weight, (as a measure of intra-uterine growth), and risk factors for CVD and T2DM were investigated, in order to obtain more insight into the genetic aspects of the fetal origins hypothesis [1,5,6,30,31](The results of the present study demonstrate that men who were carriers of one or two variant allele(s) of the IGF-I gene had significantly lower birth weights. However, this trend was not observed in women. It is not clear, why this association was absent in women, as no other study has reported gender differences in the association between IGF-I genotype and birth weight [13,32,33]. Therefore, the gender difference observed in this study might be a result of chance
So far, results on the association between IGF-I genotype and birth weight have been conflicting. Vaessen et al. reported that absence of the wild type allele (192 bp) resulted in a lower birth weight, but subjects who were heterozygous for the wild type allele did not differ in birth weight from the homozygous subjects [13]. Nevertheless, Vos et al. [33], Frayling et al. [32] and Day et al [34] could not confirm these findings. These conflicting results could be due to differences in the population backgrounds, but also to the way in which subjects were classified per genotype. In the present study, an alternative method was used, in which the allele with 194 bp was also considered as a wild type allele, based on the observations made by Rietveld et al[24]. Subjects previously categorised as VC were now categorised as WT (e.g. subjects with genotype 192 bp/194 bp, or 194 bp/194 bp). Therefore, the VC group in other studies was actually heterogeneous, which may explain discrepant observations. To investigate this possible explanation, we investigated the characteristics of the subjects who would have been categorised as VC according to the traditional classification, and were now categorised as WT (84 subjects). The men in this group had a mean birth weight of 3.63 kg (± 0.51 kg), which is comparable with the men who were categorised as WT in both classification methods (3.64 ± 0.43 kg). The women in this group had a mean birth weight of 3.44 kg (± 0.53 kg), which is exactly the same as the women classified as WT in both methods. Besides this, the three groups were also different with regard to the interaction between birth weight and SBP and DBP in a way that the relationship between birth weight and adult SBP and DBP was strongest and significant in the 'constant' VCs (β = -8.0 for SBP and β = -5.1 for DBP), and weak and not significant in the two other groups. These results suggest that the alternative method used in the present paper discriminates better between the genotypes with regard to the observed health outcomes.
The results concerning risk factors for CVD and T2DM showed an increased risk in the VC group solely for LDL concentrations, all other risk factors did not differ between WT and VC groups. That only one risk factor was significantly different between the groups might be real or a result of chance, since we tested several associations. Moreover, the relationship between IGF-I genotype and LDL concentrations disappeared after adjustment for BMI and there were no differences in HDL concentrations (p = 0.255, data not shown). Several studies have shown that lower IGF-I bio-activity is related to higher incidence of atherosclerotic cardiovascular disease, higher carotid IMT values, lower levels of HDL cholesterol, and impaired glucose tolerance [5-7,35].(However, other studies have failed to show these associations [36-38] Until now, observations have thus been inconsistent, which may be due to other factors that affect cardiovascular health, insulin metabolism and serum IGF-I levels, such as nutrition and endocrine factors. One should, however, realise that the subjects of the present study were still rather young (i.e. 36 years), and that a longer exposure to lower IGF-I bioactivity might be necessary to induce unfavourable levels of risk factors for CVD and/or T2DM.
Another aim of the present study was to investigate whether birth weight was associated with risk factors for CVD and T2DM. This was found to be the case for FFM, however this association decreased after adjustment for body weight. When studying this association within tertiles of BMI, it was found that only in the 2nd tertile the relationship between birth weight and FFM was significant (data not shown). Another significant association was found between birth weight and for SBP, which is in line with others and previously found in the AGAHLS[39,40]. No other significant associations were found, although the associations between birth weight and adult LDL, FM and waist circumference were in the expected (negative) directions [41-46] No associations were found between birth weight and resting heart rate or carotid IMT. This latter finding is in contrast with what has been reported by Leeson et al. [47]. However, their study focussed on an older population.
Birth weight is considered to be mainly dependent on the intra-uterine environment, such as the availability of nutrients and oxygen [30]. In the present population, the IGF-I genotype could only explain 6% of the variance in birth weight in men, and only 1% of the variance in birth weight in women. The magnitude of the relationship between birth weight and risk factors for CVD and T2DM, however, seems to be dependent on genes (i.e. IGF-I promoter polymorphism) (Table 4). This modification was strongest in the association between birth weight and blood pressure. In the VC group, a 1 kg lower birth weight was found to be related to an 8 mmHg increase in SBP, which is much more than has been reported in the literature (2 to 3 mm Hg) [39]. The relationship between birth weight and DBP was also stronger than was expected (as 1 kg lower birth weight was related to a 5 mmHg increase in DBP). No other study has reported on interactions between genes and birth weight in the relationship with adult blood pressure, although IJzerman et al, in twins studies, have shown that the association between birth weight and blood pressure depends on genetic factors [48,49] Interactions between birth weight and other genes have been reported before, which might suggest that some genotypes are more prone to adverse circumstances during fetal growth, under the assumption that birth weight is mainly dependent on the intra-uterine environment [15-17] On the other hand, the observed interactions between birth weight and IGF-I promoter polymorphism genotype could be a result of a gene-gene interaction, since birth weight is also determined by genes (other than the IGF-I gene).
This study was conducted in 264 subjects only, which is considered few in studies on genetic associations. This might be a reason that we did not find significant associations between genotype and the risk factors. Subjects born pre term were excluded, since gestational age may be another factor associated with risk factors for CVD and T2DM, but with another underlying mechanism [21]. We reanalysed the data including subjects born pre term but fulfilling the other inclusion criteria (N = 22), which showed some different β 's (birth weight was now significantly associated with FM, waist circumference (unadjusted model) and with DBP pressure (adjusted model)). However, the interaction between birth weight and IGF-I genotype was the same, with strong associations between birth weight and SBP and DBP in the VC (β = - 8.40 and β = -5.67, respectively). Furthermore, despite the fact that the AGAHLS is a longitudinal study, no data was available on infant growth, nor reliable data on birth length was available. If so, it was possible to study effects of IGF-I genotype on infant growth or interactions with infant growth.
Conclusion
From this study, it is concluded that IGF-I promoter polymorphism genotype is related to birth weight in men only, that this genotype is not associated with risk factors for CVD and T2DM, and, most interestingly, that the IGF-I promoter polymorphism genotype modifies the relationship between birth weight and risk factors for CVD and T2DM, especially for SBP and DBP.
List of abbreviations
β – regression coefficient
95%CI – 95% confidence interval
AGAHLS – Amsterdam Growth and Health Longitudinal Study
BMI – body mass index
bp – base pair
CA – cytosine adenine
CVD – cardiovascular diseases
DBP – diastolic blood pressure
T2DM – type 2 diabetes
FFM – fat-free mass
FM – fat-mass
HbA1c – glycated hemoglobin
IGF-1 – insulin like growth factor 1
IMT – intima-media thickness
LDL – low density lipoprotein
SBP – systolic blood pressure
VC – variant carrier
WHR – waist-to-hip ratio
WT – wild type
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
The first three authors (StV, EvR and PV) equally contributed to the paper. They were involved in the statistical analysis, interpretations of the data, laboratory work to determine the IGF-I polymorphism and writing of the paper.
JT, HD, CS, WvM, SL and HK were supervisors and involved in developing the design, assist in statistical analysis and interpretation of the results. They all have been involved in drafting the article or revising it critically for important intellectual content. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The AGAHLS is supported by multiple grants from the Dutch Prevention Fund (ZON), Dutch Heart Foundation (NHS), Dutch Ministry of Education an Science, Dutch Ministry of Well Being, Public Health and Sports (VWS), the Dairy Foundation on Nutrition and Health (ZVG), Dutch Olympic Committee / Dutch Sports Foundation (NOC/NSF), Scientific Board Smoking and Health, and Heineken Inc. We would like to thank all the men and women who participated in the AGAHLS
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BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-5-161591889210.1186/1471-230X-5-16Case ReportIdiopathic portal hypertension complicating systemic sclerosis: a case report Moschos John [email protected] Grigoris I [email protected] Clive [email protected] James [email protected] Savvas [email protected] Department of Gastroenterology, Queen Elizabeth Hospital, Sheriff Hill, Gateshead NE6 9SX, UK2 Department of Rheumatology, Queen Elizabeth Hospital, Sheriff Hill, Gateshead NE6 9SX, UK3 Department of Pathology, Queen Elizabeth Hospital, Sheriff Hill, Gateshead NE6 9SX, UK2005 26 5 2005 5 16 16 15 12 2004 26 5 2005 Copyright © 2005 Moschos et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Patients with systemic sclerosis may develop mild abnormalities of liver function tests. More serious hepatic involvement has been well documented but is rare. Idiopathic portal hypertension had been reported only in a few female patients with systemic sclerosis.
Case presentation
An 82-year-old man with known systemic sclerosis presented with melaena. Urgent gastroscopy revealed oesophageal varices, which re-started bleeding during the procedure and were treated ensocopically, with Sengstaken tube and glypressin. Liver function tests and coagulation were normal. Non-invasive liver screen (including hepatitis viral serology and autoantibodies) was negative. Ultrasound scan of the abdomen revealed a small liver with coarse texture and no focal lesion. Hepato-portal flow was demonstrated in the portal vein. The spleen was enlarged. A moderate amount of free peritoneal fluid was present. A CT scan confirmed the absence of portal vein thrombosis. One month following discharge the patient had a liver biopsy. Histological examination showed essentially normal liver tissue; there was no evidence of any excess inflammation and no features to suggest cirrhosis or drug-induced liver disease. Taking into account the above evaluation we concluded that the patient had idiopathic portal hypertension.
Conclusion
Both male and female patients with systemic sclerosis may – rarely – develop idiopathic portal hypertension.
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Background
Systemic sclerosis (SSc) is a multisystem disease of unknown cause. The incidence of the disease is 10/million population per year. The female/male ratio is 4:1. A few cases of idiopathic portal hypertension (IPH) associated with SSc have been reported, all in females.
Case presentation
We report a case of an 82-year-old man admitted to the hospital with melaena and dizziness on standing.
He had a past medical history of SSc diagnosed 3 years before and pulmonary fibrosis. He was on prednisolone and azathioprine. There was no history of liver disease or alcohol abuse.
On examination his blood pressure of 95/65 mmHg, pulse 90/min. He had bilateral fine inspiratory crackles. The abdomen was soft and non-tender. Rectal examination revealed melaena.
White blood count was 12,700/mm3, haemoglobin 10.9 g/dl, platelets 186,000/mm3 and MCV 99.4 fl. Blood urea was 12.5 mmol/l, creatinine 68 μmol/l, electrolytes were normal, glucose 6.3 mmol/l. Serum alanine aminotransferase was 12 u/l, bilirubin 12 μmol/l, γ-glutamyl transferase 54 u/l, alkaline phosphatase 35 u/l, total protein 52 g/l, albumin 27 g/l and C-reactive protein 12. Coagulation studies were normal. Factor V Leiden mutation was not present. Hepatitis viral serology and autoantibody screen were negative and α-1-antitrypsin was normal. Ferritin was 103 μmol/l, caeruloplasmin was 0.25 μmol/l and α-fetoprotein was 0.9 kU/L.
Urgent gastroscopy revealed oesophageal varices, fresh blood and clots in the stomach. During the procedure the oesophageal varices started bleeding. Haemostasis was not achieved despite sclerotherapy and therefore a Sengstaken tube was inserted. The patient was admitted to the High Dependency Unit and commenced on intravenous glypressin, antibiotics and was transfused four units of blood. The following day a repeat gastroscopy showed two columns of grade II oesophageal varices, two oesophageal ulcers (probably secondary to sclerotherapy) and mild portal gastropathy.
Ultrasound scan of the abdomen revealed a small liver with coarse texture and no focal lesion. Hepato-portal flow was demonstrated in the portal vein. The common bile duct was of normal caliber. The spleen was enlarged (bipolar length 12.8 cm). A moderate amount of free peritoneal fluid was noted. CT scan confirmed the absence of portal vein thrombosis.
The patient was discharged three weeks following admission on propranolol 40 mg twice daily, azathioprine 50 mg twice daily, spironolactone 100 mg once daily, prednisolone 7.5 mg once daily, lansoprazole, vitamin D and calcium. He had two further endoscopies, one and five weeks following discharge and his varices were successfully treated with banding. One month following discharge he had a liver biopsy. There was no evidence of any excess inflammation and no features to suggest cirrhosis or drug-induced liver disease. Overall this was essentially normal liver tissue.
Taking into account the liver histology and the absence of evidence of portal vein thrombosis on imaging we concluded that the patient had IPH. We decided not to proceed to portal pressure measurement because this would not contribute to the management of the patient.
Two months following discharge the patient was reviewed and was well. He had not had any further episodes of haematemesis or melaena. He had normal full blood count and biochemistry results.
Conclusion
Abnormalities of liver function occur frequently in patients with rheumatic conditions. Although benign extra-articular manifestations of rheumatic disease are the most common ones, more serious hepatic involvement, including hepatic steatosis, vasculitis, nodular regenerative hyperplasia and primary biliary cirrhosis, portal vein obliteration and portal hypertension and rarely portal fibrosis with abnormal lobular architecture have been observed in rheumatic diseases. Vascular disorders of the liver also have been described, such as Budd-Chiari syndrome [1]. The medical management of rheumatic disease involves potentially hepatotoxic medications. Occult hepatitis C infection and associated cryoglobulinaemia can mimic rheumatic disease and antiviral therapy may be beneficial [2].
IPH is frequently associated with autoimmune diseases in Japan. In a large survey by questionnaire 12% of patients with IPH had one or two autoimmune diseases and a quarter of them had hyperglobulimaemia [3]. This data raise the possibility of causal rather than an incidental association between IPH and autoimmune disorders. An immunological basis for IPH pathogenesis has been suggested by Terada et al in Japan; the smaller venous radicles in the small and medium-sized portal tracts may be targets of immunologic attack in IPH [4].
Liver involvement in patients with SSc has been well documented but is rare. In a post-mortem series of 57 SSc patients histological liver damage was found in 8.8% of cases [5].
There are only a few reported cases of SSc with IPH, and all have been females [6-8]. Our case is the first male patient reported with SSc and IPH.
When causes of portal hypertension like cirrhosis, extra-hepatic portal vein obstruction, and infections like Schistosomiasis have been excluded, there remains a group of patients with portal hypertension of uncertain aetiology. These patients have good parenchymal reserve and much better survival than patients with portal hypertension caused by cirrhosis. Infection or exposure to several environmental toxins such as arsenic has been suspected as a cause but not proven.
There is a striking geographic variability in incidence and manifestations of IPH. In the United States, the sex incidence is nearly equal and the patients are generally above 50 years old, with gastrointestinal bleeding being the usual presentation. Liver function tests are normal and anaemia and leucopoenia are frequent. IPH is more common in India and Japan [9,10]. The largest study from India [9] observed female preponderance (62% female); mean age of presentation was 31 years. The majority of patients in this study presented with self-felt abdominal mass (splenomegaly) and / or gastrointestinal bleeding. In Japan 75% of patients are female, and the mean age of presentation is 36 years. In these patients gastrointestinal bleeding, splenomegaly, or anaemia are equally frequent.
The liver may look nodular, but this nodularity is usually relatively superficial. The nodules are similar to those seen in nodular regenerative hyperplasia. Nodularity is more pronounced in the cases from India. Cirrhosis by definition is not present, but there is usually significant fibrosis along the portal vein and its branches particularly near the hilus. However this is difficult to be recognized by liver biopsy. The portal vein usually has a thick sclerotic wall and there may be concentric fibrosis in the portal areas in the advanced cases. There may be diminution or obliteration of portal vein branches within the triads. Inflammation and necrosis are not prominent features of this condition. Portal vein thrombosis can occur, but this is a complication rather than a cause of IPH since it usually occurs later in the course of well-established disease.
In conclusion, clinicians should be aware of the rare association of systemic sclerosis and idiopathic portal hypertention, which may develop even in male patients.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
JM and SK drafted the manuscript., GL, CK and JH participated in the manuscript preparation. All authors were involved in the management of the patient and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Written consent was obtained from the patient for publication of study.
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| 15918892 | PMC1166546 | CC BY | 2021-01-04 16:03:27 | no | BMC Gastroenterol. 2005 May 26; 5:16 | utf-8 | BMC Gastroenterol | 2,005 | 10.1186/1471-230X-5-16 | oa_comm |
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BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-6-261594388010.1186/1471-2156-6-26Research ArticleConserved genomic organisation of Group B Sox genes in insects. McKimmie Carol [email protected] Gertrud [email protected] Steven [email protected] Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK2005 19 5 2005 6 26 26 27 1 2005 19 5 2005 Copyright © 2005 McKimmie et al; licensee BioMed Central Ltd.2005McKimmie et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Sox domain containing genes are important metazoan transcriptional regulators implicated in a wide rage of developmental processes. The vertebrate B subgroup contains the Sox1, Sox2 and Sox3 genes that have early functions in neural development. Previous studies show that Drosophila Group B genes have been functionally conserved since they play essential roles in early neural specification and mutations in the Drosophila Dichaete and SoxN genes can be rescued with mammalian Sox genes. Despite their importance, the extent and organisation of the Group B family in Drosophila has not been fully characterised, an important step in using Drosophila to examine conserved aspects of Group B Sox gene function.
Results
We have used the directed cDNA sequencing along with the output from the publicly-available genome sequencing projects to examine the structure of Group B Sox domain genes in Drosophila melanogaster, Drosophila pseudoobscura, Anopheles gambiae and Apis mellifora. All of the insect genomes contain four genes encoding Group B proteins, two of which are intronless, as is the case with vertebrate group B genes. As has been previously reported and unusually for Group B genes, two of the insect group B genes, Sox21a and Sox21b, contain introns within their DNA-binding domains. We find that the highly unusual multi-exon structure of the Sox21b gene is common to the insects. In addition, we find that three of the group B Sox genes are organised in a linked cluster in the insect genomes. By in situ hybridisation we show that the pattern of expression of each of the four group B genes during embryogenesis is conserved between D. melanogaster and D. pseudoobscura.
Conclusion
The DNA-binding domain sequences and genomic organisation of the group B genes have been conserved over 300 My of evolution since the last common ancestor of the Hymenoptera and the Diptera. Our analysis suggests insects have two Group B1 genes, SoxN and Dichaete, and two Group B2 genes. The genomic organisation of Dichaete and another two Group B genes in a cluster, suggests they may be under concerted regulatory control. Our analysis suggests a simple model for the evolution of group B Sox genes in insects that differs from the proposed evolution of vertebrate Group B genes.
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Background
The family of Sox-domain containing proteins encompass a group of metazoan transcriptional regulators first identified by their similarity with the mammalian testis-determining factor SRY. Membership of the Sox family is conferred by the presence of an HMG1-type DNA-binding domain sharing greater than 60% amino-acid sequence identity to that of SRY [1]. Mammalian genome sequencing projects indicate that in humans and mice there are twenty Sox genes [2], divided into eight subgroups (A-H) on the basis of sequence identity within and outwith the HMG-domain. Aside from mammals, Sox genes have been identified in all metazoans examined to date, including birds, fish amphibians, basal chordates, insects and nematodes [3].
The B subgroup is of particular interest since members of this group are most closely related to SRY and appear to be functionally conserved during evolution. Sequence analysis and functional studies suggest that, in vertebrates, the five members of the B subgroup can be subdivided into two further groups; B1; Sox1, Sox2 and Sox3; [4] and B2; Sox14 and Sox21; [5]. It has been suggested from studies in the chick that the three group B1 proteins act as gene activators whereas the B2 proteins act as gene repressors [6]. In terms of genomic organization, all five of the group B genes are devoid of introns. Sox3 is located on the mammalian X chromosome and is believed to be the ancestor of Sry [7,8]. In humans, the remaining four autosomal group B genes are arranged in two pairs, each comprising one B1 gene and one B2 gene: Sox2 and Sox14 map together on chromosome 3 [9,10] and Sox1 and Sox21 map together on chromosome 13 [5,11]. This organization is conserved, at least in part, in other vertebrates with Sox2-Sox14 mapping together in the chick and the monotreme, O. anatinus, and Sox1-Sox21 mapping together in the chick [12,13]. There is, however, no linkage of Group B Sox genes in the mouse genome [14,15]. A model suggesting the evolution of group B genes and Sry from a single ancestor has been proposed, which suggests that pairs of B1 and B2 genes arose by a tandem duplication and then a chromosomal duplication [13].
The fruitfly, Drosophila melanogaster, has proved to be a tractable system for studying conserved aspects of eukaryotic gene function and, with the production of other insect genome sequences, a useful baseline for evolutionary studies of gene organisation [16]. Whole-genome sequence is now available for three insects, Drosophila melanogaster, Drosophila pseudoobscura (which diverged from melanogaster some 46 million years ago) and Anopheles gambiae, which diverged from melanogaster approximately 250 million years ago [17,18]. Sequencing and assembly of a further ten Drosophila species is currently underway [19] promising an unparalleled data source for evolutionary studies. In addition to the diptera, the sequencing of the Hymenoptera, Apis mellifera (honey bee ~280 million years from Drosophila), is now well underway, allowing fragments of a fourth insect genome to be assessed. In functional terms, Drosophila is a useful model for studying SOX gene function due to its genetic tractability. For example, we have previously shown that, in the case of the Drosophila group B gene Dichaete, there is functional conservation between insect and mammalian genes [20]. In addition, we, and others, have demonstrated a degree of in vivo functional redundancy between Dichaete and SoxN [21,22] as had been proposed for the mammalian group B genes [23]. Of particular interest is the fact that the expression patterns and functional studies of group B genes suggest that they participate in the earliest events of CNS differentiation in all organisms that have been studied to date including Drosophila, Xenopus, chick, mouse, ascidians and hemichordates [24].
To further explore the relationship between group B Sox genes we examined the extent and organization of the family in insects. Our studies show that group B Sox gene organisation is similar in four different insects. We find conservation in the sequence and genome organization of the group B genes in D. melanogaster, D. pseudoobscura, A. gambiae and A. melifora. In contrast to mammals and in agreement with a previous report [25], we find that two group B2 genes contain introns and are organized as a single genomic cluster along with the intronless Dichaete gene. Our studies indicate a potentially different evolutionary path for members of the group B family in insects and vertebrates.
Results
To explore the structure of the group B Sox genes in insects we first accurately determined the extent and structure of the family in Drosophila melanogaster. The group B genes, Dichaete and SoxNeuro (SoxN) have already been well described in the literature [26-28]. Two other group B gene fragments have been identified [25], Sox21a and Sox21b, but their structure and genomic organisation have not been reported. Using a combination of database searching and DNA sequencing we characterised both of these genes in detail. We find no evidence for any other group B genes in Release 3.2 or Release 4 of the Drosophila genome sequence, indicating that there are a total of four in the D. melanogaster genome.
Sox21a
Blast searches of the Drosophila genome identified a group B HMG-domain interrupted by a 1655-bp intron in the 70D region of chromosome arm 3L. Using primers designed against each of these predicted exons we amplified a fragment of 1238 bp from the LD cDNA library produced by the BDGP [29]. For reasons that are as yet unclear, we have been unable to recover a clone from this or any of the several other cDNA libraries that we have screened. The fragment amplified from the library was sequenced in its entirety and found to contain a long open reading frame encoding a 389 amino acid Sox domain protein. The predicted polypeptide initiates with a methionine and probably contains the entire coding sequence for the gene. When aligned with the genome sequence we predict a gene with two exons spanning 2.8 kb. Blast searches with the predicted protein find over 90% identity with a range of group B Sox proteins in the HMG DNA-binding domain. The best scores are with the DNA-binding domains of the vertebrate Sox21 and Sox14 proteins; however, there is little significant similarity outside of the DNA-binding domain. The Sox21a gene has previously been reported as SoxB2-3 (CG7345) and it has been suggested that it may represent a pseudogene [25]. As we show below, RT-PCR and in situ hybridisation studies indicate that Sox21a is expressed in both D. melanogaster and D. pseudoobscura indicating that it is not a pseudogene.
Sox21b
Along with Sox21a, Blast searches indicated a second interrupted Sox domain in the same region of the genome. In this case, database searches found a potential cDNA clone from the BDGP (GH07353), which was obtained and sequenced in its entirety. The sequence of the clone revealed a long open reading frame, initiating with a methionine, encoding a predicted polypeptide of 571 amino acids. Alignment with the genome sequence indicates that the gene spans 19 kb of genomic DNA and is composed of 7 exons, the first of which is non-coding. The DNA-binding domain contains two introns; the first, 6388 bp in size, is in the middle of the DNA-binding domain and the second, of 59 bp, is in the same position and frame as the Sox21a intron described above. Blast searches with the predicted amino acid sequence find greater than 90% amino acid identity the DNA-binding domains of group B Sox proteins, the highest scores being with Dichaete. The sequence indicates that Sox21b corresponds to the SoxB2-2 gene fragment previously reported [25] and whole mount in situ hybridisation with probes derived from genomic DNA and the cDNA clone confirm the pattern of expression previously reported (Figure 2). Thus, both Sox21a and Sox21b are expressed group B Sox genes that have their DNA binding domains interrupted by introns.
Figure 1 Full alignments of the insect Group B protein sequences. A) SoxN. B) Dichaete. C) Sox21a, the position of the conserved intron is indicated with an arrow. D) Sox21b, the location of the exons in the D. melanogaster sequence in indicated in italics above the alignment. Black arrowheads above the alignment indicate positions of introns conserved in all four species. The grey arrowheads indicate intron positions conserved in the diptera. The black arrow above the alignment indicates the drosophila specific intron and the grey arrows below the alignment indicates the apis-specific introns.
Figure 2 Embryonic expression of group B genes in D. melanogaster and D. pseudoobscura. A-H, D. melanogaster. A'-H', D. pseudoobscura; anterior is to the left in all cases. A-A') Lateral view of stage 5 embryos showing expression of Dichaete in a central domain and the cephalic neuroectoderm. B-B') Lateral views of stage 8 embryos showing extensive Dichaete expression in the developing CNS. C-C') Ventral view of stage 5 embryos showing SoxN expression is restricted from the presumptive mesoderm. D-D') Dorsal view of stage 8 embryos showing SoxN expression in the CNS. E-E') Lateral view of stage 9 embryos showing Sox21a expression in the anlage of the foregut and hindgut. F-F') Ventral views of stage 14 embryos showing Sox21a expression restricted to specific cells in the midline. G-G') Lateral views of stage 13 embryos showing Sox21b expression in abdominal epidermal stripes. H-H') Ventral view of stage 14 embryos showing Sox21b expression in abdominal epidermal stripes.
To verify the gene predictions and gain some insight into their possible biological functions we determined the developmental expression of each of the four group B genes by RT-PCR using RNA templates isolated from different stages of the Drosophila lifecycle. In the case of Sox21a and Sox21b, we used primers to the Sox-domain encoding exons spanning a predicted intron. All RT-PCR reactions included a reverse transcriptase minus reaction and the amplified products were verified by sequencing. The results of this analysis are presented in Table 1 and can be summarised as follows: the expression profiles of Dichaete and SoxN are very similar, they are expressed during embryonic, larval and pupal stages of development, the level of expression reducing during the later stages of pupal development. Both genes are expressed in adult male as well as female flies, with bodies showing stronger expression than heads. Sox21a is expressed throughout development and in adults it is stronger in heads than in bodies. In contrast to the other group B genes, Sox21b has a more complex expression pattern during development. It is strongly expressed in embryos but is below detectable levels for much of larval and pupal life. After eclosion it is weakly expressed in male heads but not bodies. Thus, in common with mammalian group B genes, all four D. melanogaster group B Sox are expressed during embryogenesis and at other stages throughout development.
Table 1 representation of Sox expression in during Drosophila development assayed by RT-PCR.
Dichaete SoxNeuro sox21a sox21b
Embryo + + + +
1st instar + + (+) (+)
2nd instar + + (+) -
Early 3rd instar + + (+) -
Late 3rd instar + + + -
Prepupa + + + -
12 h pupa + + + -
36 h pupa (+) (+) + -
Heads (+) (+) + (+)
Bodies + + (+) -
Male + + + (+)
Female + + + -
+ = expression, - = no expression, (+) = weak expression.
Group B genes in other insects
Our findings show that Drosophila melanogaster has four group B Sox genes compared to the five found in vertebrates and, unlike vertebrates, two of the genes contain introns. To investigate whether this particular organization is unique to D. melanogaster we searched the available genome sequence of other insects to find potential Sox domain genes. Using the Dichaete DNA-binding domain as a query, we searched the Drosophila pseudoobscura, Anopheles gambiae and Apis mellifera genome and EST sequence databases using Blast-P and Blast-N (see materials and methods for EST and genome scaffold accessions). In all three cases we found evidence for four Group B genes and were able to build gene models, from the genome sequence alone or with the addition of EST data where available. The initial characterization of the insect group B genes, based on the HMG-domain sequence, suggests that there is a single orthologue of each Drosophila gene in the other three species.
SoxN
The alignment presented in Figure 1a. shows the similarity between the insect SoxN proteins and mouse Sox1. As previously reported, conservation between vertebrate and invertebrate Sox proteins is mostly restricted to the DNA-binding domains [26,27]. Between the insect proteins there are more extensive regions of homology outwith the DNA-binding domain. The Drosophila SoxN sequences show over 90% sequence identity over their entire length and, as expected from the phylogenies based on rDNA and protein coding sequences, the other insect sequences are more diverged [18,30]. A. gambiae is overall 64% identical to the melanogaster sequence with particularly well conserved regions in the N-terminal 50 amino acids and more patchy conservation C-terminal to the DNA-binding domain. A. mellifera is further diverged (52% identity with Drosophila). Conserved regions outside the DNA-binding domains among all four sequences are restricted to a stretch of amino acids C-terminal that may represent conserved functional motifs important in transcriptional regulation.
Dichaete
The situation with Dichaete is similar to that observed with SoxN, and the figures for amino acid identity are virtually identical (Figure 1b). Outside of the DNA-binding domain the Dichaete sequences show even less similarity comparing the Drosophila species and the other two insects; conservation between all four being restricted to limited regions C-terminal to the DNA-binding domain. Interestingly, we have shown that the C-terminal region of D. melanogaster Dichaete contains sequences required for activity in a context-specific manner [20] and C-terminal regions of the mouse and chicken Sox2 protein are believed to be involved in aspects of correct Sox2 function [31].
Sox21a
This gene is the least conserved between the four species and outside of the DNA-binding domain they show little similarity with vertebrate group B2 proteins (Figure 1c). There is extensive homology between the two Drosophila species, however, the Anopheles and Apis sequences are very diverged outside of the DNA binding domain. As with D. melanogaster, there are no EST sequences available that support the structure of Sox21a in the other insects.
Sox21b
The predicted Drosophila Sox21b proteins are again very similar, over 88% identical over their length. The other insect sequences are less well conserved, although the Anopheles sequence has a block of conservation C-terminal to the DNA-binding domain, including a Glutamic acid-rich domain (Figure 1d). The predicted Apis sequence is less well conserved, we note, however, that all four proteins are identical at the extreme C-terminus. With both the Anopheles and Apis proteins we cannot confidently predict the N-terminal exons and are unable to find any regions with amino acid similarity to the first 2 coding exons of the Drosophila sequences in the Anopheles or Apis genomic sequence between the end of Dichaete and the Sox21b Sox-domain encoding exons. Our current models are, however, supported by the available EST sequences for both species although the EST sequences are not full-length. Therefore, the definitive structure of these two insect Sox21b genes will require further investigation. Nevertheless, it is clear from the available sequence that orthologues of Sox21b are present in other insects.
To confirm the identification of four group B genes in both D. melanogaster and D. pseudoobscura, we performed whole-mount in situ hybridization to embryos of both species using exon-specific probes generated by PCR from genomic DNA. In all four cases we find very similar patterns of expression during embryogenesis. In the case of Dichaete, we find blastoderm expression including a broad central domain and a region of expression in the cephalic neuroectoderm (Figure 2A and 2A'). After gastrulation there is extensive expression in the developing CNS (Figure 2B and 2B') including the midline (not shown). With SoxN we find conserved blastoderm expression, including an identical restriction from the ventral region of the embryo, followed by extensive expression throughout the developing CNS (Figure 2C to 2D'). With Sox21a, we identified conserved expression in the anlage of the foregut and hindgut at stage 12 (Figure 2E and 2E') with later expression in specific cells of the midline after stage 14 (Figure 2F and 2F'). Sox21b shows conserved expression in abdominal epidermal stripes from stage 13 (Figure 2G to 2H'). These observations indicate that all four group B genes have conserved expression patterns during embryogenesis.
Genomic organisation of group B genes in Drosophila: the Dichaete complex
In some vertebrates the two classes of group B genes, B1 and B2, are linked on the same chromosome. In contrast, with Drosophila a single gene, SoxN, maps to the second chromosome and the remaining three all map to chromosome 3. We examined the organisation of the group B genes in the other insect genomes and found that the situation was very similar to that observed in Drosophila. In melanogaster, SoxN is intronless and sits alone in the middle of an 80 Kb island with no flanking genes for 35 Kb proximal and 45 Kb distal, an unusual organisation for a Drosophila gene. We have previously shown that Dichaete is controlled by extensive 3' regulatory sequences, suggesting that perhaps the paucity of genes flanking SoxN may also indicate the presence of extensive regulatory sequences. In support of this, we find several clusters of predicted transcription factor binding sites from 35 kb upstream to 20 kb downstream of SoxN when we use a stringent search criteria with CisAnalyst analysis software [32,33] (data not shown). Similar searches with Dichaete find previously identified regulatory sequences, suggesting that SoxN may indeed be subject to complex regulation. Comparative analysis of the melanogaster and pseudoobscura genomes with the Vista genome alignment viewer [34,35] indicates that the genomic organization is very similar in the two species. The Ensemble annotation of the Anophelese genome indicates that the region around SoxN is also sparsely populated, with only 2 short stretches of EST homology in the 150 kb flanking SoxN. Therefore, it is possible that SoxN is subject to complex regulatory control in Anopheles. There is currently insufficient contiguous genomic sequence from Apis to assess the organization of the SoxN region.
In the 70D region of Drosophila melanogaster chromosome arm 3L the remaining three group B genes are clustered within an 77 kb region (Fig 3). As we have previously reported, Dichaete is an intronless gene controlled by at least 30 Kb of regulatory sequence 3' to the transcription unit [36]. 16 kb further distal to these regulatory sequences we find the start of Sox21b and a further 28 kb distal to this the start of Sox21a. The region ends with the Fat Body Protein 1 (Fbp1) gene 6 kb downstream of Sox21a. The genomic organization of Sox21b is highly unusual for a group B Sox gene. It is split into seven exons, the first of which is non-coding and exons 3, 4 and 5 contain the DNA-binding domain. All of the predicted splice junctions have consensus GT-AG sequences. Sox21a, comprises 2 exons, each containing a portion of the DNA-binding domain. As we note above, the position of the intron, which has consensus splice junction sequences, is in the same position as the second DNA-binding domain intron of Sox21b (intron 4).
Figure 3 The genomic organisation of the insect Dichaete regions. Exons are represented by shaded boxes and introns by the linking lines. A scale bar of 2 kb is indicated. The melanogaster and pseudoobscura sequences are to scale, the larger distance between Dichaete and Sox21b in Anopheles and Apis is indicated by a break in the line, the remainder of the diagram is to scale.
In the case of D. pseudoobscura, the homology extends from upstream of the Dichaete coding region to at least the Fat body protein 1 gene downstream of Sox21a. The organization of the three Sox genes is virtually identical comparing the two species and we could construct gene models including all of the Sox21a and Sox21b exons. There is absolute conservation of the intron position between both Drosophila species, furthermore, the sizes of the introns is also similar, although nucleotide similarity is lower than in coding sequences ranging from 40 – 75%. As with melanogaster, we find no evidence for additional genes in the intergenic region between Dichaete and Sox21b. We used the OWEN sequence alignment programme to plot the conservation between the Dichaete – Sox21b intergenic region in both species (Figure 4). Throughout the entire region we see that there is a high degree of sequence conservation, since we know that at least 30 kb of this region contains essential Dichaete regulatory sequences in melanogaster, we predict that regulation in the region will be similar in both species. A suggestion supported by the in situ hybridization data presented above (Figure 2).
Figure 4 OWEN alignment of the region between Dichaete and Sox21b in D. melanogaster and D. pseudoobscura showing extensive sequence similarity throughout the 45 kb region.
The organization of the Dichaete region in the Anopheles genome is very similar to that in the Drosophila species with three genes found in a 190 kb region of chromosome arm 3L. Dichaete is intronless and Sox21b is located approximately 110 kb downstream of this. There are no other predicted genes in the region. The Sox21b has a similar structure to those of the Drosophilids, however, it is not identical. We have been unable to find a 5' non-coding exon and, as we note above, the second intron found in the DNA-binding domains of the Drosophila Sox21b genes is absent in Anopheles with exons 4 and 5 fused. The other introns are, however, conserved in position (figure 1d). With the Anopheles Sox21a gene, the single intron position is conserved with the Drosophila species, however, the intron is considerably larger and contains an insertion of a Q-class retrotransposon in the sequenced strain [37]. We find no evidence for an Fbp1 orthologue in the vicinity, the nearest similar sequence being some 5 Mb away on the same chromosome arm.
The available sequence in the region is more fragmentary in the case of Apis. Here we find an intronless Dichaete gene and can define two sets of exons corresponding to the split DNA binding domains of Sox21a and Sox21b. Overall, the organization is similar to the other insects; like Anopheles, the intergenic region between Dichaete and Sox21b is large (~90 kb), however, unlike the other insects the distance between Sox21b and Sox21a is also large (~80 kb). In the case of Sox21b we have used EST sequence to support the gene model we have derived. The EST confirms the first four exons and we predict the terminal exon on the basis of homology with the other species, particularly the terminal 30 amino acids. As with Anopheles, the Apis Sox21b gene has a single DNA-binding domain intron in the same position of the first Drosophila DNA-binding domain intron. The intron immediately downstream of the DNA-binding domain is also conserved in all four insects, however, the remaining two intron positions differ between Apis and the other insects. Although the Apis assembly is preliminary in this region, with several gaps still present in the sequence, the fact that the gene models are very similar to the other insects and that Dichaete and Sox21b predictions are supported by EST data suggests that the gene models we propose are likely to be accurate for the majority of the coding sequence.
We compared the Dichaete to Sox21b intergenic regions of Anopheles and Apis to the melanogaster sequence with the OWEN alignment tool and failed to detect any significant stretches of similarity, even at relatively low stringency. This suggests that if there is conservation in gene regulatory sequences between these diverged insects it may be difficult to detect or have undergone extensive rearrangement.
Evolutionary perspective on insect group B genes
To attempt a classification of the insect group B Sox genes, we performed a multiple sequence alignment with the DNA-binding domains of the predicted proteins along with representative group B-like sequences from other organisms (Figure 5). The aligned ClustalX output suggests that the insect Sox domains may be subdivided into 3 classes. The first clearly groups the SoxN proteins from each of the insects with the mammalian Sox1, 2 and 3 proteins. Along with these we find representative sequences from nematodes (C. elegans, S. ratti, and W. bancrofti), hemichordate Acorn worms (S. kowalevski and P. flava) and the sea squirt (H. roretzi). Thus, together these are likely to represent a single class, orthologous to vertebrate group B1 proteins. The second class, the Sox21a proteins, have sequences similar to the mammalian group B2 proteins, Sox14 and 21 and may represent an insect group B2 protein. The third class, containing Dichaete and Sox21b, are clearly differentiated from all other group B proteins by the presence of a Leucine/Isoleucine residue at position 18, an Isoleucine residue at position 23 and a divergent set of C-terminal amino acids. These two insect proteins may represent an insect-specific group B family. This suggests that a single group B1 protein, represented by SoxN-Sox3 like sequences, was present in a common ancestor before the divergence of vertebrates and invertebrates. Similarly, the close association of the insect Sox21a proteins with nematode and vertebrate Sox14 proteins suggests that these were also present in a common ancestor. The alignments clearly highlight the distinction between the Dichaete-Sox21b pair and other group B proteins, emphasizing a distinct evolutionary history for these proteins in the insects.
Figure 5 Group B Sox-domain alignment. Clustal X alignment of DNA-binding domain sequences from the insect proteins and representative group B proteins from other species. The insect sequences are highlighted in grey. Accession numbers of protein sequences are as follows: SOX15 Human, O60248; SOX15 Mouse, P43267; Dichaete melanogaster, Q24533; Dichaete pseudoobscura, TR; Dichaete Anophelese, TR; Sox21b melanogaster, Q9VUD3; Sox21b pseudoobscura, TR; Sox21b Anophelese, TR; Sox21b Apis, TR; Dichaete Apis, TR; SOX1 Human, O00570; SOX1 Mouse, P53783; SOX1 Chicken, O57401; SOX3 Human, P41225; SOX3 Mouse, P53784; SOX3 Chicken, P48433; SOX3 Xenopus, P55863; SOX3 Medaka, Q9PT76; SOX2 Chicken, P48430; SOX2 Xenopus, O42569; SOX2 Human, P48431; SOX2 Mouse, P48432; SOX2 Sheep, P54231; SOX1 S. kowalevski, Q7YTD4; SOXB1 P. flava, (Taguchi et. al. 2002); SOXB1 Sea Urchin, Q9Y0D7; SoxN Apis, TR; SoxN melanogaster, Q9U1H5; SoxN pseudoobscura, TR; SoxN Anopheles, TR; SoxB s. ratti1, BI323817; SoxB s. ratti2, BI323817; SoxB W. bancrofti, CD455919; SOX2 C. elegans, Q21305; SOXB1 H. roretzi, Q86SB8; SOX19 Zebra Fish, P47792; SOX21 Zebra Fish, Q9YH21; SOX14 Chicken, Q9W7R6; SOX14 Human, O95416; SOX14 Mouse, Q04892; SOX14 Platypus, Q8MIP4; SOX21 Human, Q9Y651; SOX21 Mouse, Q811W0; SOX21 Chicken, Q9W7R5; SOXB2 P. flava, (Taguchi et. al. 2002); Sox21a melanogaster, Q9VUD1; Sox21a pseudoobscura, TR; Sox21a Anopheles, TR; Sox21a Apis, TR; SOXB2 Sea Urchin, Q9Y0D8; SoxB C. virginica, CD648628; SOX3 C. elegans, Q20201; SoxB T. spiralis, BG302262; SoxB M. hapla, BU095063; SRY Human, Q05066; SRY Sea Lion, AAR10360; SRY Mouse, Q05738. TR = This report.
Taken together, the analysis presented here shows that the genomic organization and sequence of group B Sox genes have been conserved during insect evolution. Particularly striking is the clustering of three genes in a small region of the genome. The structure of these genes and their relationship with vertebrate Group B genes suggest that SoxN and Sox21a are homologous to vertebrate group B1 and B2 genes respectively, whereas Dichaete and Sox21b may represent insect-specific group B genes.
Discussion
The sequence alignments of the HMG DNA-binding domains from insect and mammalian group B Sox proteins suggests that the insect proteins may be separated into three distinct groups. The first, containing SoxN, aligns with the vertebrate Sox1, 2 and 3 proteins and most likely represents an orthologue of the vertebrate group B1 class. This conclusion, based on sequence, is supported by the functional analysis of group B1 proteins in vertebrates and Drosophila. In both cases, group B1 genes are expressed from the earliest stages of CNS development and are implicated in regulating early neural specification [21,22,38,39]. In addition, we have evidence that mammalian Sox1 genes can rescue SoxN phenotypes in the Drosophila CNS, supporting the view that these proteins are functionally conserved (P. Overton and S.R. unpublished observations). The group B sequences isolated from the basal chordates, acorn worm and sea squirt, have also been shown to be expressed early in the specification of the CNS [40,41]. Thus, it appears that all metazoans studied to date have at least one group B gene with expression marking neural lineages early in development. Further studies of primitive invertebrates will determine whether group B Sox expression is a universal marker for CNS development.
In a previously published phylogenetic studies it was suggested that Dichaete be classified as a Group B2 protein [3]. However, while the analysis clearly differentiates between the group B proteins and other fly Sox proteins it could not unambiguously resolve the relationship between each of the group B proteins. In terms of function and expression, the Dichaete gene behaves very much like a group B1 gene, it is expressed early during CNS development and is required for neural differentiation [20,42]. We have previously shown that the mouse Sox2 gene efficiently rescues Dichaete phenotypes, further supporting a functionally similarity between Dichaete and vertebrate group B1 genes [20,42]. In contrast to the conclusion based on functional studies, the sequence analysis suggests that insect Dichaete DNA-binding domain sequences are markedly different from other group B1 proteins and are more similar to group B2 proteins. The conservation of the insect sequences indicates that a Dichaete-like sequence was present at least 300 My years ago, when Apis and the Diptera last shared a common ancestor [18]. We believe that the functional evidence is more convincing than the arguments based on sequence alignments and therefore suggest that Dichaete represents a group B1 function that has diverged from the canonical group B1 sequence, presumably due to selection for insect-specific functions. For example, Dichaete is required for early segmentation in the Drosophila embryo, a highly derived function, and it may be that sequence changes in the HMG-domain have been selected for such a function while still allowing a role in CNS-specification. As with Drosophila, both Anopheles and Apis are long germ insects that share some aspects of early development such as the early appearance of striped domains of even skipped expression [43,44]. Thus it is possible that insect Dichaete genes have a common role in early patterning events. It will be of considerable interest to examine the complement of group B Sox genes in Coleoptera, Homoptera or Orthoptera to see if the HMG domain sequence and gene organisation is the same as the insects so far sequenced. To investigate this we used the Dichaete DNA-binding domain to search the available sequence of the silk moth Bombyx mori. [45] and found a single Group B gene that was clearly an orthologue of the Dichaete genes discussed here, containing the diagnostic Leucine and Isoleucine residues described here.
As with vertebrate group B1 genes, SoxN and Dichaete are expressed in broadly overlapping domains and act partially redundantly in CNS specification [21,22]. The close similarity between the expression and function of SoxN and Dichaete in the CNS raises the possibility that they arose from a common ancestor by a duplication event and may thus share some common regulatory sequences. However, when we compared the sequences 5' or 3' to SoxN with the Dichaete 3' sequence we could not detect any sequence similarity indicating that any conservation in regulatory sequences is not visible at a large scale; this is not entirely surprising since we cannot detect any sequence similarity between the Dichaete regulatory sequences from Drosophila and Anopheles, while our analysis indicates the divergence of SoxN and Dichaete predates the Drosophila-Anopheles divergence.
Based on the sequence alignment of insect Sox21a DNA-binding domains with those of vertebrate Sox14 proteins, it is possible that Sox21a may be an orthologue of the group B2 class. It has been suggested that in chicken Sox14 and Sox21 act as antagonists of group B1 function in a subset of the developing CNS [6]. The function of Sox21a in Drosophila is not known at present, however, Sox21a is expressed late in the development of the embryonic CNS midline, a site of SoxN and Dichaete expression, indicating there is the potential for the type of antagonistic interaction proposed for vertebrates. The Sox21b DNA-binding domain sequence indicates that it is closely related to Dichaete. Both these proteins have a set of unique residues in their DNA-binding domains that are not found in any other group B proteins identified to date. The Sox21b gene is conserved between the insects and its close similarity to Dichaete suggests that both genes arose from a common origin in the ancestor of the arthropods after their divergence from the nematodes since there is no close sequence in C. elegans or its relatives. In terms of expression, Sox21b is expressed in the large hindgut along with Dichaete, supporting the possibility that it may also antagonise the activity of Dichaete. In this respect then Sox21b may represent a group B2 function. It is therefore possible that insects contain 2 group B1 class activities, involved in early CNS development, and two B2 class genes. Again we emphasise that the functional assignment of the insect genes may contrast with the data derived from sequence analysis, which predicts a single group B1 gene and three group B2 genes. We suggest that the separation of group B Sox domains into a B1 class and B2 class based solely on sequence does not reflect meaningful functional differences in insects. We have initiated a functional analysis of Sox21a and Sox21b in the hope that we can clarify this issue.
The genome organisation of the Dichaete cluster is unusual, not only are three genes clustered together in the genome but two of them, Sox21a and Sox21b, have introns within the HMG-domain. The single Sox21a intron is conserved in all four of the insect genes suggesting that it is ancestral to the insects. Sox21b is more complex, there are six introns in melanogaster and pseudoobscura, four of these are conserved in Anopheles and two are conserved in Apis. In the Drosophila species, there are two introns in the DNA-binding domain, the first of which is present in all four insects. The second intron, in an identical location to the Sox21a intron, is only found in the two Drosophila species. A simple model of a single intron loss is therefore unlikely to account for this since both Apis and Anopheles do not have the intron. It is possible that Apis and Anophelese lost the intron independently or, alternatively, that the common ancestor of the Drosophila species gained the intron, perhaps via a gene conversion event with Sox21a. Interestingly, the two group B genes from C. elegans also contain introns in the DNA-binding domain, in identical positions in both genes, but they are in different positions to the Sox21a and Sox21b introns. This suggests that the common ancestor of insects and nematodes did not contain DNA-binding domain introns and that these have been acquired independently in both lineages.
The conservation of genome structure with the insect Dichaete cluster suggests that there may be functional constraints on the organisation. We suggest that this is likely to be a reflection of shared regulatory sequence since the region between Dichaete and Sox21b in melanogaster contains extensive regulatory sequences essential for correct Dichaete expression. We note that both Sox21a and Sox21b have expression domains that overlap with Dichaete, in the midline for Sox21a and the hindgut with Sox21b. These expression domains may therefore be controlled by common regulatory sequences and the need to maintain coordinated regulation of the three genes has maintained the integrity of the cluster in the insects. The conservation in expression between D. melanogaster and D. pseudoobscura is consistent with this view; it will be of interests to examine the expression of the all of the Sox genes in Anopheles to further explore this hypothesis.
Conclusion
Taking our observation together, we propose a simple model for the evolution of group B SOX genes (Figure 6). We base our model on the proposal of Kirby et. al. [13] who suggest that a single group B gene underwent a duplication to generate two Sox3- like genes. We propose that these are represented by SoxN and Dichaete in the insects. A further tandem duplication of one of these genes generated linked group B1 and group B2 genes. We propose that this is represented by Dichaete duplicating to generate Sox21a. Sox21a would then acquire the sequence changes characteristic of the group B2 class of proteins. We suggest that these events predate the Protostome-Deuterostome divergence over 650 My ago [46] and provide the basal Sox gene complement of the Bilateria of three group B Sox genes. After the divergence of the lineages leading to vertebrates and invertebrates, Dichaete diverged from the canonical group B DNA-binding domain sequence and then underwent further duplication event, at least predating the divergence of the holometabolous insects, to generate Sox21b. An analysis of the group B family in other insects and basal chordates will be required to definitively describe the ancestral situation.
Figure 6 A model for the evolution of Group B Sox genes in insects following the proposal of Kirby et al (2002) for vertebrates. In this view an ancestral group B gene is duplicated during an ancient genome duplication event to generate Dichaete and SoxN. A tandem duplication of Dichaete generates Sox21a; these events would be common to the ancestor of vertebrates and invertebrates. In insects, a further duplication of Dichaete gives rise to Sox21b.
Methods
Genome sequences
The following sources were used to obtain genome sequence: D. melanogaster (Release 3.2, [47,48]) from FlyBase [49] and the following scaffolds were used; AE003535 for the Dichaete region and AE003622 for the SoxN region. D. pseudoobscura (Freeze_1 assembly) was obtained from the Human Genome Sequencing Center, Baylor College of Medicine (HGSC-BCM [50]) and the following scaffolds used; Contig5946_Contig6670 for the Dichaete region and Contig1741_Contig5707 for the SoxN region. Anopheles gambiae genome sequence release 19.2a.1, compiled by the International Anopheles Genome Project [51], was obtained from the Ensembl server at the Wellcome Trust Sanger Institute [52]. In the Ensemble annotation the Sox genes have the following accessions: SoxN (ENSANGG00000019842), Dichaete (ENSANGG00000010137), Sox21a (ENSANGG00000010002) and Sox21b (ENSANGG00000009947). Anopheles EST sequences representing Dichaete (TC44994) and Sox21b (TC45155) were obtained from The Institute for Genome Research [53]. Apis mellifera Genome assembly Amel_1.1 was obtained from HGSC-BCM [54] and the following scaffolds used: for the Dichaete region, Group8.12 (Dichaete and Sox21b) was found to overlap by 4.5 kb with GroupUn.570 containing Sox21a and the sequences were combined into a single contig. SoxN was contained within Group17.6. In addition a search of the Honey Bee Brain EST project [55,56] uncovered two EST sequences corresponding to Dichaete (BB170009A10D01) and Sox21b (BB170011B20A11). These were used to verify the exon predictions from the genome sequence. Vertebrate group B sequences were obtained from Uniprot. Nematode sequences were recovered by Blast searches of the EST collections at Nematode.net (Genome Sequencing Center, Washington University, St Louis, [57]).
Informatics tools
Homology searching was performed using the Blast algorithm [58] at Sanger, HGSC-BCM and Berkeley Drosophila Genome Project [59] web sites. Genomic sequences were imported into Artemis v5 [60,61] and annotated manually using the Blast output as a guide. Multiple Sequence alignments were performed locally using ClustalXv1.8 [62] and graphically represented with BoxShade [63]. The alignment of intergenic regions was performed with OWEN [64].
Molecular biology
A cDNA clone for Sox21b (GH07353) was obtained from the Drosophila gene collection [29] and sequenced on both strands using an ABI prism kit in the Genetics Department sequencing core. PCR and RT-PCR amplifications were carried out using minor modifications to standard techniques [65] using the following primer combinations:
Melanogaster primers for RT-PCR
Dichaete F ACAATCCATTCCATCAACTACC
Dichaete R TTGGTGTTCCCTCCTTACTC
Sox21B F AGTCTCATGAACAGCGGAAG
Sox21B R GGAGTTGCTCAGATACGACG
SoxN F CAGCAGCAACAGCAACACTAC
SoxN R TTTCATCGCCTCGCCACAAC
Pseudoobscura primers for in situ probes:
Dp-Dichaete F CGAACTACGGATTCCACCT
Dp-Dichaete R CATTCCGTTGGCCTGCAT
Dp-SoxN F AGCTGAGTCACCATAACCAC
Dp-soxN R GTCATGTGATGGCTACCAA
Dp-Sox21A Exon1 F GAGCATCTCGACGCTACTAC
Dp-Sox21A Exon 1 R GGAATTGGAGTGGCTATGAT
Dp-Sox21A Exon 2 F CTAAGGACATGCAGTCACAG
Dp-Sox21A Exon 2 R GACTTCACGCAGCCGTAGGAT
Dp-Sox21B F CGTCTATCCACACACCTGTC
Dp-Sox21B R GACGATGTCTGCTGCTGTT
Whole-mount in situ hybridisation to Drosophila embryos was performed using minor modifications to a standard protocol [66].
All genetic nomenclature is according to FlyBase [49].
Authors' contributions
C.McK. performed the sequencing, mapping of the Drosophila Sox21a and Sox21b genes in and the in situ hybridisation experiments. G.W. carried out the RT-PCR analysis. S.R. designed the experiments, carried out the genomic analysis and wrote the paper.
Acknowledgements
This work was supported by an UK-Medical Research Council programme grant to S.R., M. Ashburner and D. Gubb. We are grateful to M. Ashburner for comments on the manuscript and to S. Marcellini and J. Roote for assistance with the D. pseudoobscura husbandry.
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| 15943880 | PMC1166547 | CC BY | 2021-01-04 16:38:18 | no | BMC Genet. 2005 May 19; 6:26 | utf-8 | BMC Genet | 2,005 | 10.1186/1471-2156-6-26 | oa_comm |
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BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-6-291592152110.1186/1471-2156-6-29Research ArticlePotential genetic modifiers of the cystic fibrosis intestinal inflammatory phenotype on mouse chromosomes 1, 9, and 10 Norkina Oxana [email protected] Lisle Robert C [email protected] Department of Anatomy and Cell Biology University of Kansas School of Medicine Kansas City, KS 66160 USA2005 27 5 2005 6 29 29 17 1 2005 27 5 2005 Copyright © 2005 Norkina and De Lisle; licensee BioMed Central Ltd.2005Norkina and De Lisle; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Although cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the severity of disease is highly variable indicating the influence of modifier genes. The intestines of Cftr deficient mice (CF mice: Cftrtm1Unc) are prone to obstruction by excessive mucus accumulation and are used as a model of meconium ileus and distal intestinal obstruction syndrome. This phenotype is strongly dependent on the genetic background of the mice. On the C57Bl/6 background, the majority of CF mice cannot survive on solid mouse chow, have inflammation of the small intestine, and are about 30% smaller than wild type littermates. In this work potential modifier loci of the CF intestinal phenotype were identified.
Results
CF mice on a mixed genetic background (95% C57Bl/6 and 5% 129Sv) were compared to CF mice congenic on the C57Bl/6 background for several parameters of the intestinal CF phenotype. CF mice on the mixed background exhibit significantly greater survival when fed dry mouse chow, have reduced intestinal inflammation as measured by quantitative RT-PCR for marker genes, have near normal body weight gain, and have reduced mucus accumulation in the intestinal crypts. There was an indication of a gender effect for body weight gain: males did not show a significant improvement at 4 weeks of age, but were of normal weight at 8 weeks, while females showed improvement at both 4 and 8 weeks. By a preliminary genome-wide PCR allele scanning, three regions were found to be potentially associated with the milder phenotype. One on chr.1, defined by marker D1Mit36, one on chr. 9 defined by marker D9Mit90, and one on chr. 10, defined by marker D10Mit14.
Conclusion
Potential modifier regions were found that have a positive impact on the inflammatory phenotype of the CF mouse small intestine and animal survival. Identification of polymorphisms in specific genes in these regions should provide important new information about genetic modifiers of the CF intestinal phenotype.
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Background
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1]. Different mutations have a range of effects on the levels of CFTR protein and its proper functioning in epithelial transport of Cl- and HCO3- [2,3]. The severity of the pancreatic phenotype in human CF is well correlated with the extent of impaired CFTR function caused by specific mutations. Loss of CFTR function results in destruction of the exocrine tissue and eventual pancreatic insufficiency. On the other hand, the effects of CF on organs including the airways and intestines is less well correlated with specific CFTR mutations and their effects on CFTR protein function [4-8]. This indicates that other genes are likely to be important as modifiers of the CF phenotype.
With the exception of pancreatic insufficiency resulting in impaired digestion, other aspects of CF are less readily related to loss of CFTR function. Nutritional problems can persist even with adequate oral enzyme supplementation [9] and neutralization of gastric acid to improve lipase function [10], and may involve both impaired digestion and absorption of nutrients [11]. Inadequate absorption or assimilation of nutrients appears to be of greater importance because even with adequate oral enzyme supplementation nutrition is rarely fully corrected [11]. There is also excessive mucus accumulation in the CF intestine, and inappropriate inflammation is common [12]. Mucus is involved in obstruction of the gut which occurs frequently in CF infants (called meconium ileus, MI) and adults (called distal intestinal obstruction syndrome, DIOS) [11,13]. And, similar to CF airways, there is also an inflammation of the CF intestines [14,15]. These changes are less directly related to specific mutations in the CFTR gene and are likely related to other differences in individual genetic makeup.
Previous work using human patients and genetically altered mice has identified some modifier genes and have advanced our understanding of CF pathophysiology [4]. In one study using CF mice on different genetic backgrounds, a region on mouse chromosome 7 was shown to ameliorate intestinal blockage and the effect was in part due to a calcium-regulated Cl-channel which compensated for loss of CFTR function [16,17]. Marker haplotypes of the syntenic region of human chromosome 19q13 were also shown to be associated with the risk of MI in CF patients [18]. In other work, a region on mouse chr. 6 was strongly associated with lung inflammation, consisting of mononuclear cell interstitial infiltration and fibrosis in CF mouse airways; and other loci on chr. 1, 2, 10, and 17 were also linked to the airway phenotype [19].
In this work, CF mice on a mixed strain background were found to have a less severe CF phenotype compared to CF mice congenic on the C57Bl/6 background. There were no differences in the pancreatic phenotype comparing CF mice on the different backgrounds [20]. However, mice on the mixed background seemed more robust than CF mice on the B6 background which prompted us to characterize them in greater detail. Genome wide allele scanning was used to begin identification of regions associated with the less severe intestinal phenotype. Future identification of specific genes should further our understanding of the complex intestinal CF phenotype.
Results and discussion
CF mice on the mixed background have improved body weight gain
Body mass was recorded for male and female mice at 4 and 8 weeks of age. On the B6 background, female and male mice are about 30% smaller than wild type mice at both 4 and 8 weeks of age (Fig. 1A, B). By comparison, female CF mice on the mixed background were not significantly smaller than wild type mice at 4 weeks of age (Fig. 1A). The improved body weight of female CF mice on the mixed background was maintained at 8 weeks of age. Male CF mice on the mixed background at 4 weeks of age were significantly smaller than wild type and not significantly larger than CF mice congenic on the B6 background (Fig. 1B). By 8 weeks of age, CF males on the mixed background were 12% smaller but this was not significantly different compared to wild type mice (Fig. 1B).
Figure 1 Effect of genetic background on body weights of CF mice. (A) Female and (B) male mice were weighed at 4 and 8 weeks of age. Data are means ± SEM. (*) P < 0.001 vs other three groups by ANOVA with a post-hoc Tukey's test. There were no significant differences for any of the other comparisons. Data were obtained from: 65 wild type and 26 CF B6 females, 36 wild type and 8 CF mixed background females, 68 wild type and 30 CF B6 males, and 37 wild type and 6 CF mixed background males.
CF mice on the mixed background have reduced expression of inflammatory markers
Previous work showed that CF mice on the B6 background have an innate-type inflammation of the small intestine [21]. To determine whether the mixed genetic background affected expression of inflammatory marker genes, quantitative, real-time RT-PCR was used to measure gene expression. Expression of the following genes was compared in wild type and CF mice on the different genetic backgrounds. Mast cell protease 2 (Mcpt2) is a marker of differentiated mast cells [22] and mast cells are more abundant in the B6 CF mouse intestine. Leucine-rich α2 glycoprotein (Lrg1, [23]) is a marker of differentiating neutrophils, which are more numerous in the B6 CF mouse intestine. The same gene is also known as leucine-rich high endothelial cell glycoprotein (Lrhg) and has been shown to be a marker of high endothelial venules (HEV) [24] which increase in tissues during inflammation [25,26]. Hematopoietic cell transcript 1 (HemT1, [27]) is a marker of blood cell proliferation and its expression is strongly elevated in the B6 CF mouse small intestine. Serum amyloid A3 (SAA3, [28]) is an acute phase gene and its expression in villus epithelial cells is increased in the B6 CF intestine. Suppressor of cytokine signaling 3 (SOCS3, [29]) is an anti-inflammatory gene that interacts with the JAK-STAT pathway and its expression in increased in the B6 CF intestine. Muclin (also known as dmbt1, [30]) expression is upregulated in the B6 CF intestine; it is a cell surface glycoprotein postulated to be an epithelial protective molecule [21,31].
Consistent with previous results, Mcpt2 was increased in CF mice on the B6 background by over 9-fold compared to wild type (Fig. 2A). By contrast, there was not a significant difference in Mcpt2 expression between CF and wild type on the mixed background (Fig. 2A). Lrg1/Lrhg expression was increased more than 20-fold in CF mice on the B6 background compared to wild type, but there was no significant difference between CF and wild type on the mixed background (Fig. 2B). SAA3 mRNA was about 3.5-fold increased in CF mice on the B6 background, but was not significantly different compared to wild type on the mixed background (Fig. 2C). SOCS3 was more than 2-fold increased in CF mice on the B6 background compared to wild type, and on the mixed background was only 1.5-fold greater than wild type, and the difference was not significant (Fig. 2D). Muclin is overexpressed almost 3-fold in the CF intestine on the B6 background, but on the mixed background the expression level in CF mice was not significantly different than wild type (Fig. 2E). Finally, HemT1 was overexpressed almost 20-fold in B6 CF mice compared to wild type, and on the mixed background the CF expression level was not statistically different from wild type (Fig. 2F).
Figure 2 Effect of genetic background on inflammatory gene expression in CF mouse small intestine. RNA expression levels were determined by quantitative real-time RT-PCR using gene-specific primers. Data are expressed relative to GAPDH mRNA, which does not vary between wild type and CF mice. Data are means ± SEM. (*) CF vs wild type on the B6 background, P < 0.005; (+) CF on the mixed background vs CF on the B6 background, P < 0.05 by ANOVA with a post-hoc Tukey's test. There were no significant differences for any of the 6 genes comparing: wild type B6 mice vs wild type mixed background; or CF mice on the mixed background vs wild type on either background. There were 8–11 samples analyzed per group for each gene.
Because of the gender differences in body weight, the gene expression data were analyzed by gender but there was no significant difference between females and males. With the limited number of animals, there was also no evidence for imprinting.
CF mice on the mixed background have less intestinal mucus accumulation
The most striking histological difference in the CF mouse small intestine is the accumulation of mucus in intestinal crypts which is associated with the lethal obstruction that results in death of these mice on a standard solid chow diet [32]. Using periodic acid Schiff's staining for neutral mucins, histological analysis of small intestine tissues was performed. The wild type small intestine on both the B6 and the mixed genetic backgrounds were similar. The intestinal crypts were very small with only traces of PAS-reactivity in the lumen (Fig. 3A and 3C, respectively). The surfaces of the villus epithelium were moderately stained and goblet cells were strongly stained in the wild type tissues. In contrast, the intestine of CF mice on the B6 genetic background exhibited greatly dilated crypts filled with PAS-reactive mucus (Fig. 3B). CF mice on the mixed background had less mucus accumulation than CF mice on the B6 background (Fig. 3D-F). The amount of mucus in CF mice on the mixed background was variable from mouse to mouse. In some mice (Fig. 3D), only occasional crypts had accumulated mucus and the crypt lumina were not very dilated. In some mice there was more mucus in the crypts (Fig. 3E), while others had moderate mucus accumulation (Fig. 3F). A total of six CF mice on the mixed background were examined histologically, and three had little mucus, one had some mucus, and two had moderate amounts of mucus. Despite the variability, all the CF mice on the mixed background had less crypt dilation and less mucus accumulation compared to CF mice on the B6 background.
Figure 3 Histological appearance of the small intestine of wild type and CF mice on the different genetic backgrounds. Tissue was paraffin embedded and stained with PAS for neutral mucins. The sections are from the middle portion of the small intestine (A) Wild type on the B6 background. (B) CF on the B6 background. (C) Wild type on the mixed background. (D-F) CF on the mixed background. (A, C) In the wild type tissue from both backgrounds, the crypts are small and have narrow lumina (arrows). (B) In the CF tissue on the B6 background, the crypt lumina are greatly dilated and filled with PAS-reactive mucus (arrows). In some CF mice on the mixed background, the crypts are not apparently different than wild type (D), and the crypts are normal appearing (arrows). Some CF mice on the mixed background had mildly affected crypts (E), while others had greater crypt dilation and mucus accumulation (F). Overall, the CF mice on the mixed background were less severely affected compared to those on the B6 background.
Thus, it appears that whatever the nature of the genetic differences between the two background strains, they affect secretion and accumulation of mucus in the CF small intestine. To determine if the difference could be accounted for by altered mucin gene expression, quantitative RT-PCR was used to measure mRNA expression of the intestinal goblet cell mucin gene, Muc2. In wild type intestine, B6 mice had 0.079 +/- 0.024 copies of Muc2/GAPDH (n = 8), and on the mixed background the level was 0.077 +/- 0.012 (n = 11). CF mice on the B6 background had 0.059 +/- 0.024 copies of Muc2 mRNA per GAPDH (n = 11), and on the mixed background the level was 0.056 +/- 0.010 (n = 11). Despite the increased mucus accumulation in CF, there is a slight decrease in Muc2 mRNA in the CF small intestine (P < 0.0001), as previously reported [33]. However, the milder CF phenotype on the mixed background, which includes less mucus accumulation, does not involve decreased mucin gene expression.
CF mice on the mixed background have improved survival
As reported by others [34], the expected number of Cftr null mice on the B6 background that survived to weaning was significantly less than that expected from Mendelian genetics (Tables 1 and 2). The degree of significance was greater for male mice (Tables 1 and 2). In contrast, on the mixed background, the distribution of genotypes of female offspring was not significantly different from the expected (Tables 1). For male mice on the mixed background, the P-value was less significant but still different compared to the B6 males (Table 2). These data indicate that the mixed background is associated with increased survival of CF mice.
Table 1 Distribution of Cftr genotypes in female offspring from breeding Cftr heterozygotes on the B6 and mixed backgrounds.
B6 Background Mixed Background
Cftr Observed Expected Observed Expected
+/+ 207 175 57 47
+/- 371 350 88 94
-/- 122 175 42 47
P 0.00001 (0.04533)* 0.21724
Statistical analysis was by Chi-square. (*) P-value if the sample size for the B6 mice is adjusted to be equal to that of the mixed background mice, assuming the same distribution of genotypes.
Table 2 Distribution of Cftr genotypes in male offspring from breeding Cftr heterozygotes on the B6 and mixed backgrounds.
B6 Background Mixed Background
Cftr Observed Expected Observed Expected
+/+ 240 201 57 52
+/- 431 403 115 104
-/- 134 201 36 52
P <0.00001 (0.01612)* 0.03749
Statistical analysis was by Chi-square. (*) P-value if the sample size for the B6 mice is adjusted to be equal to that of the mixed background mice, assuming the same distribution of genotypes.
The major cause of death in CF mice is intestinal obstruction, and intestinal obstruction is worsened when the mice are fed standard solid mouse chow [35]. Since the CF mice on the mixed background had better weight gain than on the B6 background and less mucus accumulation in the small intestine, their ability to survive on solid chow was tested. Mice were maintained on the liquid diet until 8 weeks of age, and then switched to solid chow for up to eight weeks. As shown in Fig. 4, wild type mice had 100% survival on chow, as expected. The majority of CF mice on the B6 background died within 2–3 weeks on solid chow, with about 60% mortality over the 8 week period. In contrast, only 22% of the CF mice on the mixed background died (Fig. 4).
Figure 4 Effect of genetic background on CF mouse survival on solid chow. Mice were maintained on the liquid diet (Peptamen) until 8 weeks of age, at which time they were fed standard solid mouse chow for up to 8 additional weeks. Deaths were recorded as they occurred and mice in obvious distress were sacrificed and 'death' recorded as the subsequent day. By log-rank test, CF mice on the mixed background had significantly earlier death compared to wild type (P = 0.035), and significantly later death than CF mice on the B6 background (P = 0.025). CF mice on the B6 background also died significantly earlier than wild type (P < 0.0005).
Identification of potential modifier loci
To determine the contributions of B6 and 129 alleles to the mixed genetic background, and to identify potential modifier regions, tail DNA was analyzed by PCR for markers of polymorphisms between these two mouse strains. Mice congenic on the B6 background, which were derived from mice originally on a B6/129 mixed background, were also analyzed to confirm they are congenic B6. Twelve CF mice on the B6 background averaged 99.5% B6 alleles. One of the twelve mice had alleles from both strains at chr.9, 40 cM. All twelve mice had both B6 and 129 markers on chromosome 6, 1 cM which is probably due to the targeted Cftr gene which is at chr.6, 3.1 cM [36].
Three CF mice on the mixed background were initially analyzed and were found to be 95% B6, 5% 129. A second group of 8 CF mice was analyzed and the markers used were refined to focus on the chromosomal regions showing variations from the B6 strain. The differences found from the two analyses are combined in Table 3. The differences in the mixed strain CF mice were at chr.1, 92 cM (9 of 11 mice had both B6 and 129 alleles); chr.9, 9 cM (all 11 mice had two 129 alleles); and chr.10, 65 cM (10 mice had both B6 and 129 alleles).
Table 3 Genome scanning analysis of CF mice on the mixed background.
Chromosome cM B6/B6 129/129 B6/129 Number of Mice
1 92 2 0 9 11
1 102 3 2 6 11
2 7 2 0 1 3
9 9 0 11 0 11
9 20 8 1 1 11*
9 33 5 0 3 8
9 40 1 1 1 3
10 40 6 0 2 8
10 65 0 1 10 11
11 35 5 0 3 8
11 43 6 0 5 11
12 19 6 0 2 8
The table shows the distribution of B6 and 129 alleles in mice from the two genetic backgrounds. An initial analysis was performed on 3 samples and a second analysis on 8 more samples. Only the markers that were not uniformly B6 alleles are shown. Some of the markers were refined based on the initial analysis, so not all markers were used for all 11 mice. (*) Eleven samples were analyzed but one sample was ambiguous.
Because the spacing of markers used was about 12 cM, genes within 75% of this interval on either side of the markers were looked at for potential relevance to the milder CF intestinal phenotype. None of the known chloride channels that might substitute for the missing CFTR are in the regions of the three chromosomes associated with the milder phenotype. There are several potassium channel genes in the identified regions that potentially could affect electrolyte and fluid transport: Kcnj9 (chr.1, 94.2 cM), Kcnj10 (chr.1, 93.5 cM), Kcnj5 (chr.9, 11 cM), and Kcnc2 (chr.10, 62 cM). All gene names are from the Mouse Genome Informatics website .
Inflammation is a hallmark of CF, and whether there is an inherent defect in CF that predisposes to excessive inflammation is controversial. Several genes involved in inflammation and the immune system are located in the regions of the markers identified: TNF superfamily members Tnfsf4, 6, and 8 (chr.1, 84.9–85 cM) which are involved in T cell activation [37,38]; three selectin genes (Sele, Sell, Selp, chr.1, 86.6 cM) which are involved in immune cell infiltration into inflamed tissues [39]; several members of immune cell surface proteins of the Slam family (slamf1, 2, 5, 6, and 9; chr.1, 89.5–93.3 cM) [40]; the chemokine gene Xcl1 (chr.1, 87 cM) which is expressed by mast cells and recruits lymphocytes [41]; several immunoglobulin Fc receptor genes (Fcrl3, Fcgr2b, and Fcgr3 at chr.1, 92.3 cM; Fcer1g at chr.1, 93.3 cM; Fcer1a at chr.1, 94.2 cM); the flagellin receptor Tlr5 (chr.1, 98 cM); Mmp3 (chr.9, 1 cM) which recruits CD4+ lymphocytes [42]; Mmp7 (chr.9, 1 cM) which activates Paneth cell-derived cryptdins (α-defensins) [43]; Icam1 (chr.9, 7 cM) which is involved in lymphocyte infiltration into inflamed tissues [44]; Kitl (chr.10, 57 cM) which is also known as stem cell factor, and is crucial for mast cell differentiation [45]; Im5 (chr.10, 65 cM) which is involved in antibody-responsiveness [46]; Lyzs (chr.10, 66 cM) which is a Paneth cell product that digests cell walls of bacteria [47]; Ifng (chr.10, 67 cM) which is an important inflammatory signal in CF as well as other conditions [48]; Il22 (chr.10, 67 cM), a member of the anti-inflammatory IL-10 interleukin family [49]; and the Stat2 and 6 genes (chr.10, 70 cM) which are important components of intracellular signaling pathways [50].
Also near the identified markers are a number of QTL associated with body weight: Cfbw1, CF mouse body weight at chr.1, 85 cM; Obq9, obesity 9 at chr.1, 88 cM; Bw8q1, body weight 8 at chr.1, 100 cM; Lbm6, lean body mass 6 at chr.9, 7.7 cM; Bwtq4, body weight 4 at chr.9, 8 cM; Bgeq8, body growth early 8 at chr.10, 57 cM; and Pbwg5, postnatal body weight growth 5 at chr.10, 68 cM.
Clearly, there are numerous genes in the three regions identified in this study. Because the CF mouse intestinal phenotype is characterized by an innate type immune response, with increases in mast cells and neutrophils, the genes that affect these cells are of special interest. The Kitl gene is crucial for differentiation of mast cells, and CF mice on the mixed background have much fewer mature mast cells than on the B6 background as revealed by less expression of Mcpt2. Similarly, for neutrophils the selectins and Icam1 are of interest, as these proteins are required for extravasation of neutrophils from the circulation into the inflamed tissue.
Altered immune responses may also relate to excessive mucus accumulation in the CF intestine. It is unclear why mucus accumulates to high levels in CF tissues. In part it may be due to reduced fluid secretion and a more acidic environment in the lumina of affected organs. However, there is also evidence for hypersecretion of mucus in CF [12], and it is likely that effector molecules released by mast cells and neutrophils (histamine, proteases, prostaglandins) have an important role in stimulating mucus secretion.
Conclusion
This work demonstrated that the CF inflammatory phenotype is much less severe in mice with a small contribution of 129/Sv alleles. A preliminary analysis identified regions on chr.1, 9, and 10 are that are potentially associated with the milder phenotype. Because of the inflammation of the CF small intestine, and the possible effects of immune cells on mucus secretion, the genes in the identified regions which are involved in mast cell and neutrophil differentiation and behavior are of special interest as potential CF modifiers. Future work should focus on narrowing down these regions and determining if there are polymorphisms that affect expression of specific genes that make the CF intestinal phenotype less severe.
Methods
Animals
Wild type and Cftr null mice on two different genetic backgrounds were used in this study. One group was congenic on the C57Bl/6 (B6) background, as previously described [21]. The other group was on a mixed background of about 95% B6 and 5% 129/Sv (129). The mice on the mixed background originated as part of a recently published study [20] as follows. Mice carrying a targeted mutation of the gastrin gene on a mixed B6/129 background [51] were bred for eight generations onto the B6 background. The gastrin(+/-) mice were then crossed for six generations with Cftr(+/-) mice congenic on the B6 background. A genome scan at this point showed that the mice were about 95% B6 and 5% 129 (see below). These mice were bred to obtain mice wild type for both gastrin alleles and either Cftr homozygous wild type [Cftr(+/+)] or Cftr homozygous null [Cftr(-/-)].
Mice were genotyped at 2 weeks of age by PCR as previously described [21]. Unless otherwise stated, mice were maintained on a complete elemental liquid diet (Peptamen; Nestle Deerfield, IL) to prevent intestinal obstruction that occurs in CF mice [52]. Wild type littermates were maintained on the same diet. In some experiments, 8 week old mice were transferred onto solid mouse chow instead of Peptamen, and survival was recorded. Mice with apparent distress were sacrificed and survival on chow was recorded as the following day. Male and female mice were used at 6–16 weeks of age. Mice were kept in a specific pathogen-free facility in barrier-top cages. All procedures were approved by the University of Kansas Medical Center IACUC.
Genetic background determination
The Genome Scanning Service of The Jackson Laboratory (Bar Harbor, ME) was used to determine the contributions of C57Bl/6 and 129/Sv strains in the interbred mice. Pieces of mouse tail were sent to The Jackson Laboratory for simple sequence length polymorphism (SSLP) PCR analysis with the DMit primers specific for B6 and 129 strain alleles . The SSLP panel consists of 108 mapped markers designed to distinguish between B6 and 129 strains. The markers are spaced 12–13 cM apart and span the nineteen autosomes.
Measurement of gene expression
Total RNA was extracted from the entire small intestine as previously described [21]. Quantitative, real-time RT-PCR was used to measure expression of specific genes using the previously described primers [21]. Values were normalized to GAPDH mRNA and expression of this housekeeping gene is not altered in the CF mouse small intestine [21]. Expression of the major intestinal mucin, Muc2, was also measured using the forward (5'-GAC TTC GAT GGA CAC TGC TC-3') and reverse (5'-CAC GGT GTT TAT CTA CCA AC-3') primers.
Histology
The small intestine was flushed with phosphate buffered saline and immersion fixed overnight in 4% paraformaldehyde. The tissues were then prepared for paraffin embedding and sectioning by a commercial service (HSRL, Woodstock, VA). Sections (5 μm) were stained with periodic acid Schiff's (PAS) for neutral mucins.
Statistics
Gene expression and body weight data were compared by ANOVA with a post-hoc Tukey's analysis (Systat software, Chicago, IL). Survival data were analyzed by a log-rank test for P values (PEPI software, ). The distributions of genotypes of pups surviving to weaning from breeding Cftr(+/-) mice were compared to that expected by Mendelian genetics using Chi-square analysis. For all statistical tests, P < 0.05 was considered significant.
Authors' contributions
RCD oversaw the project, performed the histological analysis, performed statistical analyses, and contributed to writing the manuscript. ON performed the quantitative RT-PCR analysis, and contributed to writing the manuscript.
Acknowledgements
We thank Larysa Stroganova for maintenance of the mouse colonies and PCR genotyping. Supported by National Institutes of Health grant DK 56791.
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| 15921521 | PMC1166548 | CC BY | 2021-01-04 16:38:18 | no | BMC Genet. 2005 May 27; 6:29 | utf-8 | BMC Genet | 2,005 | 10.1186/1471-2156-6-29 | oa_comm |
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-741594387510.1186/1471-2164-6-74Research ArticleLinkage mapping bovine EST-based SNP Snelling Warren M [email protected] Eduardo [email protected] Roger T [email protected] John W [email protected] Gregory P [email protected] Gary L [email protected] Timothy PL [email protected] US Meat Animal Research Center, Agricultural Research Service, US Department of Agriculture, Spur 18D, Clay Center, Nebraska 68933-0166, USA2005 19 5 2005 6 74 74 21 12 2004 19 5 2005 Copyright © 2005 Snelling et al; licensee BioMed Central Ltd.2005Snelling et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Existing linkage maps of the bovine genome primarily contain anonymous microsatellite markers. These maps have proved valuable for mapping quantitative trait loci (QTL) to broad regions of the genome, but more closely spaced markers are needed to fine-map QTL, and markers associated with genes and annotated sequence are needed to identify genes and sequence variation that may explain QTL.
Results
Bovine expressed sequence tag (EST) and bacterial artificial chromosome (BAC)sequence data were used to develop 918 single nucleotide polymorphism (SNP) markers to map genes on the bovine linkage map. DNA of sires from the MARC reference population was used to detect SNPs, and progeny and mates of heterozygous sires were genotyped. Chromosome assignments for 861 SNPs were determined by twopoint analysis, and positions for 735 SNPs were established by multipoint analyses. Linkage maps of bovine autosomes with these SNPs represent 4585 markers in 2475 positions spanning 3058 cM . Markers include 3612 microsatellites, 913 SNPs and 60 other markers. Mean separation between marker positions is 1.2 cM. New SNP markers appear in 511 positions, with mean separation of 4.7 cM. Multi-allelic markers, mostly microsatellites, had a mean (maximum) of 216 (366) informative meioses, and a mean 3-lod confidence interval of 3.6 cM Bi-allelic markers, including SNP and other marker types, had a mean (maximum) of 55 (191) informative meioses, and were placed within a mean 8.5 cM 3-lod confidence interval. Homologous human sequences were identified for 1159 markers, including 582 newly developed and mapped SNP.
Conclusion
Addition of these EST- and BAC-based SNPs to the bovine linkage map not only increases marker density, but provides connections to gene-rich physical maps, including annotated human sequence. The map provides a resource for fine-mapping quantitative trait loci and identification of positional candidate genes, and can be integrated with other data to guide and refine assembly of bovine genome sequence. Even after the bovine genome is completely sequenced, the map will continue to be a useful tool to link observable phenotypes and animal genotypes to underlying genes and molecular mechanisms influencing economically important beef and dairy traits.
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Background
Quantitative trait loci (QTL) have been described for a number of economically important traits in cattle [1,2]. Previous genetic maps [3,4] were sufficient to map QTL to broad regions. Narrowing those regions to fine-map the QTL, and eventually identify specific genes affecting a trait requires denser genetic maps with markers that can be associated with genes. Recently, 2277 microsatellite markers were added to the bovine genetic map [5] jointly developed by the Shirakawa Institute of Animal Genetics (SIAG) and the United States Meat Animal Research Center (MARC), reducing the average interval between markers from 3.0 cM to 1.4 cM. The updated map provides a tool to refine QTL locations, but, because it predominately represents anonymous markers, provides limited information about genes underlying the QTL. A second-generation radiation hybrid (RH) map of the bovine genome, representing 1564 gene markers (1463 with human homologs) and 349 microsatellite markers that have also been placed on linkage maps has also been described [6]. The utility of this gene-rich physical map is limited, however, by relatively sparse connections to genetic markers that can be associated with animal performance. Dense genetic and physical maps (ultimately fully annotated genomic DNA sequence) are needed to efficiently identify genes, and sequence variation, responsible for phenotypic variation. Dense connections between the physical maps and genetic marker maps are also needed, to associate animal phenotypes with the underlying genes and genomic sequence.
Recent efforts have added some gene-specific markers to bovine genetic maps. Several single-nucleotide polymorphism (SNP) markers have been developed to map specific targeted genes [7,8], or positional candidate genes near QTL [9,10]. Seventy SNP markers, developed using randomly selected bovine EST with human orthologs, were added to the bovine linkage map via twopoint linkage [11]. Two-point linkages for thirty other EST-based SNPs, selected to refine the comparison of bovine chromosome (BTA) 19 with human chromosome 17, were also obtained [11]. The present study extends EST-based SNP mapping to develop a linkage map representing nearly 1000 SNPs throughout the bovine genome. This map will provide a resource for gene-based genome-wide QTL scans and fine-mapping QTL. Only a few of the SNPs added to the linkage map are likely to represent genes directly influencing production. These SNP markers will, however, further define comparative relationships between the bovine linkage map and the well-annotated human and model organism genome sequences. These EST-based SNPs may facilitate identification of positional candidate genes that could ultimately affect costs of production and consumer acceptability of meat and milk products.
Results
Cattle genotyped for the SIAG-MARC linkage map represent four-breed crosses and backcross families [12]. F1 dams were produced by mating Piedmontese, Longhorn or Nelore bulls to non-inbred Hereford and Angus dams. The F1 dams were mated to two paternal half-sib Gelbvieh × Simmental bulls, producing full-sib four-breed cross calves by multiple ovulation embryo transfer (MOET). The backcross families were produced by mating a Nelore × Hereford bull to non-inbred Hereford dams, and a Brahman × Angus bull to non-inbred Angus dams, with full-sib backcross calves also produced by MOET. This population allows a potential of 412 informative meioses for autosomal markers [4].
The current SIAG-MARC bovine linkage maps represent 4779 markers, with 4585 markers on autosomes (Additional file 1). All of the 913 SNPs on the linkage maps (Table 1) are assigned to autosomes, including the 735 mapped in this work. In addition to the newly developed and mapped SNPs, 100 other markers including 76 previously described with two-point positions [11] were added to the multipoint autosomal linkage maps.
Table 1 Markers represented on SIAG-MARC bovine autosome linkage maps.
Informative Meiosesa,b
Marker type N Mean Minimum Maximum
microsatellite 3612 207 11 366
single nucleotide polymorphism
new SNPc 735 48 10 149
other SNPd 178 52 13 200
othere 60 69 16 307
All types 4585 174 10 366
aTotal informative meioses.
b 412 potential informative meioses from bovine reference population.
c newly developed and mapped EST- and BAC-based SNP.
d SNP marker data contributed by other projects.
e other types include sequence tagged allelic restriction sites (31), erythrocyte antigens (10), single strand conformational polymorphisms (6), serum protein markers (5), phenotypic observations (2) and sequence tagged site (2) markers from previous bovine maps[4,5,12].
A total of 918 SNP were identified in this study; 799 from EST sequences [GenBank Accessions BV103715 to BV106354] and 119 from BAC subclone sequence [Genbank Accessions BV445418 to BV446557; [13]]. One or both of the half-sib Bos taurus (Gelbvieh × Simmental) sires are heterozygous for 46% (380/834) of the SNPs genotyped in the two sires, and one or both of the Bos taurus × Bos indicus (Brahman × Angus; Nelore × Hereford) sires were heterozygous for 78% (706/908) of the SNPs genotyped (Additional file 2). Given costs of sequencing and genotyping, not every sire was sequenced or genotyped for every SNP. Because of an early observation that SNP were more prevalent in the Bos taurus × Bos indicus sires, there was a tendency to examine those bulls first, so the Bos taurus sires, progeny and mates may or may not have been genotyped for SNPs detected in Bos indicus cross animals.
Eighty percent of the SNPs developed in this study (Additional file 2) were positioned on the linkage maps. The 183 unmapped SNPs include 47 that did not have significant two-point linkage (lod > 3.0) to markers on the 1997 MARC linkage map [4], and 136 that were assigned to linkage groups but not placed on the multipoint map because they increased length of the linkage group excessively. A similar percentage of attempted SNP were placed on the multipoint swine linkage maps swine using similar genotyping and map construction strategies (G. Rohrer, personal communication). Unidentified genotyping errors are one possible cause of failure to map markers, both failure to detect significant twopoint linkage and rejecting markers inflating the map. Re-genotyping and verifying genotypes by sequencing, however, did not reveal systematic errors with the genotyping system. A more probable cause of individual marker failure may be the limited ability of the software to solve positions for biallelic markers with a small number of informative meioses encompassing a high percentage of like-heterozygote parents and offspring. Maps will inflate if a marker is placed incorrectly relative to other markers on the map, and if the most likely placement solvable by CRIMAP [14] results in an inflated map, that marker is rejected rather than allowing it to remain on a potentially distorted map. More correct placements may not be solvable by CRIMAP on 32-bit processors because the computations require more memory than can be addressed by 32-bit processors.
The 4585 autosome markers (Table 1) are placed in 2475 unique positions, with a mean (maximum) of 1.2 cM (9.1 cM) between markers. This is only slightly more dense than the maps without these SNPs [5], which contained 3755 markers in 2306 positions covering 3013 cM on autosomes (1.3 cM spacing). The new SNP markers occupy 511 distinct positions, 176 positions only represent these SNP and 335 are shared with other markers. The mean interval between the new SNP markers is 4.7 cM, with intervals up to 59.3 cM (Figure 1; Table 2). Each autosome contains at least one gap of 8.5 or more cM between SNP positions. SNP markers are spaced evenly along some chromosomes, other chromosomes contain clusters of several SNPs within a few cM. (The coefficients of variation in Table 2 provide relative measures of marker spacing; low values are indicative of even spacing, high values indicate uneven spacing with clusters of close markers separated by gaps.) Total length of the autosomes is 3058 cM, 45 cM longer than the 2004 microsatellite map [5], and 294 cM longer than the 1997 map [4]. The increased length, relative to the 2004 map, is due to recombinations introduced by new marker genotypes. These apparent recombinations may largely be attributed to incorrect ordering, when possibly more likely orders could not be solved by CRIMAP [14].
Figure 1 Marker positions on bovine autosomes. Marker positions on bovine autosomes. Vertical lines represent an individual chromosome. Red ticks to the left of each vertical line represent positions occupied by one or more newly developed SNP markers. Blue ticks to the right of each vertical line indicate positions occupied by other markers.
Table 2 Distribution of markers placed on bovine autosomes.
Markersa Positionsb Human Connectionsc Mean Intervald Maximum Intervale Marker Spacing CVf
BTA New SNPg Otherh New SNP Other New SNP Other New SNP Other New SNP Other New SNP Other
1 28 263 21 150 18 30 6.8 1.1 59.3 5.7 1.9 .9
2 39 188 31 112 33 34 4.4 1.2 17.5 6.3 .9 .9
3 45 174 29 99 37 25 4.2 1.3 16.5 5.4 1.1 .8
4 28 131 18 82 21 21 6.1 1.5 16.2 4.6 .7 .7
5 62 185 36 114 40 37 3.8 1.3 22.8 4.7 1.3 .8
6 15 230 14 115 7 35 8.6 1.2 16.7 9.1 .6 1.0
7 45 142 28 88 39 31 4.6 1.6 34.3 8.5 1.5 .9
8 24 125 21 85 19 11 5.6 1.6 18.5 7.9 .9 1.0
9 5 126 5 79 5 10 16.3 1.5 42.7 4.9 1.1 .8
10 39 131 24 87 34 25 3.7 1.4 8.6 3.8 .7 .7
11 28 199 22 106 23 28 5.4 1.3 35.5 7.6 1.6 1.0
12 4 122 4 79 3 10 22.9 1.4 47.9 8.9 1.0 .9
13 26 116 17 75 21 17 3.9 1.4 12.1 4.9 .9 .7
14 6 133 6 77 6 21 18.0 1.4 49.5 5.1 1.2 .9
15 28 145 19 92 27 38 5.5 1.2 16.4 5.6 .9 .9
16 27 101 20 66 19 6 4.7 1.5 28.5 5.2 1.3 .7
17 23 107 15 70 16 15 6.5 1.4 24.4 5.5 1.1 .7
18 49 114 27 69 39 20 2.7 1.3 10.8 5.3 .9 .8
19 39 166 32 94 32 81 2.4 1.2 12.7 6.7 1.2 1.1
20 11 119 8 64 8 12 6.8 1.3 16.1 5.2 .8 .8
21 20 123 16 71 19 15 4.8 1.2 26.3 4.0 1.5 .7
22 25 84 12 57 20 21 5.4 1.6 18.7 7.2 1.1 .8
23 29 84 16 46 25 29 5.0 1.8 30.8 6.3 1.6 .7
24 10 95 8 62 7 9 9.5 1.3 17.3 3.8 .6 .6
25 29 74 25 51 21 12 2.7 1.4 11.3 7.1 1.3 1.1
26 12 64 4 44 11 8 9.4 1.9 14.5 7.2 .6 .9
27 5 72 5 41 1 6 10.5 1.7 15.9 6.2 .5 .8
28 9 79 9 44 7 10 5.7 1.4 19.0 4.7 1.0 .7
29 25 158 19 80 24 27 2.8 .9 13.6 3.3 1.2 .8
aNumber of markers.
b Number of distinct positions.
c Number of markers with homologous human sequence, determined by BLAT alignments between marker sequences and UCSC hg17 sequence..
d Mean interval between distinct positions.
e Maximum interval between distinct positions.
f Coefficient of variation for marker spacing. Low values indicate relatively even space between markers; high values indicate uneven spacing with clusters of markers separated by gaps.
g Newly developed and mapped EST- and BAC-based SNP.
h All other markers, including markers from previously described maps and newly mapped markers with data contributed by other projects.
Adding SNP markers resulted in minimal rearrangement of the linkage map. Correlations between marker positions on the 2004 [5] and current maps were near unity (r > .99) for all autosomes. Eighty-nine percent of the markers on autosomes from the 2004 map remained in the same index position (markers indexed 0,1,2 ... n on each autosome). Eight percent were shifted by a single position, and four percent moved by two to five positions. The most substantial shifts were with ambiguously placed markers, which can be included at any one of several positions without affecting map likelihood.
Adding EST- and BAC-based SNPs substantially increased comparative points defined by alignment of marker sequences against the human genome. Human homologues were identified for 1159 markers, including 498 markers from the 2004 map [5]. Newly developed SNP add 582 homologies, and 79 are from other markers added to the multipoint maps.
Utility of the current map to explore regions of the genome that influence animal performance can be demonstrated by examining the regions surrounding previously described QTL. A region of BTA 5, from 73.5 to 77.6 cM on the 1997 MARC map [4], bounded by microsatellite markers IGF-1 and BM1819, has been associated with preweaning gain [15]. Allowing for changes in the map, the region defined by these two markers now spans 76.9 to 84.6 cM. The two markers are separated by one marker on the 1997 map. The 2004 SIAG-MARC map [5] placed nine microsatellites in the region; and the current map shows the same nine microsatellites as well as five SNPs. The recent bovine RH map [6] contains four gene markers and the BM1819 microsatellite in the corresponding region, from 326.8 to 337.8 cR5000. Three of the gene markers (IGF1, TU12B1-TY, SYCP3) were aligned with human chromosome (HSA) 12 sequence, from 100.6 to 102.7 Mbp. The fourth gene marker (TIMP3) aligned with HSA 22 at 31.5 Mbp. Sequences associated with linkage markers also align with HSA 12 and 22. Sequence associated with microsatellite DIK4787 aligns with HSA 12 at 105.2 Mbp. Sequences containing an SNP (17019_98) and two microsatellites (BMS1216, DIK5165) align with HSA 22, from 31.2 to 32.0 Mbp. These alignments suggest that the QTL region contains a break in synteny, in agreement with the break described by the bovine RH – human comparative map [6].
Agreement in comparative alignments between the current linkage and RH maps suggests either map might be used to identify positional candidate genes underlying this QTL. Taken together, information from both maps may provide more complete coverage than that indicated by either map alone. The polymorphic linkage markers will be more useful to identify relationships between marker genotype and phenotype and fine-map the QTL, although further marker development will be necessary to isolate causative polymorphisms.
Accuracy of placing SNPs on the linkage map was a concern, because bi-allelic SNPs are generally less informative than highly polymorphic microsatellite markers (Table 1). Most markers (80%) with three or more alleles have unambiguous placement, while a single, most likely, position can be determined for only 36% of the bi-allelic markers, and the mean 3.6 cM three-lod confidence interval for multi-allelic markers is narrower than the 8.5 cM confidence interval for bi-allelic markers.
Discussion
The current linkage map, representing over 4000 anonymous markers and several hundred gene-specific SNPs, provides a resource to link genetic variation in animal performance to underlying DNA sequence variation. The procedures used to construct this map were designed to allow frequent updates, so new markers can easily be added. The reduction of mean space between markers, from 3.0 cM to 1.3 cM, primarily because of recently added microsatellites [5], increases opportunities to fine-map QTL. Addition of EST-based SNPs increases connections between the genetic map and gene maps, including currently available bovine RH maps, annotated human and model organism sequence, and eventually bovine sequence [16]. These connections may increase efficiency of identifying genes and causal mutations affecting animal performance. Where QTL regions can be narrowed, and the regions include markers connecting the region to annotated sequence, the list of positional candidate genes that might partially explain phenotypic variation may be shortened considerably.
The current or future versions of the bovine genetic map will be useful to assemble and validate bovine genomic sequence. The genetic map represents the intact living genome, so it is not subject to cloning and assembly problems associated with other mapping and sequencing techniques [17]. However, resolution of the genetic map is limited, and markers that were not separated by recombination in the experimental population cannot be correctly ordered on the linkage map. Genetic mapping data can be combined with bovine RH [6,18] and BAC map data [19], exploiting resolution characteristics of both genetic and physical mapping data [20,21] to obtain a high resolution, well ordered consensus map useful to guide and refine bovine sequence assembly [17,22], and anchor QTL on the draft sequence [23]. Even after complete assembly of the bovine genome, the sequence will not replace the genetic map. The genetic map, especially if it is continually updated to represent new SNPs and other markers, should provide valuable links between phenotypes and associated marker genotypes, in order to identify and exploit genomic variation influencing economically important traits.
Conclusion
More than 700 EST- and BAC-based SNP markers were added to the bovine linkage map. Order of previously mapped markers was largely unaffected. The SNPs increased the density of the map somewhat, and substantially increased connections to gene-rich physical maps, including annotated human sequence. The number of linkage markers with human homologues was more than doubled by addition of these SNP and other markers. The map provides a resource for fine-mapping quantitative trait loci and identification of positional candidate genes, and can be integrated with other data to guide and refine assembly of bovine genome sequence. The map can easily be updated with a cyclic map construction process, and it will continue to be a useful resource connecting observable phenotypes and animal genotypes to underlying genes and molecular mechanisms influencing economically important beef and dairy traits.
Methods
Marker development
Single nucleotide polymorphism markers were developed from bovine EST sequence as described [11]. Briefly, tentative consensus (TC) clusters of bovine EST were obtained from The Institute for Genomic Research (TIGR) Bovine Gene Index[24]. Repeat elements in the TC were masked using RepeatMasker [25] and aligned with the human genome draft sequence via BLASTN [26]. Alignments were checked for the presence of apparent introns using a perl script that computed the predicted intron size. Primers were designed from the bovine EST sequence in such a way as to cross introns and produce products of approximately 800–1300 bp, while including at least 100 bp of putative exon sequence to allow confirmation that the primers targeted the desired gene.
Alternatively, SNP for some genes were developed from sequence associated with bacterial artificial chromosome (BAC) clones carrying all or part of the target gene(s) essentially as described [27]. Briefly, high density filters representing the CHORI-240 BAC library (P. deJong, personal communication) were screened with radiolabeled insert from EST clones representing target genes as recommended by the manufacturer (BACPAC resources, Oakland, CA). Positive clones were partially digested using the restriction enzyme Sau3AI (Promega, Madison, WI) by incubation of isolated BAC DNA. Aliquots of the reaction were removed at 10, 20 and 30 minutes of incubation into 10 mM final concentration EDTA on ice, desalted by gel filtration column (Axygen, Union City, CA), and separated on a 0.8% agarose gel. Fragments of 0.8–1.5 kilobase (kb) were excised from the gel and purified by ion exchange column as directed by the manufacturer (Marligen, Ijamsville, MD). Isolated fragments were cloned into pBluescript vector (Clonetech, Palo Alto, CA) prepared by digestion with restriction enzyme BamHI, and 192 randomly selected subclones for each BAC were picked into 80 ul LB media supplemented with ampicillin at 50 ug/ml in 384-well plates for sequencing from both ends with vector primers. A total of 134 BACs were screened, representing 122 loci (approximately 0.3% of the bovine genome).
Resulting sequences were analyzed using Phred, Phrap, and Consed programs [28] and amplification primers were designed using the autofinish and primer3 options of Consed. Primers derived from EST sequence or BAC subclones were used to amplify DNA from four bulls that were the sires (two Bos taurus × Bos taurus; two Bos taurus × Bos indicus) of the MARC reference panel mapping families [12], and the PCR products were sequenced with the amplification primers to identify heterozygous positions (SNP) in the amplicons using Polyphred and Consed. The SNPs were genotyped in progeny and mates of heterozygous sires from the MARC reference population using the Sequenom MassArray System [29] following established procedures [11].
Map construction
Software and procedures
Map construction was an iterative process completed in cycles. Cycles were initiated when genotypes for new markers, or corrections to previously genotyped markers, were recorded for animals in the MARC reference population. CRIMAP 2.4 [14], modified to reduce occurrence of unsolvable marker orders and controlled by a series of Perl scripts, was used to assign markers to linkage groups, order markers in each linkage group, and identify possible genotyping errors. All markers genotyped in the reference population were considered in map construction, including SNPs developed for this project as well as microsatellites, SNPs and other types of markers with recorded genotypes.
The modifications to CRIMAP included redimensioning arrays to accommodate a larger number of markers, and using logarithmic arithmetic for intermediate calculations to avoid values exceeding the precision limits of the 32-bit CPUs used to solve the maps. The perl scripts were developed to construct sets of alternative marker orders necessary at each step, distribute the needed CRIMAP fixed executions to nodes of a Linux cluster, and identify the most likely order from the set of orders evaluated in each step.
Mapping data sets
Two data sets were used to construct maps. Final marker order and map distances were computed from the complete reference population pedigree and genotype data. A reduced data set was used to initially place new markers and order the maps. The reduced data set was constructed by eliminating progeny genotypes where the progeny and both parents had identical heterozygous genotypes. These ambiguous, like-heterozygous, genotypes provided little information about recombination, although phase of inheritance and recombination are inferred from linked markers with unambiguous genotypes. Genotypes for about one-half of the markers included at least one ambiguous genotype, but fewer than 2.5% of the total number of observed genotypes were eliminated from the reduced data set. Including these ambiguous genotypes increased the number of calculations and computer memory necessary to compute likelihood of a particular marker order. Likelihoods of certain marker orders, usually involving several markers with ambiguous genotypes ordered consecutively, could not be solved using the complete data set. When the reduced data set was employed, uncomputable orders were eliminated and the time required to solve individual likelihoods reduced, making it feasible to evaluate a larger number of alternative marker orders.
Linkage group assignments
A map construction cycle began by extracting genotypes and pedigree data from the MARC database, and formatting the full and reduced data sets. Genotypes exhibiting non-Mendelian inheritance patterns were detected by the CRIMAP prepare option. Two-point analyses, with the complete data, were conducted to assign newly genotyped markers to linkage groups representing entire chromosomes. The two-point analyses established linkage between the new markers and subset of markers from the 1997 MARC map [4], selected to contain the most informative marker within each 5 cM interval. A LOD score greater than 3.0 was required to assign a new marker to a linkage group. Markers were assigned to the linkage group containing the marker with the highest two-point LOD score with the lowest recombination fraction.
Initial ordering of markers within linkage group
Markers assigned to a linkage group were initially ordered using the reduced data set. Starting from the existing order of a linkage group, each marker assigned to that linkage group by two-point analysis, but not present on the multi-point map, was tested in every possible location. These unmapped markers were evaluated in order of decreasing informativeness; the marker with the greatest number of informative meioses was tested first, and the marker with the least informative meioses was the last evaluated in each round of marker insertion. Markers were inserted at the location with the highest log-likelihood, if placement at that position did not increase map length by more than 0.75 cM. This somewhat arbitrary limit on increases in map length was imposed to minimize distortion that can result from errors in genotypes and marker order. Once a set of markers meeting the allowable increase in map length was inserted, alternative marker orders were evaluated in an iterative procedure until a more likely order could not be identified. Log-likelihoods were computed with each pair of adjacent markers interchanged. The current order was then replaced by the order of the switched pair showing the greatest improvement in likelihood, and the process was repeated until switching pairs of adjacent markers did not improve likelihood of the map. Map lengths with each marker temporarily removed from the linkage group were then determined. If removing a marker reduced map length, that marker was evaluated in all possible positions and reinserted at the position with the highest likelihood. If order was changed by removing and reinserting markers into more likely positions, the reordering process, starting with interchanging adjacent pairs of markers was repeated. If the resulting marker order was different than the initial order from the previous attempt to insert markers, and some markers assigned to the linkage group remained unmapped, processing continued with another attempt to insert markers.
Finalizing maps
After no more markers could be inserted that satisfied the criteria defined above, and the algorithms did not reveal a more likely marker order from the reduced data, the full data set was used to finalize the set of markers on the multi-point map, then order and position those markers. Inferred inheritance of ambiguous genotypes, included in the complete data set, suggested some map inflation and marker rearrangements that were not apparent in the reduced data set. Lengths of the map with markers individually removed were determined, and interior markers that stretched the map more than 0.75 cM were eliminated from the set of mapped markers. At this stage, the limit prevented map inflation caused by placing markers in the most likely solvable order, when possibly more likely orders, that did not increase map length, could not be solved using the complete data. The process of switching adjacent marker pairs was repeated with the full data set to establish a final marker order, and marker positions were computed from this order. The final maps represent the most likely marker order identified with the complete data set, although an exhaustive search of all possible marker orders was not conducted (and is not feasible).
After determining the final marker order, the chrompic option of CRIMAP was used to determine likely grandparental origin of marker alleles and identify recombination along each progenys' chromosomes. Several chrompic analyses were conducted for each linkage group; one for the final map and one for each marker that increased map length by more than 0.75 cM. Possible genotyping errors, indicated by two or more recombinations in an individual's chromosomes, were identified and suspicious genotypes were checked by manual inspection of the raw spectrographic data. Where no apparent error in the assay could be detected, animals were genotyped a second time using the Sequenom system, and in selected cases, genotypes were verified by sequencing. Corrections were entered in the database, and used in subsequent map construction cycles.
Confidence interval estimation
Confidence intervals around marker positions were estimated by computing the likelihoods of each marker in all possible positions, while preserving the final order of remaining markers in the linkage group. These likelihoods were computed with CarthaGene [20] using output translated from the CRIMAP [14]chrompic analysis of the final marker order. CarthaGene was used primarily for speed; the CarthaGene analyses ignored the distinction between probable and known allele phase, but yielded similar results in substantially less time and eliminated any occurrence of uncomputable orders. For a given LOD threshold, alternate positions yielding a log-likelihood difference less than the threshold were determined, as well as map positions (cM) of markers holding that order. Confidence intervals were computed from map positions corresponding to marker order. A map containing n markers ordered 1, 2, ... n had corresponding positions p1, p2, ... pn. An individual marker m holding order i could be placed anywhere between the position corresponding to the left index li to the position corresponding to the right index ri (li <= i <= ri) without reducing log-likelihood more than some threshold t. Confidence intervals were estimated from the equations:
CImt = pli + 1 - pri - 1 for li > 1 and ri <n
CImt = pli - pri - 1 for li > 1 and ri = n
CImt = pli + 1 - pri for li = 1 and ri <n.
The estimated confidence intervals are bounded by 0 and pn, so intervals including either end of the linkage group may be underestimated.
Comparative mapping
The collection of bovine sequences in GenBank sequence tag site (STS), mammal (MAM), patent (PAT), EST, and genome survey sequence (GSS) divisions, excluding sequences from the ongoing bovine sequencing effort [16], were obtained. Bovine sequences associated with markers were identified with e-PCR [30]. Where the primer pair for a marker matched multiple sequences, a consensus sequence representing that marker was obtained with phrap[28], and repetitive sequence was masked [25]. BLAT [31] was used to align resulting sequences to the May 2004 human genome assembly [32]. The highest scoring alignment for each marker sequence was identified, and was considered comparative only if all high scoring alignments for that marker consistently aligned with the same region of the human genome. Marker-human alignments were discarded if the marker sequence aligned with two or more regions of the human genome with similarly high BLAT scores.
Authors' contributions
WMS developed map construction procedures, conducted linkage analyses and drafted a major portion of the manuscript. EC organized DNA sample collection and storage. RTS and TPLS developed SNP markers and genotyped animals. JWK developed procedures to record and manage genotypes. GPH conducted sequence analyses, including masking repeats in EST sequence, BLAST alignments, and primer design. GLB oversees linkage data collection and curates the maps. All authors were involved in conceiving and planning the research, which was coordinated by TPLS. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Excel spreadsheet containing linkage maps of bovine autosomes. Data include marker name, locus, and type; PubMed or other reference, chromosome, position on chromosome, 3-lod confidence interval and comparative position on human sequence.
Click here for file
Additional File 2
Excel spreadsheet containing details about each newly developed SNP. Data include MARC id number, marker name, GenBank STS id, GenBank Accession, primer sequneces, and results from twopoint and multipoint linkage analyses.
Click here for file
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| 15943875 | PMC1166549 | CC BY | 2021-01-04 16:39:53 | no | BMC Genomics. 2005 May 19; 6:74 | utf-8 | BMC Genomics | 2,005 | 10.1186/1471-2164-6-74 | oa_comm |
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-761590720810.1186/1471-2164-6-76Research ArticleIdentification, characterization and metagenome analysis of oocyte-specific genes organized in clusters in the mouse genome Paillisson Amélie [email protected]é Sébastien [email protected] Isabelle [email protected] Martine [email protected]ès-Tran Rozenn [email protected] Daniel [email protected] Philippe [email protected] Physiologie de la Reproduction et des Comportements, UMR 6175 INRA-CNRS-Université François Rabelais de Tours-Haras Nationaux, 37380 Nouzilly, France2 Département de Biologie Structurale, LMCP, CNRS, UMR 7590, Universités Paris6 et Paris 7, case 115, 4 place Jussieu, 75252 Paris Cedex 05, France3 U709 – INSERM, Pavillon Baudelocque, Hôpital Cochin, 123, Boulevard de Port-Royal, 75014 Paris, and INRA, Département de Génétique animale2005 20 5 2005 6 76 76 25 1 2005 20 5 2005 Copyright © 2005 Paillisson et al; licensee BioMed Central Ltd.2005Paillisson et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Genes specifically expressed in the oocyte play key roles in oogenesis, ovarian folliculogenesis, fertilization and/or early embryonic development. In an attempt to identify novel oocyte-specific genes in the mouse, we have used an in silico subtraction methodology, and we have focused our attention on genes that are organized in genomic clusters.
Results
In the present work, five clusters have been studied: a cluster of thirteen genes characterized by an F-box domain localized on chromosome 9, a cluster of six genes related to T-cell leukaemia/lymphoma protein 1 (Tcl1) on chromosome 12, a cluster composed of a SPErm-associated glutamate (E)-Rich (Speer) protein expressed in the oocyte in the vicinity of four unknown genes specifically expressed in the testis on chromosome 14, a cluster composed of the oocyte secreted protein-1 (Oosp-1) gene and two Oosp-related genes on chromosome 19, all three being characterized by a partial N-terminal zona pellucida-like domain, and another small cluster of two genes on chromosome 19 as well, composed of a TWIK-Related spinal cord K+ channel encoding-gene, and an unknown gene predicted in silico to be testis-specific. The specificity of expression was confirmed by RT-PCR and in situ hybridization for eight and five of them, respectively. Finally, we showed by comparing all of the isolated and clustered oocyte-specific genes identified so far in the mouse genome, that the oocyte-specific clusters are significantly closer to telomeres than isolated oocyte-specific genes are.
Conclusion
We have studied five clusters of genes specifically expressed in female, some of them being also expressed in male germ-cells. Moreover, contrarily to non-clustered oocyte-specific genes, those that are organized in clusters tend to map near chromosome ends, suggesting that this specific near-telomere position of oocyte-clusters in rodents could constitute an evolutionary advantage. Understanding the biological benefits of such an organization as well as the mechanisms leading to a specific oocyte expression in these clusters now requires further investigation.
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Background
Mammalian oocyte is the only known cell able to activate the zygotic genome after fertilization. Its cytoplasm is also able to reprogram the nucleus of a differentiated cell in cloning experiments. Therefore, it is likely that several genes specifically expressed in the oocytes are responsible for this ability to program diploid genomes. It is the case for the so-called maternal genes such as Maternal Antigen That Embryo Required (MATER), Zygotic Arrest 1 (Zar1), Stella and nucleoplasmin 2 (Npm2) that are all required for normal embryonic development beyond the 1- or 2-cell stage [1,2]. Moreover, new observations have shown that loss of function mutations in the two oocyte-specific genes GDF-9 and BMP-15 are responsible for severe alterations of ovarian folliculogenesis, these alterations being different depending on the hetero- or homozygous state of the individuals in the ovine species [3,4]. So genes specifically expressed in the oocyte seem to play key roles in oogenesis, ovarian folliculogenesis, fertilization and/or early embryonic development. In an attempt to identify novel oocyte-specific genes, several groups have used both mRNA differential display [5] and in silico subtraction approaches [6]. By using the latter, we have recently identified six genes of the oogenesin family [7], these genes being present on chromosome 4 in a cluster of almost 1 Mb composed of twelve oogenesin paralogous genes. We have also identified six genes presenting similarities with NALP5/MATER [8] which are specifically expressed in the mouse oocyte, three being clusterized on chromosome 7. We have experimentally validated the specificity of expression for three oogenesin and five NALP genes [7,8]. The fact that these two groups of genes are localized in two clusters indicates that they likely originate from duplications of an ancestral gene.
In the present work, we have used the same in silico approach to identify new oocyte-specific genes that are also localized in clusters in the mouse genome. In particular, we have identified three new loci in the mouse genome containing several genes specifically expressed in the oocyte, and two loci containing genes specifically expressed in male and female germ cells, which may thus be called "germ cell loci". We have experimentally verified the specificity of expression for eight of them by RT-PCR and five of them by in situ hybridization. Moreover, we have compared the map position of all the "oocyte-clusters" identified so far with that of the other "isolated" oocyte-specific genes. We could show that oocyte-specific clustered genes are significantly closer to telomeres than oocyte-specific isolated genes. Our results emphasize a new vision of gene regulation, where large-scale genome organization likely plays an important function.
Results
In silico identification of oocyte-specific genes
By using the Digital Differential Display (DDD) software, and as previously described [7,8], we found in silico a list of approximately 60 genes for which ESTs were exclusively detected in mouse egg libraries. After localization on the mouse genome [9], we noticed that several of these genes were neighbored by genes for which ESTs were exclusively present in egg and/or testis libraries as well. Finally, the number of oocyte-specific genes approached one hundred. Amongst these genes were found most of the well-known oocyte specific genes such as GDF-9, Mater or Zar1, validating the methodology, as well as the 12 oogenesins and the 6 NALP/Mater-like genes [7,8]. In the present work, we have focused our attention towards five other clusters of genes (Fig. 1).
Figure 1 Localization of germ-cell-specific cluster in mouse chromosome 9, 12, 14 and 19 [9]. The presence of mRNAs or ESTs of these different genes in male and female germ cells was assessed by experimental (RT-PCR, Southern blot and in situ hybridization) and/or in silico methodologies as described in Methods. O stands for Ovary/Egg; T stands for Testis.
The first cluster is localized on chromosome 9 and is composed of thirteen genes corresponding to the GenBank accession numbers AK054339, AK087710, AK087709, AK087808, XM_284540, XM_486447, XM_486263, XM_488195, XM_356253, AK054298, AK087669, XM_356191 and AK007274. All of these genes encode proteins that contain an N-terminal F-box domain as well as WD40 repeats in the C-terminal region. As the gene corresponding to the GenBank accession number AK087709 is named "F-box only protein 12" (FBXO12), we have renamed the twelve other genes FBXO12A, FBXO12B, FBXO12C, FBXO12D, FBXO12E, FBXO12F, FBXO12G, FBXO12H, FBXO12I, FBXO12J, FBXO12K and FBXO12L (Table 1 & Fig. 1).
Table 1 In silico informations available on Unigene about the genes, which are present in the five clusters studied here.
Name chromosome GenBank cDNA source
FBXO12A 9 F2 AK054339 Ovary
FBXO12B 9 F2 AK087710 ovary; in vitro fertilized eggs
unfertilized egg
FBXO12 9 F2 AK087709 ovary, pituitary gland, egg, pre-implantation
embryo, mid-gestation embryo
FBXO12C 9 F2 AK087808 ovary, egg
FBXO12D 9 F2 XM_284540 in vitro fertilized eggs ;
Unfertilized Egg
FBXO12E 9 F2 XM_486447 Ovary, Egg, Pre-implantation Embryo
FBXO12F 9 F2 XM_486263 Egg, Pre-implantation Embryo
FBXO12G 9 F2 XM_488195 Kidney, Egg, Pre-implantation Embryo, Neonate
FBXO12H 9 F2 XM_356253 Egg
FBXO12I 9 F2 AK054298 Ovary, Egg, Pre-implantation Embryo
FBXO12J 9 F2 AK087669 Brain, Ovary, Skin, Egg, Pre-implantation Embryo
FBXO12K 9 F2 XM_356191 Ovary, Thymus, Egg, Pre-implantation Embryo,
FBXO12L 9 F2 AK007274 Brain, Testis, Pre-implantation Embryo
Tcl1b2 12 E NM_013775 Egg, Pre-implantation Embryo
Tcl1b1 12 E BC052337 Egg, Pre-implantation Embryo
Tcl1b5 12 E NM_013776 Pituitary Gland, Egg, Pre-implantation Embryo, Mid-gestation Embryo
Tcl1b3 12 E NM_013772 embryo ; egg
Tcl1b4 12 E NM_013774 embryo ; egg
Tcl1 12 E BC052336 Unfertilized Egg ; in vitro fertilized eggs
embryo; egg; retina
Speer A 14 A3 XM_138939 unfertilized egg; in vitro fertilized eggs
embryo; egg; ovary; round spermatids
14 A3 AK076711 testis
14 A3 AK005833 testis
14 A3 AK016429 testis
14 A3 AK019699 testis
Oosp2 19 A XM_129237 Ovary, Pre-implantation Embryo
Oosp1 19 A AF420487 Egg, Pre-implantation Embryo, Mid-Gestation
embryo
Oosp3 19 A XM_129191 unfertilized egg; in vitro fertilized eggs
ovary; embryo
trik channel 19 D3 XM_285304 in vitro fertilized eggs; egg
unknown 19 D3 AK015359 testis
The second cluster contains the so-called Tcl1 (T-cell leukemia/lymphoma protein 1) and Tcl1b genes, and maps on chromosome 12. This cluster contains six genes related to Tcl1 gene and is predicted to be oocyte-specific in silico (Fig. 1 & Table 1). The protein encoded by this gene shares similarities with the protein encoded by the gene MTCP1, also involved in chromosomal translocations in T-cell proliferative disease. Its structure consists of an eight-stranded β-barrel with a particular topology, with the N- and C-terminal halfs being linked by a pseudo dyad [10].
We have identified another cluster on chromosome 14, which was composed of five genes. One of them encoded for a protein that belongs to the Speer (SPErm-associated glutamate (E)-Rich protein) family, characterized by a very high proportion of alpha-helical secondary structure. We have thus renamed this gene SpeerA (Genbank identification number XM_138939). The corresponding Speer proteins are weakly similar to proteins that contain interacting domain with cytoskeletal as well as nuclear matrix proteins such as actin and dynein. In silico, ESTs of SpeerA are present in egg libraries but also in round spermatid libraries (Table 1). The ESTs corresponding to the four nearest neighboring genes, corresponding to the GenBank identification numbers AK076711, AK005833, AK016429 and AK019699, have been exclusively detected in silico in testis libraries (Fig. 1 & Table 1). The gene corresponding to the AK016429 accession number encoded for a protein that presented no clear similarity with any known protein. The three other genes likely encoded for untranslated RNAs, as no long ORF could be detected in their sequence.
Two other clusters map to chromosome 19. The first one is composed of three genes (Genbank identification numbers XM_129237, AF420487, and XM_129191). The corresponding three protein sequences share about 47% of similarity and can be aligned with the 150 first amino acids of domains of the Zona-Pellucida family (Fig. 2). Secondary structure predictions indicate mainly a β-fold for this domain, in which the conserved Cys78 and Cys98 might form a disulfide bond, according to experimental data obtained on the corresponding Cys61 and Cys81 of ZP3 [11]. The C-terminal ends of Oosp proteins (not represented) are highly variable and cannot be aligned with the much more large C-terminal part of ZP domains. The gene corresponding to the GenBank accession number AF420487 is the well-known "oocyte secreted protein 1" (Oosp1) [12]. So we have renamed the two other genes Oosp2 and Oosp3 (Table 1 & Fig 1).
Figure 2 Alignment of Oosp protein sequence with the N-terminal part of the ZP domain (150 amino acids) of ZP3. Identities and similarities are shown with black boxes and black frames, respectively. An hypothetical disulfide bond is suspected between Cys78 and Cys98 according to experimental data obtained on the corresponding Cys61 and Cys81 of ZP3 [11].
The second cluster on chromosome 19 is composed of an oocyte specific gene, corresponding to the XM_285304 GenBank identification number (Fig. 1), the protein of which is similar to a TWIK-Related spinal cord K+ (Trik) channel. The other gene, corresponding to the AK015359 number, was predicted to be testis-specific in silico (Table 1), and encoded for an unknown protein (Fig. 1).
Expression analysis
By analysis of RT-PCR products on BET-stained gels, FBXO12B, FBXO12D, Tcl1, Tcl1b3, Tcl1b4, SpeerA and Oosp3 appeared to be exclusively expressed in the ovary, whereas the gene corresponding to the AK076711 GenBank identification number appeared to be exclusively expressed in the testis (data not shown). By contrast, actin was readily amplified in all tissues. To increase the sensitivity of detection of these transcripts, Southern blot experiments were performed on RT-PCR products, confirming these results, except for Tcl1 and SpeerA genes for which a slight expression was also detected in the testis (Fig. 3).
Figure 3 Expression analysis of FBXO12B, FBXO12D, Tcl1b3, Tcl1b4, Tcl1, SpeerA, AK076711, Oosp3 and actin by RT-PCR in mouse tissues. Except for actin, total RNA from different tissues were subjected to Southern blot analysis of the RT-PCR products as described in Methods.
In situ hybridization confirmed that within the ovary, SpeerA, FBXO12B, FBXO12D, Tcl1 and Oosp3 were exclusively expressed in oocytes (Fig. 4). Transcripts were detected in primary follicles, as well as in early and large antral follicles (Fig. 4), except for FBXO12D and Tcl1, for which transcripts were only slightly if at all detected in large antral follicles (Fig. 4).
Figure 4 Localization of SpeerA, FBXO12B, FBXO12D, Tcl1 and Oosp3 mRNAs by in situ hybridization. Black arrows point: oocytes; gc: granulosa cells
Metagenome analysis
Altogether, the chromosome localization of 92 oocyte-specific genes was known, amongst the ~100 in silico-predicted oocyte-specific genes recovered. Amongst these 92 genes, 48 were organized in 7 "oocyte-clusters" (Table 2). We did not consider the two "germ-cell clusters" on chromosomes 14 and 19 in this analysis, but we included the oogenesin and Nalp9 clusters previously described by our group [7,8], as well as the Obox cluster described by Rajkovic et al. (2002). The other 44 oocyte-genes were considered as "isolated" in the mouse genome. For these latter genes, the average distance expressed in relative distance from the chromosome end ranged from 2.5% to 49.8% (average = 24 ± 16%). By contrast, the 48 oocyte-specific genes organized in clusters mapped between 5.6% and 20.6% from the closest chromosome end (average = 12.6 ± 5.3%). A Student t test was performed to compare the two categories, considering only one position for each cluster, and resulted in a value of 0.035. Similar analysis was performed taking into account the number of genes before and after the locus/cluster under scrutiny. This analysis gave rise to similar results. Therefore, oocyte-specific genes that are organized in clusters map significantly nearest chromosome ends than those that are "isolated".
Table 2 description of the 7 clusters of oocyte-specific genes that were considered for the metagenome analysis: clusters of the oogenesin proteins [7], the Oas proteins [38], the Obox proteins [39], the Nalp9 proteins [8], the FBXO12 proteins, the Tcl1 proteins and the Oosp proteins.
chromosome name of the cluster number of genes localized in the cluster
9 E1 Oogenesin 12
5 F Oas 3
7 A1 Obox 8
7 A3 Nalp9 3
9 F2 FBXO12 13
12 E Tcl1 6
19A Oosp 3
Total 48
Discussion
The present work and two previous identifications of the oogenesin genes [7] and of the mater family genes [8] demonstrate that the in silico subtraction is an efficient and reliable methodology to identify new oocyte-specific genes. By localizing these genes on the mouse genome, we have discovered other "germ-cell" specific genes, organized in clusters. RT-PCR as well as in situ hybridization experiments confirmed their specificity of expression in the ovary and/or in the testis. As for GDF-9, Mater, Zar1 or Stella, these genes may play important roles in folliculogenesis, fertilization and/or early embryonic development.
Amongst the "oocyte-specific clusters" presented here, the Tcl1/1B has previously been described. Indeed, in a previous work on human TCL1 and TCL1B genes, the six murine Tcl1 and Tcl1b1-5 orthologous genes were already shown to be expressed in oocytes and two-cell embryos but not in other adult tissues except in lymphoid cell lines, where overexpression of Tcl1 in these cells leads to leukemia or lymphoma [13]. Tcl1 was also shown to be expressed in testis, as previously described [14,15], its overexpression inducing the formation of testicular seminomas [16]. Interestingly, Tcl1-/- female mice are sterile, as early embryos are unable to undergo normal cell cleavage up to 8-cell stage, the majority of embryos being even unable to proceed beyond the 4- to 8-cell stage [16]. Therefore, like Mater and Zar1, Tcl1 can be considered as a maternal effect gene as well. It is possible that part of the effects of this gene family is to promote cell survival and/or proliferation by activating the Akt kinase as previously observed [17,18].
The F-box proteins, encoded by the thirteen genes localized on chromosome 9 cluster, might specifically interact with other proteins in the oocyte. Indeed, the F-box motif is present in numerous proteins known to serve as a link between a target protein and an ubiquitin-conjugating enzyme. In particular, it has been shown that the Early Mitotic Inhibitor (Emi)-1, which contains an F-box domain, is able to regulate mitosis progress by inhibiting premature anaphase promoting complex/cyclosome (APC) activation [19], the APC complex being composed of an ubiquitin ligase coupled with the SCF (Skp1-Cullin-F-box protein) complex [20]. So it is possible that the oocyte-specific F-Box proteins would participate in the stabilization of the meiotic process at the prophase I stage.
Concerning the cluster on chromosome 14, in situ hybridization showed a high level of expression of SpeerA gene in the mouse oocyte, corroborating previous results from Matzuk's group [6]. By analyzing the RT-PCR results, we found that this gene was also expressed in the testis, likely in male germ cells. The Speer family has recently been described as a new family of 14 genes. Interestingly, SPEER-1, SPEER-2, and SPEER-4D were recently shown to be expressed specifically in the mouse testis [21]. These genes have open reading frames of approximately 200-220 amino acids, and encode for proteins that exhibit a high proportion of alpha-helical secondary structure, comprising approximately 15% glutamate residues. According to their possible interaction with cytoskeletal proteins such as actin or dynein, it has been hypothesized that the SPEER may be nuclear matrix proteins involved in the reorganization of the spermatocyte/spermatid nucleus [21]. SpeerA, discovered in our study (XM_138939), shares about 34% amino acid sequence similarity with SPEER-4A gene, which could suggest that it plays a similar role in the oocyte. Of note, in contrast to most of the oocyte clusters, the four testis-specific genes neighboring the SpeerA gene were not structurally related to each other.
One of the clusters on chromosome 19 contains the well-known oocyte-specific gene Oosp-1 [12], and two structurally similar genes, these three genes clearly sharing a ZP-like domain. This suggests that these genes encode for structural proteins involved in the formation of certain components of the zona pellucida. A structural analysis of the sequences of these proteins is underway in our laboratory to further investigate the structural specificity of these proteins. The biological roles of the two genes present in the other cluster on chromosome 19, as well as the role of the testis-specific genes present on the chromosome 14 cluster are unknown.
Overall, for all of these genes, further experiments as well as structural analysis are now necessary to investigate their biological roles in oocyte and follicle maturation. Investigating such a role will require functional studies such as identification of partners and/or targeted invalidation.
As we previously discussed, the targeted invalidation of MATER gene leads to female infertility due to a blockage of early embryonic development [22]. This suggests that the oocyte-specific MATER-like genes, i.e. the Nalp9A-F genes that we have identified [8] are not able to compensate the absence of the MATER/Nalp5 product in Mater-/- mice. Similarly, invalidation of Tcl1 gene in the mouse was not clearly compensated by the five oocyte-specific Tcl1B genes. Overall, this suggests that despite an apparent redundancy in the expression of these structurally similar genes, every member of the oocyte Nalp and Tcl1 family of paralogous genes seem to have very specific biological roles in female reproduction. Invalidation of each of these genes is now necessary to study their specific biological role. Moreover, as expected, most oocyte-specific genes organized in clusters are paralogous genes, originating presumably from a common ancestral gene by successive duplication events. It is the case for the oogenesin, Nalp9, Fbxo12, Tcl1 as well as the Osp1-3 genes. It is clearly not the case for the clusters localized on 14A3 and 19D3 (Fig. 1). Nevertheless, such an organization of "oocyte- or germ-cell clusters" suggests the presence of specific genomic regulatory regions in the vicinity of these genes.
As we depicted in figure 1, the SpeerA gene is localized on a cluster on chromosome 14, in the vicinity of four unknown testis-specific genes. On chromosome 19, the oocyte-specific gene related to TWIK-Related spinal cord K+ channel is localized near the gene corresponding to AK015359 GenBank number, which is predicted in silico to be testis-specific as well. Interestingly, we have previously shown that in the oogenesin cluster on chromosome 4, the oogenesin-4 gene is also slightly expressed in the testis. Of note, Pramel1 gene, which is localized near oogenesin4, has also been described as a male germ-cell specific genes [7,23]. Similarly, in the oocyte-specific Nalp cluster on chromosome 7, we have shown that Nalp9D is also clearly expressed in the testis, presumably in germ cells. So the notion of oocyte-specific cluster would not be systematically restricted to female germ cells, and would be extended in some cases to "germ-cells cluster" as suggested in this work.
One striking observation of our work is the difference in chromosome localization between oocyte-specific genes organized in clusters and oocyte-genes that are not. If genes were located at random on the chromosome and that the distance to the closest end is measured, then the expected range would be 0% to 50%, and one would expect to reach an average distance of 25% if enough genes are taken into consideration. In our study, isolated genes were very close to this value (24%) whereas clusters were located in average at half of this distance (12%). Gene clusters presumably originate from local gene duplications [24], which are now recognized as efficient motors of diversification in metazoans. Of note, the location of gene families near telomeres has a deep sense in terms of rapid evolution. Indeed, subtelomeric sequences are recognized as the most rapidly evolving regions in the genome, maybe due to the high level of recombination present in these regions [25]. On the other hand, clusters of genes are the entry gates for accumulation of mutations, without the risk of noxious consequences for the organism, and therefore enable the development of new functions, as clearly illustrated by the variants of the human hemoglobin beta chain, which adapts the foetus to successive oxygen environments during development [26,27]. For mammals with a high reproductive potential, such as mice and other rodents, the possibility of rapid evolution for genes involved in ovarian function may be a great advantage. Therefore, one can hypothesize that the specific near telomere position of oocyte-specific gene clusters corresponds to an evolutionary advantage.
Interestingly, the genes that we have described here are strictly silenced in non-ovarian tissues, and one may hypothesize that the specific near telomere position of oocyte clusters would contribute towards such a silencing. In particular, it has been shown that telomeric and pericentric regions of chromosomes are mainly composed of heterochromatin in most eukaryotic genomes. Moreover, euchromatic genes relocated by chromosomal rearrangement or by transposition in the neighbourhood of heterochromatin can be silenced in drosophila [28]. In yeast, a similar phenomenon of reversible gene silencing near telomere has been called Telomere Position Effect (TPE) [29,30]. The presence of TPE has also been observed in human cells [31]. Actually, a very specific nuclear topology would explain such a phenomenon. Indeed in yeast, the 32 telomeres cluster at the nuclear periphery in 8 to 10 groups, organized in compartments rich in histone-binding silencing factors [32]. Testing if the telomeres would be organized in very specific compartments in oocyte and in other tissues requires further investigations.
Conclusion
We have studied five clusters of genes specifically expressed in female, some of them being also expressed in male germ-cells. Moreover, contrarily to non-clustered oocyte-specific genes, those that are organized in clusters tend to map near chromosome ends, suggesting that this specific near-telomere position of oocyte-clusters could result in an evolutionary advantage. Understanding the biological roles as well as the mechanisms underlying such a specific expression now requires further investigation.
Methods
In silico identification of oocyte-specific genes
As previously described [7,8] three cDNA libraries derived from mouse unfertilized eggs (dbEST library ID.10029), 2-cell egg (dbEST library ID.5391) and in vitro fertilized eggs (dbEST library ID.2589) were submitted to digital differential display analysis [33] to identify oocyte-specific ESTs that are not found in several non normalized cDNA libraries from different adult somatic non-tumoral tissues (brain, kidney, stomach, liver, lung, spleen, muscle, heart, skin, bone marrow, adipose tissue, adrenal gland). The physical localization of identified genes on the mouse chromosomes was retrieved from [9]. The presence of specific domains within the corresponding proteins was determined on [34] as well by a Blast search of homologous proteins [35]
RT-PCR analyses
Total RNA was extracted from whole adult tissues (ovary, liver, kidney, testis, heart, brain, muscle, adrenal gland and spleen) using RNAble reagent according to the manufacturer's procedure (Eurobio, Les Ulis, France). Reverse transcription was performed for 1 hour at 42°C in a total volume of 20 μl with 2 μg total RNA per sample following standard procedure. Five μl of the cDNA product was amplified by PCR using primers described in Table 3. RT-PCR products were also analyzed by Southern blot. Briefly, the RT-PCR products were fractionated on 1% agarose gel, transferred to Hybond-N+ membrane (Amersham-Pharmacia) and hybridized with the corresponding cDNA fragment labeled by random priming (1 × 106 cpm/ml) as described previously [36].
Table 3 Primers used for RT-PCR amplification of cDNA fragments of FBXO12B, FBXO12D, Tcl1b3, Tcl1b4, Tcl1, SpeerA, AK076711, Oosp3 and actin.
Gene Primers annealing temperature fragment size (pb)
FBXO12B Upper ATTTGCCTCGTTTGCCTCTGA 49°C 890 pb
Lower GGTTATCCTGTCTTCCCTTCT
FBXO12D Upper AGCCCTGTCCTTTTCCTGTCA 50°C 491 pb
Lower AATAGTCTGGTTTCCCCTCAC
Tcl1b3 Upper TGGTAGACAGTGGGTAGTTGC 54°C 585 pb
Lower CAGGTGGAGGAGTTTGAATGG
Tcl1b4 Upper TAAGAAGGCAGCCAACCAGAC 54°C 838 pb
Lower CTACCCAGCACCAGGCGAACT
Tcl1 Upper TTTTATCACGGACTGGCATTG 54°C 1208 pb
Lower CCCTCATTTATTGGCATCTCG
SpeerA Upper TGAAGCAGGAAAATAGGAGA 51°C 900 pb
Lower CCAAACAGAAAACCAAACAC
AK076711 Upper GGCTCCCAGAGGCTGAATCA 61°C 769 pb
Lower TAAGGGTGTCCAAAGAATCA
Oosp3 Upper TATTTGGGATGTGGAGATGTT 52°C 416 pb
Lower TTGAGGAGGAGGGTGGAAGTC
Actin Upper ACGGAACCACAGTTTATCATC 60°C 180 pb
Lower GTCCCAGTCTTCAACTATACC
PCR products were purified from the agarose using the gel extraction kit QIAEX II (Qiagen, Hilden, Germany) and inserted into pGEM-T vector (Promega, Madison, WI, USA). Identity of the selected clones was checked by sequencing.
In situ hybridization
Frozen ovaries from 3 female mice in di-estrus, 3 female mice in proestrus and 3 female mice in estrus were serially sectioned (10 μm) with a cryostat to perform in situ hybridization experiments using 35S-labeled cRNAs probes as previously described [37]. The specificity of the hybridization signals was assessed by comparing the hybridization of the cRNA antisense with the corresponding sense probes. Histological determination of follicular size and degree of atresia was performed on adjacent sections stained with Feulgen [37].
Metagenome analysis
For the metagenome analysis, we have taken account for all the oocyte-specific genes identified so far by the in silico methodology (data not shown), including well known genes (Mater, Zar1), as well as the 12 genes of the oogenesin family [7], and the 6 genes similar to NALP5/MATER [8] that we have previously identified. A total of 92 genes were analyzed, 44 of which are isolated on the chromosomes while 48 were organized in 7 clusters (Table 2). To study the relative distribution of these two subgroups of genes, their relative distance to the closest chromosome end was calculated, and a global Student t-test was calculated to compare the two subgroups. To avoid any bias due to the fact that genes in clusters often comes from duplication of ancestral genes, bias which would artificially increase their coefficient in such a test, we have considered only one position for each cluster. Moreover, although genes are not strictly homogenously distributed along the chromosomes, the clusterized and the isolated oocytes genes considered for the analysis do cover all the mouse chromosomes, thereby limiting the risk of a bias.
Authors' contributions
AP and SD performed most of the in silico investigation, as well as the molecular biology and the in situ hybridization experiments. IC contributed to the structural analysis of the Oosp-cluster genes. MB participated to the in situ hybridization experiments and DV performed most of the metagenome analysis of the oocyte-genes organized in clusters. PM was assisted by RD-T to coordinate this work.
Acknowledgements
We wish thank Claude Cahier and his team as well as Eric JeanPierre for expert animal care. We wish also to acknowledge Joëlle Dupont for advices and Thierry Delpuech for bacterial technical assistance. We thank Pascal Mermillod, Svetlana Uzbekova, and Sophie Pennetier for helpful discussion, as well as Alice Pierre for her appreciated help.
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| 15907208 | PMC1166550 | CC BY | 2021-01-04 16:39:53 | no | BMC Genomics. 2005 May 20; 6:76 | utf-8 | BMC Genomics | 2,005 | 10.1186/1471-2164-6-76 | oa_comm |
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-771590720010.1186/1471-2164-6-77Methodology ArticleA generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data van Hijum Sacha AFT [email protected] Jong Anne [email protected] Richard JS [email protected] Harma A [email protected] Naomi E [email protected] Rasmus [email protected] Hengst Chris D [email protected] Casper J [email protected] Jan [email protected] Oscar P [email protected] Department of Molecular Genetics, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, the Netherlands2 Groningen Bioinformatics Centre, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, the Netherlands2005 20 5 2005 6 77 77 1 11 2004 20 5 2005 Copyright © 2005 van Hijum et al; licensee BioMed Central Ltd.2005van Hijum et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
In research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed.
Results
A number of validation experiments were performed on Lactococcus lactis IL1403 amplicon-based DNA-microarrays. Using the validation scheme and ANOVA, the factors contributing to the variance in normalized DNA-microarray data were estimated. Day-to-day as well as experimenter-dependent variances were shown to contribute strongly to the variance, while dye and culturing had a relatively modest contribution to the variance.
Conclusion
Even in cases where 90 % of the data were kept for analysis and the experiments were performed under challenging conditions (e.g. on different days), the CV was at an acceptable 25 %. Clustering experiments showed that trends can be reliably detected also from genes with very low expression levels. The validation scheme thus allows determining conditions that could be improved to yield even higher DNA-microarray data quality.
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Background
The development of DNA-microarray technology has enabled genome-wide expression profiling to become a valuable tool in the investigation of an organisms' gene regulation [1-3]. For our studies on gene regulation in Gram-positive bacteria [4] we use in-house developed DNA-microarrays containing amplified DNA fragments of the annotated genes of Lactococcus lactis ssp. lactis IL1403 [5], L. lactis ssp. cremoris MG1363 [6], Bacillus subtilis 168 [7], Bacillus cereus ATCC 14579 [8], and Streptococcus pneumoniae TIGR4 [9].
Standardization of every step in the DNA-microarray procedure is crucial to correctly and efficiently perform DNA-microarray experiments, and to obtain reproducible data [10-13]. In the process from manufacturing DNA-microarrays to performing the actual experiments, systematic errors and / or bias in the data are introduced in each of the different steps. The effects of various factors (e.g. dye and slide) on the quality of DNA-microarray data have been studied quite extensively albeit for experiments performed with eukaryotic systems [14-20]. In contrast, no data quality determination has yet been performed on DNA-microarray data from experiments with bacterial cultures. Furthermore, the effects of different array batches or the influence of the experimenter on data quality have not been included in the previous mentioned experimental designs. Here, we show that the latter factors are indeed important for optimizing DNA-microarray data quality.
In order to assess the reproducibility of, and factors involved in, DNA-microarray data produced in our laboratory during transcriptome analyses by a number of researchers, a validation experiment was designed and implemented. This validation scheme is routinely applied to validate the DNA-microarrays of the various organisms under study in our group. In addition, it allowed to set a quality standard as well as to assess sources of errors in the expression data.
We discuss a novel validation scheme and assess data quality of a number of validation experiments performed on amplicon-based DNA-microarrays of L. lactis IL1403. For any laboratory in which DNA-microarray experiments are performed on a regular basis, the validation scheme will provide at the cost of only a few hybridizations, valuable information on the DNA-microarray data quality. Combining multiple validation experiments allows estimation of the main sources of errors.
Results
DNA-microarray quality assessment
Six researchers working with L. lactis IL1403 slides performed nine validation experiments (see Methods and Figure 1). General statistics on these validation datasets are listed in Table 1. One has to bear in mind that DNA-microarrays with lower signals will yield more noisy data, and thus higher coefficients of variance (CVs). Since these lower signals might also contain valuable information, they are included in the analyses described here.
Figure 1 The validation procedure. It consists of 4 steps: (i) cell culturing, (ii) cell pelleting and RNA isolation, (iii) cDNA labeling, and (iv) hybridization, scanning, image- and data analysis.
Table 1 General statistics on data obtained from the validation experiments (Figure 1 and supplementary Table S1 [21]).
Validation Validation slide 5 % low spot filter 40 % low spot filter
1 2 3 CV (%) Spots (%) CV (%)
A-I x x x 27.3 89.5 17.4
A-I x 21.6 88.7 13.4
A-I x 26.1 88.9 17.3
A-I x 24.6 91.1 16.5
A x x x 16.4 86 12.5
B x x x 13 84.3 9.4
C x x x 14 94.5 8.6
D x x x 16.7 92.1 11.1
E x x x 9.2 90.4 6.7
F x x x 27.5 87.3 20.2
G x x x 16.5 88.5 10.3
H x x x 23.8 91 15.1
I x x x 18.1 92.2 12.4
No differentially expressed genes were detected
Differential expression tests were performed for the factors (supplementary Table S1 [21]; e.g. spot-pins, experimenters, and validation experiments), but no genes meeting the criteria were observed. No differential expression was expected because the hybridizations were performed with cDNA derived from cells grown under (very) similar conditions. The resulting expression ratios were thus close to 1.
CV comparison
The CVs of the validation experiments range from 9 % to 28 % with an average of 17 % and using about 90 % of the spots. The lower CVs of the 40 % low-intensity-spot-filtered data (Table 1) indicate that a significant part of the variance originates from genes with low expression. Slides 2 and 3 of each validation experiment (S2 and S3, respectively) examine biological replicates of independent comparisons between the cultures A and B (Figure 1). Their data quality is thus a "worst case scenario" estimate of the quality to be expected from "real" DNA-microarray experiments as the validation experiments were performed with a large number of differing parameters: (i) different researchers performed the experiments, (ii) on different days, while, lastly, (iii) the cells were harvested in a growth phase in which small changes in culture optical density will result in relatively large differences in expression levels (see below). Table 1 shows, as expected, that data from the pooled slides 1 of all validation experiments (S1) have a smaller average CV (22 %) than those of S2 (26 %) and S3 (25 %). The CV frequency distribution for S1 is shifted towards zero while S2 and S3 have quite similar distributions (supplementary Figure S1 [21]) because of intra-culture differences (Ba or Bb; Figure 1).
Detailed comparison of two slides
The two representative validation experiments, i.e. E and H, showed clear differences in data quality (supplementary Table S1 [21]). Box plots of data before the Lowess grid-based normalization show clear spot pin-dependent patterns in average signal levels (supplementary Figure S2 [21]). A non-linear intensity-dependent dye-effect in data from slide E3 (supplementary Figure S2 [21], Graph E2, (i) is evident from the curved Lowess fits. The Lowess curves (one curve fitted for each spotted grid; supplementary Figure S2 [21]) (ii) of slides E3 and H2 are "stacked", indicative of a grid-dependent gradient of ratios. The above-mentioned effects are normalized by using the Lowess grid-based normalization method (supplementary Figure S2 [21], Graph V).
Gene-dependent fluctuations in ratios and signals
Clustering was performed on the SDs of the ratio-data to investigate gene-dependent behavior across the validation experiments (Figure 2). Cluster 1 contains more strongly expressed genes than cluster 4, with clusters 2 and 3 encompassing genes with intermediate expression levels.
Figure 2 Sammon projection of the clustering of validation data using a self-organizing Kohonen map. Validation experiments (A-I) are shown as well as the clusters (1 – 4; consisting of 761, 230, 227, 886 genes, respectively). Operon names, the number of members, and their (putative) functions are listed to the right of the corresponding clusters. The minimum number of genes in an operon of which all members should be in a certain cluster was determined at a probability of 0.02 or lower for clusters 1 (4 genes), 2 (2 genes), 3 (2 genes), and 4 (5 genes).
The clustering results were simplified by grouping genes
A first selection of genes was based on the L. lactis IL1403 genome annotation with the underlying assumption that related genes (either by function or because they are part of the same operon) are expected to show similar expression behavior. Only related genes with all members occurring in the same cluster (probability lower than 0.02) were considered.
Cell growth-related genes show large fluctuations
Clustering revealed that genes with similar SD fluctuations were involved in (i) amino acid biosynthesis, (ii) energy metabolism, (iii) cell-wall synthesis, and (iv) salvage of nucleosides and nucleotides (Figure 2). Genes showing highest ratio and signal CVs (supplementary Table S2 [21]): (i) are of unknown function, (ii) are (pro) phage-derived, (iii) encode proteins involved in transport of various compounds, or (iv) encode transcriptional regulators.
Some genes with low expression show correlated expression fluctuations
Figure 3 clearly illustrates that (i) the genes with low expression have significantly higher CVs than the highly expressed genes, which is most probably due to their lower signals, and (ii) the related genes (clustered in Figure 3) showing similar expression behavior have average expression levels varying from very low (1.7 % of the maximum intensity) to relatively high (65 % of the maximum intensity). After a close inspection of these (mostly low-intensity) spots, the fluctuations in ratio and / or expression levels did not appear to be correlated to spot quality (data not shown).
Figure 3 Plot of percentage of maximal intensity versus CV values calculated for the expression levels of genes in the 9 validation datasets (dark-blue small squares). Purple solid triangles show the top 40 genes with highest variability in ratio and signals (supplementary Table S2 [21]). Functionally related genes showing validation experiment-dependent SDs (Figure 2) are indicated by cluster 1 (solid yellow circles), cluster 2 (open light-blue triangles), cluster 3 (open red squares), and cluster 4 (open green circles).
ANOVA
A clear correlation between CVs (data quality) and e.g. array batches or experiments could not be determined. For instance, validation experiments H and I were performed on the same DNA microarray batch by the same experimenter, but yielded different CVs. The ANOVA technique allowed estimating the contribution of several sources of errors to the total variance in the DNA-microarray data of all slides (Figure 4; S = 1v2v3). The following factors contributed significantly to the total variance: G (gene; 5 %; Table 2), VG (validation experiment and gene interaction; 27 %), SG (slide and gene interaction indicative for dye-effects; 4 %; Table 2), and VSG (validation experiment, slides, and gene interactions; 31 %).
Figure 4 ANOVA results. Each bar represents averages (with error bars signifying the standard deviations for the respective interactions) for 10 random samples of ratio data obtained for the indicated slide combinations (1, 2, and 3; Figure 1). E.g. S = 1v2 indicates a comparison of data from slides 1 with data from slides 2. The interactions (indicated by the colored bars as detailed in the inset) and "Error" (residual variance) amount to 100 % (the total variance present in the data).
Table 2 Contribution of sources to the variance estimated for the nine validation experiments (Figure 4) and contribution of individual factors to the VG interactiona.
Variance source Contribution to the variance (%)
Gene (G) 5.0
Dye (SG) 4.2
Gene × Arrayb 7.8
RNA isolation and labelingc 1.5
Sampling 7.1
VGd 26.9
Day × Gene 19.7e
Experimenter × Gene 17.3e
Array batch × Gene 14.9e
Spot pins × Gene 4.5f
a The degrees of freedom results in the separate ANOVAs are listed in the supplementary web-site [21].
b Assumed to consist of hybridization effects and signal-to-noise differences per slide.
c Derived from the variance observed between Ba and Bb cultures (Figure 1).
d Variances that are dependent on the validation experiment performed and due to day-to-day differences, identity of the experimenter, and DNA microarray batch differences.
e Due to overlap in levels, the contribution of these interactions were individually determined.
f A change from 8 to 12 spot-pins used for array spotting coincided with a switch in the RNA isolation method.
The VSG interaction detailed
In order to distinguish the separate sources of errors in the VSG interaction, additional variance analyses were performed with combinations of 2 slides: (i) by omitting slide 1 (S1; containing a self-hybridization) the VSG interaction (S = 2v3) decreased with 7.8 %; (ii) by omitting slides 2 or 3 (S2 or S3; containing inter-culturing hybridizations) the VSG interaction (S = 1v2 or S = 1v3) decreased with 9.4 % and 9.1 %, respectively; and (iii) the decrease in the VSG interactions coincides with an increase of the VG interaction. This leads to the conclusion that variances occur on each slide (Gene × Array; Table 2) and may, in part, be due to hybridization effects. Since the variance for a particular slide (7.8 %) is omitted from the variance analyses, the VSG interaction will decrease, but the VG interaction will increase (the 7.8 % variance was specific for the slide that was omitted from the analyses). This 7.8 % variance is assumed to be the same for each of the three slides. The larger effect of S2 and S3 compared to S1 in the VSG interaction is probably caused by the fact that on these slides inter-culture comparisons were performed. Since dye-effects are assumed to be global, it can be concluded that the intra-culturing differences (differences between the Ba and Bb cultures) account for the 1.6 and 1.3 % larger decrease in the VSG interaction (by omitting S2 or S3, respectively). The variance introduced by the Ba and Bb cultures is quite reproducible (1.3 – 1.6 %) and is caused by RNA isolation and labeling (Table 2).
Slide and sampling differences can be determined from VSG
The variance of S1 versus the pooled S2 and S3 (S = 1v23) in the VSG interaction decreased with 16.1 % to 14.9 %, with the variance in the VG interaction remaining virtually unchanged. By combining S2 and S3, the Gene × Array interactions occurring specifically on S2 and S3 are pooled. They are, thus, not accommodated in the VG interaction, but rather in the residual error. The remaining 14.9 % variance in the VSG interaction still contains the Gene × Array interactions for S1 (7.8 %) and sampling differences (7.1 %; Table 2).
Day-to-day differences are most prominent in the VG interaction
The VG interaction contains differences between validation experiments (Figure 4): the DNA microarray batch used (BG), day-to-day differences (AG), the researcher performing the experiment (PG), and spot-pin / RNA isolation method used (DU). Due to confounding of these factors, a less efficient estimation of their relative contributions was unavoidable. However, the contributions of BG, PG, AG, DU in relation to the VG interaction could be determined (Table 2). The day-to-day differences were estimated to have the largest contribution to the variance, followed by experimenter, the DNA microarray batch, and lastly a relatively low contribution of switching the RNA isolation method (coinciding with a change from 8 to 12 spot-pins).
Discussion
The validation procedure presented here was implemented to provide a standardized method to assess DNA-microarray data quality generated in our laboratory and should be well-suited for use in other laboratories. A workable trade-off between costs, time investment, and data-quality was obtained by using only three DNA-microarray slides for each validation experiment. This scheme is suitable for identifying factors that yield "unreliable" data (i.e. data with ratios that deviate from 1 due to, for instance, outliers). In a number of cases, the validation experiment even identified experimenters who did not flag bad spots stringently enough.
Assessment of high-throughput gene expression data quality is a challenging task. A potential problem arises from the fact that many studies do not describe in detail the resulting amount of data on which statistic analyses was based. This information is, however, crucial to determine data-quality. To demonstrate the effect of filtering on data quality, statistics were also calculated for data in which 40 % of the lowest intensity spots were removed (Table 1). These rigorously filtered data do show improved data quality, but at the expense of many measurements that could contain valuable information. The 5 % low-intensity spot filter employed in our study was selected after careful examination of data from various DNA-microarray experiments performed in our laboratory. Some targets with low expression levels allowed grouping genes by function, revealing trends that would have been difficult to discern with more rigorous filtering. A thorough discussion of these results is, however, outside the scope of this study.
The data quality of the validation experiments described in this paper proved to be satisfactory, while at same time a maximum amount of data was preserved. One has to bear in mind that a significant part of the variance in our data is caused by varying factors (e.g. differences in the days on which the experiments were performed; discussed in more detail below). In addition, the quality of the glass surfaces used in this study was lower than that of presently used superamine glass slides (Telechem International Inc.). Together with recently implemented increased stringency of clean-room rules, this will increase data-quality even more. The average CV value for the validation experiments was 26.1 % and 24.6 % for S2 and S3 with use of 90 % of the spots (Table 1). These results are comparable to CVs, ranging from 11 to 23 %, reported for a number of studies using cDNA derived from eukaryotic cell cultures hybridized on various DNA microarray platforms [20,22,23]. For other DNA-microarray experiments performed in our laboratory the data quality is considerably higher (average CVs of under 20 %) stipulating that in effect, the average CV of about 25 % described in this study is an underestimation of the data quality one could obtain.
By mining the data from several validation datasets it was possible to determine which factors contribute to the variance in normalized DNA-microarray data. The following factors were identified (Figure 4 and Table 2): (i) validation experiments (VG; 27 %), (ii) sampling (7 %), (iii) Array × Gene (8 %), gene variances (5 %), and dye-effects (4 %). The contributions of RNA isolation and labeling to the variance were quite low (1.5 %; Table 2). Additional variance analyses showed that the day-to-day differences contribute most to the 27 % variance observed for the VG interaction, followed by the experimenter, the DNA microarray batch, and lastly a change in the RNA isolation method (coinciding with the use of arrays spotted with 12 instead of 8 spot-pins). The contribution of dye-effects was determined to be only 4 %, which is low compared to the contribution of dye-effects determined for in studies from Chen et al. and Dombrowski et al. [18,24]. The latter study describes the use of a direct labeling kit. In contrast, indirect labeling was used in our study, in which differential hybridization of Cy3 and Cy5-labeled cDNA is anticipated. Direct-labeling adds, next to this differential hybridization, (i) preference of the reverse transcriptase enzyme for the Cy3 label and (ii) prolonged exposure to air and light of the dyes increasing the chance of oxidation and / or bleaching. The main contributing factors identified in this study are in agreement with a number of studies involving cDNA derived from eukaryotic tissue cultures [18,19,25]. In contrast to these studies, we were able to attribute a relatively large contribution of the total variance to specific sources of errors (67 %) because of the efficient design of the validation experiment described here. Since the contributions of day-to-day variation, DNA microarray batch differences, and the experimenter to the variance amounted up to 27 %, it can be concluded that even higher data-quality can be obtained when experiments are performed under identical conditions.
The ANOVA model used does not account for gene-to-gene variances. Additional variance analyses were performed with datasets of which the 10 % most noisy genes (with highest CVs) were omitted. In these experiments, the relative contribution of the various factors identified above remained unchanged (results not shown), indicating that the proposed procedure is robust and that its results are not dependent on a relatively small portion of noisy genes.
In this paper, data from hybridizations with RNA derived from the same experimental conditions were used. To examine whether the probes used on the slides are correct and whether observed gene expression levels are accurate, experiments should be carried out which measure known differentially expressed genes. A number of such studies in which targets were identified by DNA-microarray experiments (e.g. on arginine and glucose metabolism and on nisin resistance development), and subsequently verified by alternative techniques (real-time PCR, gene knock-out and / or overexpression studies), have successfully been performed in our laboratory (results not shown).
The validation experiments described in this study were designed to be a "worst case scenario." Data quality proved to be good even though they were obtained at challenging conditions: (i) flask-grown cells, (ii) harvesting in a growth phase in which relatively large changes in gene-expressions occur, and (iii) change of factors (e.g. day). These factors represent the conditions under which DNA microarray experiments are performed in our laboratory. Another laboratory could have different factors and levels: e.g. only one researcher that performs the experiments or a different organism under study. Such a laboratory should perform the validation experiments to determine the contribution of the factors that play a role in their particular case. The results of clustering indicate that functionally related genes share specific behaviour across the validation experiments (Figure 3). The significant expression levels and relatively large fluctuations in ratios of the ybg, ybj, and yia gene groups are probably due to biological variations (growth-phase and medium-batch related). Furthermore, one can conclude that data from even genes with very low expression can reveal interesting trends. By preserving the maximum amount of data, one might be able to discern more subtle differences in expression levels of genes with low expression.
Conclusion
In this paper a novel validation scheme was employed to assess data quality and sources of errors of DNA-microarrays. Even in the case that 90 % of the data were preserved and the experiments were performed at challenging conditions, the coefficient of variance was at an acceptable 25 %. Clustering experiments showed that trends could be detected from genes with very low expression. Using ANOVA, day-to-day as well as experimenter-dependent variances were found to contribute strongly to the variance, while dye and culturing contributions to the variance were relatively modest. The validation scheme thus allows determining conditions that could be used to obtain DNA-microarray data of improved quality.
Methods
DNA-microarray experimental procedures
DNA-microarrays were prepared from amplicons of 2108 genes in the genome of Lactococcus lactis ssp. lactis IL1403 (Genbank accession number NC_002662; its annotation is based on the B. subtilis genome, Genbank accession number NC_000964). Primers were designed to amplify unique regions of these genes [26]. Generation of the amplicons, slide spotting, slide treatment after spotting, and slide quality control were performed as described [4] with modifications (see protocols at supplementary web-site [21]). Samples for RNA isolation were taken by rapid sampling of exponentially growing cultures of L. lactis. Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA (target) synthesis, indirect labeling, hybridization, and scanning are described in the supplementary web-site [21].
Validation experiment
The validation experiment (Figure 1) was designed as follows: two independent cultures of L. lactis ssp. lactis IL1403 were grown at 30°C to an optical density at 600 nm (OD600) of 2.0 / cm (corresponding to end-log phase) in standing flasks with 50 mL M17 medium [27] containing 0.5 % glucose (w/v). A 10 mL sample was taken from one of these cultures, while from the other culture two samples of 10 mL were withdrawn. For the validation experiments (supplementary Table S1 [21]), total RNA was extracted using the RNA isolation methods with and without macaloid, for slides made with 12 spot pins and 8 spot pins, respectively. The cDNAs were labeled according to the scheme in Figure 1. The mRNA derived from the A culture was labeled once with Cy3 and three times with the Cy5 dye. The mRNA derived from the Ba and Bb cultures were both labeled with the Cy3 dye. Finally, the labeled cDNAs were hybridized on L. lactis IL1403 DNA-microarrays (Figure 1).
Data processing
Slide data were processed by using MicroPreP [28,29]. (i) spots that were bad (for instance due particles on the slide surface) were manually flagged (for an example see supplementary Figure S3 [21]). These flagged spots were deleted from the datasets because they yield unreliable measurements; (ii) since the spotting buffer contains small random DNA fragments, spots will always have a base signal, particularly in the Cy3 channel, due to autofluorensence of these fragments. The spot backgrounds in each grid for both channels were corrected for this autofluorescense by subtracting the intensity of the weakest spot; (iii) the 5 % or 40 % weakest spots (sum of Cy3 and Cy5 net signals) were deleted. The effect of filtering low-intensity spots from the datasets is demonstrated in supplementary Figure S4 [21]. The 5 % cutoff was determined empirically: the most noisy data is removed from the datasets without removing reliable data; (iv) normalization was performed (the ratios were made comparable across slides) using a grid-based Lowess transformation [30] with f = 0.5 (fraction of genes to use); (v) for both channels the intensities of the "Lowess" fraction of genes were added to yield a total signal, and all intensities were divided by this total signal, yielding scaled, arbitrary expression levels. One has to bear in mind that the scaling procedure affects the signals, but not the ratios. Since the statistical procedures in this paper are based on the ratios, scaling does not affect these analyses; (vi) tables for variance analyses were made. These tables list for each measurement the factors and their levels (see also supplementary Table S1 [21]). For example: spot 1 of slide 1 of validation experiment 1 is gene X (from the gene factor), was obtained by experimenter Y (from the factor experimenter), on day Z (from the factor day).
The scanned images, data, and experimental conditions were stored in the MIAME-compliant Molecular Genetics Information System (MolGenIS) [31].
Statistical procedures and clustering
The quality of the validation datasets discussed in this paper are presented by coefficient of variance (CV). CVs are calculated by dividing the standard deviation (SD) by the mean ratio of a gene and multiplying by 100 %. The minimum and maximum numbers of measurements for each gene were 13 and 54 (i.e. 9 validation experiments × 3 slides per validation experiment × 2 technical replicates per slide), respectively. For single validation experiments, CVs and differential expression levels were determined for genes for which at least 4 measurements were available.
Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test [32]. These tests yield for each gene the probability that it has a significantly different ratio than 1. Due to that multiple tests for differential expressions were performed, the false discovery rate (FDR) was determined. The FDR represents the probability that a significant differentially expressed gene is in fact false-positive. FDRs were calculated by (i) ranking the genes by p-value, (ii) multiplying the p-values with the number of tests performed (similar to Bonferroni correction), and (iii) dividing by the number of genes with lower p-values. Genes were considered differentially expressed at both p < 0.01 and FDR < 0.01.
The SDs of log (base 2)-transformed ratios were used for clustering purposes. The clustering technique groups genes which SDs are similar across the validation experiments. The values of SDs for genes with less than four measurements were interpolated by using the K-nearest two neighbours approach using Engene [33]: only four genes which lacked the first or last SD had to be omitted. For each gene, SDs were centered after which clustering was performed using the Kohonen self-organizing map (SOM) algorithm (2 × 2 matrix) in the Engene clustering package.
ANOVA
The statistical software package SPSS (version 11; SPSS Inc., Chicago, IL) was used to perform variance analyses (ANOVA). ANOVA determines the contributions of factors (e.g. day) and their levels (e.g. an experiment performed on Monday) to the total variance observed in the datasets. Supplementary Table S1 [21] presents factors and their levels used for ANOVA.
ANOVA is robust with respect to violations
The assumptions of ANOVA that (i) error variances are equal and (ii) the residuals of the model are normally distributed generally do not hold for DNA microarray data. However, the sole purpose of ANOVA for this paper was to estimate the relative contributions of the various factors, a purpose for which ANOVA is extremely robust. If the error variances are not equal, the estimators for the type III sums of squares of the various factors, although less efficient, are still valid and unbiased [34]. Furthermore, the efficiency reduces most when the ANOVA design is very unbalanced and/or random factors are implemented [35]. In our case, the design is quite balanced and a fixed-factors model is used. The relative sums of squares are used instead of p-values, because the latter might be violated by deviations from the assumptions.
A whole-slide model was chosen over a gene-by-gene model
When performing variance analyses on DNA-microarray data, one can either use a whole-slide model or a more complicated model that allows for gene-to-gene differences. Gene-by-gene models can deal better with variances that are gene-dependent (due to differences in gene expression levels). However, as each of the three hybridized slides (Figure 1) contains different combinations of cDNAs derived from the A and B cultures, the gene expression levels are expected to differ from slide-to-slide, rendering the gene-by-gene method less effective than our whole-slide model.
Genes were randomly selected for ANOVA
The software could not handle a gene factor of 2108 levels (genes) and additional interactions in model (1). To reduce data dimensions, we chose to randomly select genes instead of other methods (e.g. grouping of genes based on clustering or function) because the latter depend on assumptions of which the validity for the datasets are difficult to determine. The selection was repeated 10 times (with 5 % or 105 random genes each time) yielding 1050 genes of which 196 were drawn two or more times. These 854 uniquely selected genes (40.5 % of the total genes) corresponded well to the predicted 40.0 % (calculated by [1 - (((2108-105) / 2108)10)]). The sums of squares were averaged for the sources (i.e. factors) contributing significantly to the variance (α = 0.05).
The ANOVA model uses log-transformed ratio data
Attempts to identify the sources of errors and their contributions to the variance based on signal data, proved to be unsuccessful due to large differences in gene expression levels. A similar observation has been made for oligonucleotide-based DNA-microarrays hybridized with liver tissue RNA [17]. For this reason, we used the following ANOVA model:
rigpbtv = μ + Si + Gg + Aa + Pp + Bb + Tt + Vv + Uu + (VG)vg + (SG)ig + (VSG)vig + εigpbtv (1)
where rigpbtv is the log (base 2)-transformed ratio of gene g, which is the tth replicate spot on slide i performed by experimenter p on array batch b which was spotted with u spot pins (either 8 or 12) in validation experiment v. rigpbtv is determined by μ (the mean ratio across all the factors) and the global factors slide (S), experimenter (P), array batch (B), day (A), the validation experiment (V), replicate spot (T; 1 or 2), the number of spot pins used (U), and a residual error (εigpbtv). Dye-effects are assumed to be in the SG interaction: they are global although the relative contributions of slides 1 – 3 might differ since only slide 1 contains a self-hybridization. The VSG interaction contains variances due to hybridization and sampling.
Some factors are confounded
Due to the fact that in our DNA-microarray laboratory validation experiments are only performed when necessary (i.e. to introduce a new scientist (experimenter) in the laboratory) confounding of some factors could not be avoided. Therefore, variance analyses were performed by employing the validation experiment (VG) interaction which incorporates: experimenter (PG), array batch (BG), day (AG), and the number of spot pins, coinciding with a change in RNA isolation method (GU).
Authors' contributions
SvH, AdJ, RB, JK, and OK have designed the validation scheme. SvH, HK, NK, RL, and CdH have conducted the DNA-microarray experiments. SvH, AdJ, and CA analyzed the data and generated the figures. SvH, RB, AdJ, and CA drafted the paper. CA provided guidance with the statistical analysis. JK and OK critically read, revised and approved the final manuscript. All authors read and approved the final manuscript.
Acknowledgements
The authors would like to acknowledge Aldert Zomer, Wietske Pool, and Ite Teune for their valuable contributions and suggestions to this study. Work performed by SvH was supported by grant QLK3-CT-2001-01473 under the EU programme 'Quality of life and management of living resources – the cell factory'. The work of RB, HK, and CdH was supported by SENTER, Ministry of Economic Affairs, in the form of a BTS project. The work of NK was supported by NWO-STW, grant 349-5257.
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| 15907200 | PMC1166551 | CC BY | 2021-01-04 16:39:54 | no | BMC Genomics. 2005 May 20; 6:77 | utf-8 | BMC Genomics | 2,005 | 10.1186/1471-2164-6-77 | oa_comm |
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-821593264210.1186/1471-2164-6-82Research ArticleLarge-scale genomic correlations in Arabidopsis thaliana relate to chromosomal structure Kendal Wayne S [email protected] Brian P [email protected] Division of Radiation Oncology, The Ottawa Hospital Regional Cancer Centre, Ottawa, Ontario, K1H 1C4 Canada2 The Ottawa Hospital Research Institute, Ottawa, Ontario, K1H 8L6 Canada3 Ontario Genomics Innovation Centre, Ottawa, Ontario, K1H 8L6 Canada2005 2 6 2005 6 82 82 27 3 2005 2 6 2005 Copyright © 2005 Kendal and Suomela; licensee BioMed Central Ltd.2005Kendal and Suomela; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The chromosomes of the plant Arabidopsis thaliana contain various genomic elements, distributed with appreciable spatial heterogeneity. Clustering of and/or correlations between these elements presumably should reflect underlying functional or structural factors. We studied the positional density fluctuations and correlations between genes, indels, single nucleotide polymorphisms (SNPs), retrotransposons, 180 bp tandem repeats, and conserved centromeric sequences (CCSs) in Arabidopsis in order to elucidate any patterns and possible responsible factors for their genomic distributions.
Results
The spatial distributions of all these elements obeyed a common pattern: the density profiles of each element within chromosomes exhibited low-frequency fluctuations indicative of regional clustering, and the individual density profiles tended to correlate with each other at large measurement scales. This pattern could be attributed to the influence of major chromosomal structures, such as centromeres. At smaller scales the correlations tended to weaken – evidence that localized cis-interactions between the different elements had a comparatively minor, if any, influence on their placement.
Conclusion
The conventional notion that retrotransposon insertion sites are strongly influenced by cis-interactions was not supported by these observations. Moreover, we would propose that large-scale chromosomal structure has a dominant influence on the intrachromosomal distributions of genomic elements, and provides for an additional shared hierarchy of genomic organization within Arabidopsis.
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Background
With recent advances in molecular biology we have begun to appreciate both the diversity of genomic elements contained within chromosomes, and the complexity of their distribution. For example, within plant chromosomes there is often a centromeric region of tandemly repeated satellite sequences that is flanked by transposons, retroelements, middle-repetitive elements, and pseudogenes. Beyond this, the genes tends to become more concentrated[1,2]. Other genomic elements like SNPs tend to form smaller scale-invariant clusters within chromosomes, a finding presumably attributable to the random and independent assortment of haplotype blocks, each with distinctive genealogical histories[3,4]. Genes exhibit a clustering similar to SNPs, although the mechanisms at play here are less well understood[5]. Housekeeping genes cluster differently, within regions of high GC content[6,7], and there is evidence that other co-expressed genes cluster within specific chromosomal domains[8].
There are other examples of such non-random positional associations within chromosomes. For example, indels have been reported to collocate with SNPs[9], and transposable elements with regions of low gene density[10]. Eukaryotic genomes appear to be both structurally and functionally organized at multiple hierarchical levels, and these non-random patterns presumably are manifestations of this organization[11].
We performed a detailed analysis of the intrachromosomal distributions of 6 different genomic elements within the Arabidopsis genome. In addition to genes, indels, SNPs, and retrotransposons, we studied a number of plant-specific elements catalogued through The Arabidopsis Information Resource (TAIR)[12], The Institute for Genomic Research (TIGR)[13] and the Arabidopsis genomic repeat database, AtRepBase. This included a class of 180 bp tandem repeats found in the Arabidopsis genome[1] and provided by AtRepBase, as well as a class of CCSs as identified by The Arabidopsis Genome Initiative [14-19] and catalogued within the TIGR plant repeat database.
Results
Figure 1 provides the density profiles of these elements within chromosome 1, as enumerated from both 50 and 1000 kb bins. The smaller bins revealed numerous high-frequency fluctuations; the larger bins more smoothed density fluctuations. In several of the smoothed profiles there were comparatively large density fluctuations coincident with their known positional associations with centromeres[1]. The profiles corresponding to indels and SNPs, however, did not reveal such major centromeric associations. Parenthetically, we should mention that there remain relatively large gaps in the Arabidopsis centromeric regions that have not been completely defined.
Figure 1 Density profiles for genomic elements along the length of Arabidopsis chromosome 1. Local densities were estimated based upon 50 kb (blue lines) and 1 Mb (red lines) enumerative bins and the density profiles were plotted as the number of structures per 50 kb length of chromosome vs. physical distance as measured from the p-terminal of the chromosome. (a) Genes. (b) Indels. (c) SNPs. (d) Retrotransposons. (e) 180 bp repeats. (f) CCSs. The centromeric region was located at about 15 Mb from the p-terminus. The discontinuity indicated along each X-axis is indicative of the gap in the physical map at this region. Although the lines and axes are drawn continuously in these graphs we must remember that there remains large gaps within the centromeric regions of each chromosome which have not been completely defined.
We performed spectral analyses of these profiles. Briefly, spectral analysis is a computational method used to study data that fluctuates about a mean value over either time or position. It is based on the mathematical theory of Fourier series, and allows one to resolve density fluctuations like those we observed here into their component harmonics. For each set of density fluctuations we were able to deduce a spectrum, where the intensity (or power) of each harmonic could be plotted vs. its particular frequency. Such power spectra can often be exploited to characterize the underlying mechanisms of the fluctuations.
Figure 2 gives the power spectrum for each genomic element, averaged over all 5 chromosomes. Remarkably, each different spectrum shared a qualitatively similar pattern. The greatest intensity of fluctuation was at the low-frequency end of each spectrum. This low-frequency activity corresponded to the large-scale fluctuations apparent to the smoothed density profiles (Fig. 1). (It would be helpful remind the reader here that frequency in these spectra was inversely related to the scale of the measurement bin size.) Further inspection of these density profiles revealed that the centromeric region had provided a major contribution to these low-frequency density fluctuations for genes, retrotransposons, 180 bp repeats and CCSs.
Figure 2 Power spectra for the fluctuations in positional density from genomic elements in Arabidopsis chromosomes. Mean spectral densities for the fluctuations of each element within individual chromosomes were calculated, normalized, averaged over all 5 Arabidopsis chromosomes, and then plotted vs. frequency. The power spectra for all 6 genomic elements were qualitatively similar on this log-log plot: the most intense fluctuations were located at the low-frequency ends of the spectra. (a) Genes. (b) Indels. (c) SNPs. (d) Retrotransposons. (e) 180 bp repeats. (f) CCSs. (g) Simulated data from a Poisson distribution. (Insert) Mean Spectral Densities plotted with linear scales to emphasize the concentration of density fluctuations at low frequency.
The low-frequency activity associated with indels and SNPs was admittedly less intense than that observed with the other elements. Yet the highest peaks from each of these power spectra were both qualitatively similar to those demonstrated with the other genomic elements, and distinguishable from simulated random background noise (P < 0.006). On the basis of the qualitative similarity between the 6 different power spectra we concluded that the major underlying process(es) that governed the density distributions of the different genomic elements had a similar stochastic basis.
These low-frequency density fluctuations, as observed from 6 genomic elements, indicated a non-random clustering of the individual elements over comparatively large chromosomal regions. Since the indels and SNPs had not exhibited such major concentration changes in the centromeric regions, and since genes, indels, SNPs, and retrotransposons had exhibited low-frequency density fluctuations in non-centromeric regions (Fig. 1), we concluded that non-centromeric chromosomal features may have also contributed to the low frequency activity.
Next we sought to determine whether local concentrations of the different elements might correlate with each other. We calculated the Pearson correlation coefficient r between the paired density profiles of the different elements. Given 6 elements, this yielded 15 different permutations. Table 1 provides r for all these different permutations, as assessed at 1 Mb intervals. Thirteen of these correlations were statistically significant. Six exceeded |r|>0.6 and could be thus considered relatively strong.
Table 1 Correlations between different genomic elements in Arabidopsis.
Genomic elements compared ra P valueb
retrotransposons vs. CCSs 0.87 < 0.00001
retrotransposons vs. 180 bp repeats 0.75 < 0.00001
indels vs. SNPs 0.68 < 0.00001
180 bp repeats vs. CCSs 0.66 < 0.00001
genes vs. retrotransposons -0.65 < 0.00001
genes vs. CCSs -0.63 < 0.00001
SNPs vs. retrotransposons -0.46 0.00005
SNPs vs. CCSs -0.42 0.0004
genes vs. 180 bp repeats -0.41 0.001
indels vs. CCSs -0.40 0.001
SNPs vs. 180 bp repeats 0.40 0.002
indels vs. retrotransposons -0.32 0.03
indels vs. 180 bp repeats -0.31 0.05
genes vs. SNPs 0.30 NSc
genes vs. indels 0.23 NS
athe correlation coefficient r given here represents the quadratic mean taken from all five Arabidopsis chromosomes using 1 Mb enumerative bins; bP value, probability value; cNS, not significant;
Statistically significant and strong correlations between the positions of indels and SNPs were apparent from our analysis, in agreement with observations from other systems[9]. We also observed a statistically significant and strong negative correlation between genes and retrotransposons, a finding similar to that reported between genes and transposable elements[10].
We then sought to determine how changes in measurement scale might affect r. In Fig. 3, several of the strongest correlations are plotted over a range of measurement scales. Each of these relationships was statistically significant for the full range of scale. Five of these correlation profiles, however, showed marked weakening (r ≤ 0.2) at bin sizes below 200 kb. The remaining correlation profile, between indels and SNPs (Fig. 3b), however did not reveal as pronounced a decrease at the lower bin sizes. This latter case was an exception to a general trend – the envelope from all 15 correlation pairs (Fig. 3, insert) revealed that the majority of correlations weakened considerably, yet remained statistically significant, at the smaller scales. Because these correlations tended to be strongest at large scales, the associations between elements implied by these correlations were presumably relatively long-ranged. Localized cis-interactions thus did not appear to have a major influence on the intrachromosomal distribution of these elements.
Figure 3 Mean correlation coefficient vs. measurement bin size. The quadratic mean of r, from all 5 Arabidopsis chromosomes, was plotted vs. a range of measurement bin sizes. The correlations provided here represent the 10 of the stronger relationships: (a) Retrotransposons vs. CCSs. (b) Indels vs. SNPs. (c) Retrotransposons vs. 180 bp repeats. (d) 180 bp repeats vs. CCSs. (e) Genes vs. retrotransposons (f) Genes vs. CCSs. The broken red lines represents the critical values corresponding to P = 0.05 and obtained by simulation. (Insert) Mean correlation vs. measurement bin size for the envelope of all 15 comparisons. Here the solid red lines represent the critical values corresponding to P = 0.05.
Discussion
To summarize, the density profiles of the 6 diverse genomic elements examined here revealed numerous high frequency and localized fluctuations upon which was superimposed a low frequency and large scale pattern of fluctuation. For 4 of these elements these low frequency fluctuations were concentrated at the centromeric regions of the chromosomes. Spectral analysis confirmed the presence of a dominant low frequency, large scale, component to these density fluctuations in each of the 6 elements, and these fluctuations tended to correlate with each other at large genomic scales. It was apparent on comparative analysis of these three sets of figures that the large scale density fluctuations seen within the density profiles of Fig. 1 were related to the dominant low frequency components evident from the power spectra of Fig. 2, and to the relatively strong large scale correlations seen within Fig. 3. It was also reasonably obvious from Fig 1 that a major (but not exclusive) component to these large scale density fluctuations was derived from centromeric structures.
These large scale correlations were demonstrated with several different genomic elements, which were themselves identified and mapped through an assortment of different methods. The possibility that some form of selection bias might have influenced the results should be considered. It would, however, be difficult to postulate a spurious source of bias to explain such fluctuations and correlations for which the predominant component was evident not only at low frequency and large scales but also with each of the different elements examined. Such a putative bias would necessarily have to be associated with the centromeres. Despite the large undefined gaps in centromeric regions that remain, sufficient portions of the centromeres have been defined so as to provide us with strong evidence for the associated centromeric density changes for gene structures, retrotransposons, 180 bp repeats and conserved centromeric sequences[1,2]. We would propose instead that the most plausible explanation for these low frequency density fluctuations, and large scale correlations, would be itself the large scale structural features of Arabidopsis chromosomes.
The persistent correlation between indels and SNPs at smaller measurement scales (Fig. 3c) warrants further consideration. These two elements have been shown to correlate with each other within the HLA region of the human genome, a observation that might be attributed to repetitive insertions and deletions, imperfect segmental duplications or adjacent nucleotide changes[20] – all of these presumably localized processes. Our observation of a modest 20% increase in the respective value of r with increased bin size indicated that in Arabidopsis a component of this correlation could be attributed to large-scale chromosomal features. At the same time, we could not exclude the possibility that the persistence of this correlation at small scales might not be attributable to localized interactions, but rather to methodological artifact since the positional cloning used to detect both SNPs and indels for the TAIR database might have allowed selection bias.
We also observed that the densities of genes and retrotransposons exhibited statistically significant and strong negative correlation, a finding consistent with previous observations with transposable elements. This has been explained by a presumed selection against the disruption of gene expression by transposable elements[10]. As with the other associations discussed here, it was difficult to reconcile explanations based upon localized processes with the decreased strength of the correlation at small measurement scales. The hypothesis that large-scale chromosomal structure could influence the spatial distribution of a variety of chromosomal elements seemed to provide a more plausible explanation.
Conclusion
We have demonstrated that the physical distributions of genomic elements within Arabidopsis chromosomes were highly heterogeneous, yet shared a common distributional pattern. The density profiles of each element exhibited low-frequency fluctuations indicative of regional clustering, and these density profiles tended to correlate with each other at large measurement scales. This was the dominant pattern underlying the positional distributions of all 6 genomic elements examined. Localized cis-interactions between different elements had a comparatively minor, if any, influence on the intrachromosomal distribution of genomic elements. This study demonstrated an additional hierarchy of eukaryotic genomic organization that was both common to a diverse set of genomic elements, and associated with major chromosomal features.
Methods
Data abstraction
The Arabidopsis thaliana data used in this study was obtained from a variety of sources: The positions of genes were obtained from the National Center for Biotechnology Information (NCBI) website and localization of indels and SNPs was provided by TAIR . TIGR provided sequence information for Arabidopsis pseudochromosomes ) as well as for retrotransposons and CCSs . Sequences for the 180 bp repeats were obtained from AtRepBase . Localization of retrotransposons, CCSs and the 180 bp repeats was performed by running BLASTN as provided at the TIGR website. In order to be as inclusive as possible, the stringency of these matches was kept low; as well, filters were used to exclude redundant matches. We thus accessed the positions of 29,826 genes, 1,732 indels, 20,008 SNPs, 20,564 retrotransposons, 14,064 tandem repeats, and 3,896 CCSs.
Since TAIR and TIGR act as repositories for the publicly available Arabidopsis data, a number of different methods had been used by many different investigators to identify the positions of the various genomic elements: SNPs and indels were identified by positional cloning of the available bacterial artificial chromosomes (BACs)[21], and by the large-scale analysis of expressed sequence tags from different accessions of Arabidopsis[22].
In our analysis the 5 Arabidopsis chromosomes were divided into non-overlapping, equal-sized, sequential bins and the numbers of p-termini for the structure of interest were enumerated for each bin. A range of bin sizes was employed, from 10 to 1,000 kb. Density profiles for the individual structures thus obtained were subjected to the additional analyses detailed below.
Spectral analysis
Density profiles for each genomic feature were parsed into 50 kb bins and padded with zeros to a length of 1024 data points. A 15% split-cosine-bell taper was employed on the data at the beginning and end of each sequence, the mean was subtracted, the data de-trended, and a 15-point Hamming data window was employed for data smoothing. The spectral density of each structure was calculated using a Cooley-Tukey fast Fourier transform and then normalized. The spectral densities were averaged over all 5 Arabidopsis chromosomes and then plotted vs. frequency. As a control for these analyses we performed Monte Carlo simulations for a random (Poisson) distributed genomic element within five chromosomes with a density comparable to that observed with genes. These data were then processed as above to yield a spectral density. A more extensive set of simulations was done to estimate the critical values for the amplitude of individual peaks expected from a random distribution of elements, and with parameters to emulate both the chromosomal densities of elements and the lengths of the individual chromosomes.
Correlation analysis
The Pearson correlation coefficient r was calculated between different density profiles over a range of bin sizes (10 to 1000 kb), and then plotted vs. bin size. We also performed scatter plots between the density profiles to exclude pronounced nonlinear relationships between variables (data not provided). Because r2 is additive, whereas r is not, the quadratic mean was used to average correlations from the 5 Arabidopsis chromosomes. Critical values for the quadratic means were estimated by Monte Carlo simulations, based upon the premise of a t-distribution for the quantity, , where n represents the number of data points[23].
List of abbreviations used
SNPs – single nucleotide polymorphisms; CCSs – conserved centromeric sequences; NCBI – National Center for Biotechnology Information; TAIR – The Arabidopsis Information Resource; TIGR – The Institute for Genomic Research; AtRepBase – Arabidopsis genomic repeat database; BACs – bacterial artificial chromosomes; P value – probability value; NS – not significant; vs. – versus; bp – base pairs; kb – kilobases; HLA – human leukocyte associated antigens; GC – guanidine cytosine; BLASTN – basic local alignment search tool for nucleotides ; PCR – polymerase chain reaction; r – Pearson correlation coefficient.
Authors' contributions
WSK conceived this study, conducted the spectral and correlation analyses, and drafted the manuscript. BPS participated in the design of the study, and conducted the bioinformatic data abstraction. Both authors read and approved the final manuscript.
Acknowledgements
The authors thank Dr. Miguel Andrade for his helpful advice in the preparation of this manuscript.
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| 15932642 | PMC1166552 | CC BY | 2021-01-04 16:39:53 | no | BMC Genomics. 2005 Jun 2; 6:82 | utf-8 | BMC Genomics | 2,005 | 10.1186/1471-2164-6-82 | oa_comm |
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BMC Health Serv ResBMC Health Services Research1472-6963BioMed Central London 1472-6963-5-401592150910.1186/1472-6963-5-40Research ArticleGeneral practitioners with a special interest in respiratory medicine: national survey of UK primary care organisations Pinnock Hilary [email protected] Gopalakrishnan [email protected] David [email protected] Aziz [email protected] Division of Community Health Sciences: GP Section, University of Edinburgh, 20, West Richmond St, Edinburgh, Scotland. EH8 9DX. UK2 Department of Primary Care and Social Medicine, 3rd Floor, The Reynolds Building, St Dunstan's Road, London, England. W6 8RP. UK3 Department of General Practice and Primary Care, University of Aberdeen, Foresterhill Health Centre, Westburn Road, Aberdeen, Scotland. AB25 2AY. UK2005 27 5 2005 5 40 40 9 1 2005 27 5 2005 Copyright © 2005 Pinnock et al; licensee BioMed Central Ltd.2005Pinnock et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
To meet the universally recognised challenge of caring for people with long-term diseases many healthcare cultures are encouraging family physicians to develop specialist skills. We aimed to determine the major factors influencing the appointment of respiratory General Practitioners with a Special Interest (GPwSI) in the UK, and to determine the priority attached to the potential roles, perceived barriers to implementation, and monitoring planned.
Methods
We sent a piloted semi-structured questionnaire to a random sample of 50% of English and Welsh primary care organisations (PCOs) (n = 161) during winter 2003. In addition to descriptive statistics, we used hierarchical cluster analysis to classify service priorities. Free-text responses to open-ended questions were analysed qualitatively by a multidisciplinary group to identify emerging themes.
Results
Of the 111 (69%) PCOs who responded, 7 (6%) already have, and a further 35 (32%) are planning, a respiratory GPwSI service. This proportion is considerably lower than in specialities linked to National Health Service clinical priorities. Local needs and pressure on hospital beds were the main described motives for developing a service. Stated service priorities were to relieve pressure on secondary care and to improve quality of care, including the strategic planning of respiratory services within PCOs.
Conclusion
The relatively few respiratory GPwSIs currently in post reflects the lack of government prioritisation of respiratory care. However, respiratory GPwSI services are increasingly being considered as a local strategy for reducing pressure on secondary care respiratory services and raising standards of chronic disease management in primary care.
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Background
The care of people with long-term disease is universally recognised as a major challenge, and national healthcare services around the world are reconfiguring to meet the demand [1,2]. Chronic respiratory disease is projected to rank as the fifth leading cause of morbidity by 2020 [3]. Increasingly, specialist roles are being devolved to family physicians, echoing recent global recognition of the contribution of primary care expertise to the management of common conditions such as respiratory disease [4]. Within the UK, the strong links between community-based practitioners and hospital specialists have long been valued and services increasingly draw on the resources of the two traditions to the mutual advantage of patients and clinicians [5].
General Practitioners with a Special Interest (GPwSIs), a key component of the UK National Health Service modernisation agenda [6], challenge traditional models of specialist care. The key policy driver is the imperative to reduce waiting lists for specialist opinions in areas such as ophthalmology, orthopaedics, dermatology, ear nose and throat surgery, and for specific procedures such as endoscopy [6,7]. The emphasis is on maintaining a family care perspective while developing defined specialist competencies to meet local healthcare need [7-9]. Primary care interest societies have broadened the potential remit of GPwSIs, delineating roles in a wider range of clinical areas and involving a more strategic role than was originally envisaged [10,11].
Despite being responsible for nearly a third of general practice consultations [12], one in eight emergency hospital admissions [13], and the major contributory factor in the winter bed crises [14], respiratory disease did not feature in any of the official documents [6-9]. This lack of national prioritisation of respiratory care is reflected internationally [15,16], with notable exceptions such as Finland and Australia [17]. The General Practice Airways Group (a UK charity focusing on delivering optimal respiratory care in community settings) considered this issue in a discussion paper outlining a number of potential roles for a respiratory GPwSI: leading the strategic planning from a primary care perspective, setting quality standards for respiratory care and providing clinical expertise for conditions most common in general practice [10]. These concepts, now embodied in a guideline [18], establish the potential of a respiratory GPwSI service to address many of the recognised unmet needs of people with respiratory and allergic conditions [19,20].
UK primary care organisations (PCOs) which manage care for populations of approximately 100,000, are charged with meeting the competing healthcare demands of their local community against a background of political pressures imposed by national policies and service frameworks. It is not known how many view positively the potential of respiratory GPwSIs. Our survey of primary care organisations in England and Wales aimed to determine the major factors influencing the appointment of respiratory GPwSI, and to determine the priority attached to potential roles, the perceived barriers to implementation of a GPwSI service and the monitoring planned.
Methods
Ethics
Grampian Research Ethics Committee advised that our survey of current practices and future plans did not require ethical approval. [MacLeod K, personal communication, July 2003]
Questionnaire design
Our questions were based on a detailed review of the literature on the GPwSI initiative, and designed with the advice of health service administrators and clinicians with a specialist interest in primary care respiratory disease from all regions of the UK [21]. Minor adjustments were made after feedback from pilot PCOs. Closed questions included an option for adding additional responses; free-text comments were invited throughout. The relative importance of the potential issues that a respiratory GPwSI might address was assessed by asking respondents to rate priorities on a 5-point Likert-scale [5 = Top priority; 0 = No priority].
Sampling procedures
During the winter of 2003/4 we sent the questionnaire to the chief executive of a 50% random sample (n = 161) of English and Welsh PCOs (Primary Care Trusts (PCTs) in England; Local Health Boards (LHBs) in Wales) asking them to forward the questionnaire to the person within their organisation best placed to complete the questionnaire. We phoned non-responding PCOs to identify the contact details of the person responsible for developing GPwSI services, to whom we e-mailed an electronic version of the questionnaire. Non-responders were sent a further postal reminder.
Sample size calculation
To estimate the frequency of PCOs with an interest in appointing a respiratory GPwSI with 95% confidence, assuming an expected frequency of 10%, with a precision of 5%, we needed 108 usable responses. We anticipated a 70% response rate and therefore sampled 50% of the 322 PCOs in England and Wales.
Data analysis
Responses to closed questions were treated as nominal data, whereas priority ratings were treated as on a linear scale. In addition to descriptive statistics, we looked for correlation between the different service priorities using Pearson correlation coefficients and used hierarchical cluster analysis to classify the priorities. All analyses were conducted using SPSS version 11.5.
Free-text responses to open-ended questions were thematically analysed by a multidisciplinary group involving practising and academic GPs, a health services manager, and a qualitative researcher. Using the principles of qualitative content analysis, we developed a coding frame and identified key emerging themes [22].
Results
We received responses from 111/161 (69%) of the England and Welsh PCOs. Seven (6%) PCOs reported that they already had a respiratory GPwSI in post and a further 35 (32%) indicated that they were considering developing a respiratory GPwSI service.
Comparison with other GPwSI services [Figure 1]
Figure 1 Current and planned GPwSI services
Ninety-eight PCOs gave information on existing GPwSIs or those under consideration in different specialities. Most commonly, PCOs tended to have three to five GPwSIs (46%) and a significant proportion (21%) had 10 or more. The top five GPwSIs in terms of frequency were respectively dermatology, minor surgery, coronary heart disease, ear, nose and throat surgery and drug misuse – areas highlighted by National Health Service policy [6,23]. Respiratory medicine ranked twelfth.
This early focus on nationally prioritised areas, with respiratory GPwSI services a later consideration, is reflected in the comments made by several PCOs:
"The PCT [Primary Care Trust]has developed GPwSI led services for dermatology, ENT [Ear, Nose and Throat], orthopaedics and ophthalmology and is developing services for mental health, diabetes and emergency care in 2004/05. Following this, the PCT is considering the development of further GPwSI led services – including respiratory services" (PCO-39: considering a respiratory GPwSI service)
"Focusing on other areas of work at the moment. Principally the NHS [National Health Service] plan, cancer plan + NSFs [National Service Frameworks]" (PCO-119: no plans for a respiratory GPwSI service)
Major factors influencing the decision
Of the 42 English and Welsh PCOs who had, or were considering developing a respiratory GPwSI service, 33/42 (79%) stated that they were responding to local needs, often identified by audits of hospital activity:
"Audit has shown that COPD [chronic obstructive pulmonary disease]/ asthma are causes of significant repeat admissions to hospital." (PCO-54: considering a respiratory GPwSI service)
"High level of deprivation and corresponding high levels of asthma morbidity. Have had asthma & respiratory as a local priority since ... late 90's." (PCO-134: existing respiratory GPwSI service)
The prime motives for considering appointing a respiratory GPwSI are given in Table 1. Reducing pressure on secondary care, particularly admissions for chronic obstructive pulmonary disease, was the most commonly cited motive and improving chronic disease management in primary care was seen as a means to achieve this – possibly even diverting costs from in-patient to community care.
Table 1 Motivating factors and barriers identified by PCOs.
Question Options N (%)
PCO-s that have (or are considering) a respiratory GPwSI service (n = 42)
What are the factors motivating your PCO to have/or consider a respiratory GPwSI service? (n = 41)* Identified local needs 33 (80)
Winter pressures on hospital beds 19 (46)
New GP contract targets 13 (32)
Government directive 5 (12)
Influence of local personalities 5 (12)
Drug cost containment 4 (10)
Secondary care initiative 4 (10)
Local patient pressure 1 (2)
There's a pot of money available 1 (2)
Pharmaceutical company influence 0
What are the major problems you will have to overcome? (n = 39)* Competition with other local priorities 25 (64)
Inadequate funding for respiratory GPwSI 20 (51)
Inadequate infra structure support funding 15 (38)
Respiratory disease is not a national priority 8 (21)
Lack of local interest/expertise from GPs 6 (15)
PCOs that are not considering a respiratory GPwSI service (n = 69)
Why is a respiratory GPwSI service not a priority in your PCO? (n = 67)* Lack of local interest/expertise from GPs 24 (36)
We have a respiratory specialist nurse 24 (36)
Inadequate funding for respiratory GPwSI 13 (19)
Inadequate infra structure support funding 13 (19)
Respiratory disease is not a local priority 10 (15)
Lack of local patient pressure 7 (10)
Respiratory disease is not a national priority 6 (9)
Opposition from secondary care 1 (1)
Winter pressures are not a problem locally 0
* Not all PCOs answered all the questions..
"Likely to be a growth area in our PCT in terms of better care leading to reduced admissions" (PCO-72: considering a respiratory GPwSI service)
"Improve appropriateness of secondary care referrals" (PCO-127: considering a respiratory GPwSI service)
"Funding has been an issue – we have always been keen. Now identified reducing hospital admissions as a way of freeing up funds." (PCO-85: considering a respiratory GPwSI service)
The inclusion of respiratory targets in the General Medical Services contract for GPs [24] and the Primary Care Collaborative (a UK initiative to facilitate development in primary care) programme on chronic obstructive pulmonary disease [25] have given added impetus to the development of respiratory services.
"We have become very motivated on this subject recently as we currently have 15 practices participating in Phase 3 of the Primary Care Collaborative. We are aware that we have neglected this area locally, as have many PCTs, however there is now real enthusiasm for change" (PCO-33: no plans for a respiratory GPwSI service)
"We do not currently have a GPwSI in respiratory service currently, but that does not mean we do not consider it as a priority. There is scope to develop this role to meet the requirements of nGMS [new General Medical Services contract]" (PCO-7: no plans for a respiratory GPwSI service)
The 69 PCOs not planning a respiratory GPwSI service at this time cited local workforce issues as the main barrier [Table 1]: 24/69 (35%) felt that local GPs did not have the interest or necessary expertise to undertake the role whilst 24/69 (35%) already had a specialist respiratory nurse who was addressing the local needs appropriately. These issues echoed the problems that needed to be overcome by PCOs who were planning a service.
"We do not have any GPs who presently have the skills (and just as importantly, the time) to give to this work. However we do have a specialist nurse led team working across primary and secondary care in respiratory illness." (PCO-64: no plans for a respiratory GPwSI service)
"We are currently recruiting for clinical leads in all areas – GP's w special interest in Respiratory medicine has not been filled" (PCO-38: considering a respiratory GPwSI service)
Conversely, the presence of an 'existing respiratory champion' was a positive motivating factor.
"Local need..... plus local GP with an interest" (PCO-105: existing respiratory GPwSI service)
"GP with expertise moved into area" (PCO-85: considering a respiratory GPwSI service)
The importance of specialist teams
The free text comments elaborated on the respective roles of GPwSIs and specialist nurses and emphasised the importance attached to team work, though the potential role of the GPwSI within that team varied.
"However the LHB [Local Health Board] is in the process of implementing an integrated COPD team (with local trust) (Primary / Community care and secondary care). This team is lead by a consultant with links to GP's, Practice nurses, therapists and community nurses. The second phase will be to develop a GPwSI." (PCO-158: considering a respiratory GPwSI service)
"I think that for GPwSI's to be fully effective, robust support from respiratory nurses is essential. We have one F/T [full-time]lead nurse plus 2 P/T [part-time] Nurses" (PCO-134: existing respiratory GPwSI service)
"It is anticipated that the GPwSI role would support the provision of a nurse-led spirometry service" (PCO-28: considering a respiratory GPwSI service)
Priorities for a respiratory GPwSI service
In line with the factors motivating PCOs to develop respiratory GPwSI services, reducing hospital admissions was the top priority, with raising standards of respiratory care also highlighted. In contrast, allergy services were rarely prioritised [Table 2].
Table 2 Priorities for a GPwSI service. Based on the question "Please rate the priority of the following specific issues a respiratory GPwSI might address?" Score: 0 is no priority, 5 is top priority
Priorities Ratings
N* Mean 95% CI
Reducing acute respiratory admissions 38 4.5 4.3 to 4.7
Raising standards of respiratory care in practice 37 4.3 4.0 to 4.5
Reducing respiratory outpatient referrals 37 4.0 3.7 to 4.3
Strategic planning of respiratory services 34 3.9 3.6 to 4.3
Reducing respiratory outpatient referral waiting times 35 3.8 3.5 to 4.1
Improving access to spirometry 36 3.6 3.3 to 3.9
Coordination of General Medical Services quality framework for asthma and chronic obstructive pulmonary disease 34 3.6 3.4 to 3.9
More appropriate home oxygen use 33 3.2 3.0 to 3.5
Development of management templates/coordinated data collection and extraction 34 3.2 2.8 to 3,6
More appropriate home nebuliser use 32 3.2 2.9 to 3.5
Provision of an allergy service 30 1.9 1.7 to 2.2
* Not all PCOs answered all the questions
We found that some priorities tended to be highly correlated; for example PCOs prioritising reduction in the number of outpatient referrals also tended to prioritise reduction in outpatient waiting times (r = 0.8). Other highly correlated priorities were appropriate usage of home nebulisers and oxygen (r = 0.9), strategic planning of respiratory services and development of management templates/coordinated data collection and extraction (r = 0.8). Applying hierarchical cluster analysis, we were able to group the priorities into three main areas of consideration:
1) Relieving pressure on secondary care, including reducing admissions, outpatient referrals and waiting lists.
2) Improving quality of care, including strategic planning of respiratory services, raising standards of respiratory care in practice, coordination of GMS quality framework for asthma and chronic obstructive pulmonary disease and development of management templates/coordinated data collection and extraction. More specifically, improving access to spirometry and more appropriate home oxygen and nebuliser use also correlated with quality of care.
3) Providing allergy services.
Infrastructure, support and monitoring planned
Monitoring focussed on the impact of a respiratory GPwSI service on secondary care, especially chronic obstructive pulmonary disease admissions, though nearly half the PCOs planned to assess patient satisfaction. Most PCOs appreciated the need to provide infra-structure support for a GPwSI and two thirds acknowledged the importance of supporting on-going professional development [Table 3].
Table 3 Infrastructure, support and monitoring planned for GPwSI service.
Question Options N (%)
What infra-structure/support have you got / do you plan for a respiratory GPwSI service? (n = 34)* Clinical support – e.g. nurses, physiotherapists 30 (88)
Medical equipment: e.g. spirometer, oximeter 23 (68)
Training and on-going continuing professional development for the GPwSI 22 (65)
Office support – e.g. room, desk, computer, etc. 17 (50)
Administrative support – e.g. secretary 15 (44)
What monitoring are you undertaking/planning? (n = 38)* Admission rates for chronic obstructive pulmonary disease 37 (97)
Accident and emergency attendances 21 (57)
Quality of respiratory care 19 (51)
Practice prescribing for respiratory disease 19 (49)
Patient satisfaction 19 (49)
Admission rates for asthma 18 (46)
Waiting times for chest clinic referrals 16 (43)
Home oxygen use 5 (14)
* Not all PCOs answered all the questions
Discussion
Although currently few in number, appointment of respiratory GPwSIs is currently being considered by nearly a third of PCOs in the UK: still however considerably less than in specialities prioritised by government policy. Reducing pressure on secondary care services is the key motive for appointing a respiratory GPwSI, a top priority for the role, and the aspect of the service most likely to be monitored. Improving the quality of respiratory care is also highlighted, both as a means of reducing referrals and admissions and also in line with the increasing emphasis on chronic disease management in primary care [24].
Limitations of study
We achieved a response rate of 69% and our results may not reflect the situation in the non-responding PCOs. It is probable that PCOs most interested in appointing a respiratory GPwSI will have responded promptly; those who had not yet focussed on the potential of a respiratory GPwSI service may have been less motivated to respond, indeed in some organisations with few plans for GPwSI services it may not have been clear who would be best placed to answer the questionnaire. This would result in an overestimate of the interest in a respiratory GPwSI.
GPwSI services are a rapidly developing initiative and despite extensive background reading, wide consultation and piloting the questionnaire it is likely that the closed question format did not predict all possible responses. Each question, therefore, offered the opportunity to add additional responses. Reassuringly, for most questions the space was used to clarify the closed responses rather than add new options. The exception was the very wide range of innovative GPwSI services listed. Those offered most frequently have been included in Figure 1, though the frequency cannot be directly compared with those specialities for which a prompt was given.
The answers to closed questions and free text responses can only provide limited insight into the development of respiratory GPwSI services. We did not attempt to define a respiratory GPwSI because the absence (at the time of the survey) of agreed accreditation processes and the concept of a locally developed service would have made that difficult, so it is likely that there was some variation in the interpretation of the question. However, the responses do provide a quantitative assessment of the interest in this initiative, and the comments indicate the potential value of a follow-on in-depth exploration of perspectives on the development of respiratory GPwSI services.
Main strengths of study
We achieved our anticipated response rate and therefore exceeded our intended sample size. Our piloted questionnaire appeared to be acceptable to PCOs and we identified no problems with completion. Our random sampling strategy should ensure national generalisability across England and Wales. We used an integrated quantitative and qualitative analytical paradigm, thus increasing the validity of our findings [26].
Interpretation of findings in relation to previously published work
Primary care organisations in England and Wales have adopted the concept of GPwSIs initially focusing on areas driven by government policy [6,23], but increasingly as an option for developing a wider range of specialities in order to meet local needs. Our data suggest that, although only 6% of PCOs currently have a respiratory GPwSI in post, there may be welcome interest in respiratory disease with nearly a third of PCOs considering a respiratory GPwSI service.
In keeping with national policy [6], the priority for respiratory GPwSI services in most PCOs is to reduce pressure on secondary care. Admission rates are a key target, especially for chronic obstructive pulmonary disease where in-patient stays are often prolonged and an important factor in winter pressure on hospital beds [14]. GPwSIs may also contribute to local strategies designed to meet government targets for a 3.5% per year shift of outpatient consultations to primary care [27-29]. Experience from government-driven initiatives in Finland and Australia exemplify the importance of engaging primary care specialists as care is shifted to the community [17].
The increasing global emphasis on chronic disease management [1,2] and empowering patient self-management [30] may have influenced the priority attached to the strategic role, seen by many PCOs as potentially within the remit of a respiratory GPwSI. In the UK, the new primary care contract, with a focus on respiratory chronic disease provides an important context for this initiative [24]. Internationally, the challenge of managing long-term conditions may be an useful argument for both primary and secondary care specialists, campaigning nationally to encourage governments to prioritise respiratory care and locally to ensure that the needs of respiratory patients are met [17].
The lack of priority attached by PCOs to allergy services reflects the concern expressed by the House of Commons Health Committee report on the current under-provision of allergy services in the UK [31]. This report recommends the development of a cadre of GPwSIs to give focus and expertise to the treatment of allergy in primary care.
It is encouraging that two-thirds of the PCOs indicated that their planned infra-structure for a respiratory GPwSI service included support for on-going training and professional development; however, there should be concern that this was not universally prioritised. Agreed procedures for accrediting a respiratory GPwSI have now been agreed, and emphasise the importance of appropriate training, mentoring and accreditation required to assure quality [9,32]. Concerns have already been expressed that the locally defined contracts could lead to unacceptable variations in the contractual obligations, remuneration and support [11]. Whilst diversity in locally defined roles is to be encouraged, training tailored to meet that role is a universal requirement [33].
Our survey focused specifically on the recently defined GPwSI initiative. However, there is already a strong tradition both in primary and secondary care of specialist respiratory nurses [34-36] with many well established teams leading, for example 'Hospital at Home' schemes [37]. Not surprisingly, therefore, nearly 90% of PCOs envisaged the development of specialist teams to support their planned GPwSI and a third of PCOs in our survey indicated that they either had or were considering a specialist nurse rather than a GPwSI appointment. Local factors, such as availability of GP or nurse expertise, were important in determining the planned workforce configuration.
Conclusion
Healthcare organisations in the UK are responding positively to the challenge of reconfiguring the workforce to meet local needs. Although the initial focus has been on areas highlighted by National Health Service policy, respiratory GPwSIs are increasingly being considered, both as a means of reducing pressure on secondary care, and also raising standards in primary care to meet the challenge of chronic disease management.
Abbreviations and explanation of terms
COPD Chronic obstructive pulmonary disease
ENT Ear. Nose and Throat surgery.
GMS General Medical Services. The GMS contract, recently updated as the 'new' GMS contract (nGMS), governs provision of primary care services.
GPwSI General Practitioners with a Special Interest.
LHB Local Health Board
PCO Primary Care Organisations. These organisations, known as PCTs in England and LHBs in Wales, commission local healthcare services.
PCT Primary Care Trust
NHS National Health Service
NSFs National Service Frameworks. These NHS documents set national standards for the provision of care for a range of disease areas.
Primary Care Collaborative is a UK initiative to facilitate development in primary care. Phase 3 of this initiative includes a focus on chronic obstructive pulmonary disease.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
HP initiated the idea for the study and with AS led the development of the protocol, securing of funding, study administration, data analysis, interpretation of results and writing of the paper. GN provided statistical advice and DP provided advice on the development of the protocol and the interpretation of results. All authors reviewed the final manuscript. HP and AS are study guarantors.
Funding
General Practice Airways Group.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We acknowledge the support of Ms Sian Williams who provided helpful advice from the perspective of a health service manager, and colleagues from the General Practice Airways Group whose experience of specialist GP roles was invaluable.
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| 15921509 | PMC1166553 | CC BY | 2021-01-04 16:31:50 | no | BMC Health Serv Res. 2005 May 27; 5:40 | utf-8 | BMC Health Serv Res | 2,005 | 10.1186/1472-6963-5-40 | oa_comm |
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BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-5-391591346110.1186/1471-2334-5-39Technical AdvanceA sensitive flow cytometric methodology for studying the binding of L. chagasi to canine peritoneal macrophages Gonçalves Ricardo [email protected] Etel R [email protected] Maria N [email protected] Kenneth J [email protected] David M [email protected] Wagner L [email protected] Faculdade de Medicina – DAPML – Universidade Federal de Minas Gerais (UFMG), Brazil2 Departamento de Bioquímica e Imunologia – Instituto de Ciências Biológicas (ICB), Universidade Federal de Minas Gerais (UFMG), Brazil3 Departamento de Parasitologia, Instituto de Ciências Biológicas (ICB), Universidade Federal de Minas Gerais (UFMG), Brazil4 Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA5 Departamento de Patologia Geral, Instituto de Ciências Biológicas (ICB), Universidade Federal de Minas Gerais (UFMG), Brazil2005 24 5 2005 5 39 39 31 8 2004 24 5 2005 Copyright © 2005 Gonçalves et al; licensee BioMed Central Ltd.2005Gonçalves et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces. The success of the parasite infection is dramatically dependent on this early interaction in the vertebrate host, which permits or not the development of the disease. In this study we propose a novel methodology using flow cytometry to study this interaction, and compare it with a previously described "in vitro" binding assay.
Methods
To study parasite-macrophage interaction, peritoneal macrophages were obtained from 4 dogs and adjusted to 3 × 106 cells/mL. Leishmania (Leishmania) chagasi parasites (stationary-phase) were adjusted to 5 × 107 cells/mL. The interaction between CFSE-stained Leishmania chagasi and canine peritoneal macrophages was performed in polypropylene tubes to avoid macrophage adhesion. We carried out assays in the presence or absence of normal serum or in the presence of a final concentration of 5% of C5 deficient (serum from AKR/J mice) mouse serum. Then, the number of infected macrophages was counted in an optical microscope, as well as by flow citometry. Macrophages obtained were stained with anti-CR3 (CD11b/CD18) antibodies and analyzed by flow citometry.
Results
Our results have shown that the interaction between Leishmania and macrophages can be measured by flow cytometry using the fluorescent dye CFSE to identify the Leishmania, and measuring simultaneously the expression of an important integrin involved in this interaction: the CD11b/CD18 (CR3 or Mac-1) β2 integrin.
Conclusion
Flow cytometry offers rapid, reliable and sensitive measurements of single cell interactions with Leishmania in unstained or phenotypically defined cell populations following staining with one or more fluorochromes.
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Background
Leishmaniasis is a disease resulting from infection by protozoa of the genus Leishmania, which affects man and several other species of mammals. In the new world Leishmania is transmitted to man and animals by blood-sucking sandflies of the species Lutzomyia longipalpis [1-3]. Infection is initiated when the sandfly regurgitates infectious forms of promastigotes, termed "metacyclics" [4], into a small pool of blood formed by the blood-sucking female [5]. Upon inoculation of the host, promastigotes are phagocytized by skin macrophages, where they transform into aflagellar ovoid bodies known as amastigotes. In mammals, Leishmania are dimorphic obligate intracellular parasites, which reside and multiply mainly within the phagolysosomal compartment of mononuclear phagocytes [6-8]. Depending on the species, the parasite or infected macrophages, may spread and give rise to secondary infections in distant organs, producing distinct clinical forms of the disease [9]. Thus, the pathogenesis of Leishmania infections depends to a large extent on the relationship between the parasites and host macrophages.
The interaction of Leishmania promastigotes with mononuclear phagocytes has been characterized, and multiple receptor-ligand interactions have been implicated to play a role in the attachment to, and uptake of, promastigotes by macrophages [8]. These interactions include the binding of parasite surface molecules (lipophosphoglycan and gp63) or host derived opsonins (complement, fibronectin and immunoglobulin) to multiple macrophage receptors. The best characterized of these systems is the binding of serum complement opsonized promastigotes to macrophage receptors for complement [10]. Complement-dependent opsonization of parasites not only improves their adhesion to macrophages, but also enhances their intracellular survival [11]. Although the direct interaction of promastigotes with macrophages, in the absence of opsonization, has been well established for several species of Leishmania, the identity of the macrophage receptors involved in this interaction, and to a lesser extent, the ligands on the surface of promastigotes, remains unresolved [12]. The macrophage complement receptor Mac-1 (CD11b/CD18 or CR3) has been demonstrated to be crucial for the interaction of serum opsonized promastigotes with both human and murine macrophages [10,13-15].
In the literature, there are many "in vitro" methods describing the Leishmania-macrophage interaction. These techniques are performed quantifying parasites and macrophages over coverslips where parasites can be counted by conventional microscopy, immunofluorescent microscopy (immunolabelled parasites) or radioactivity associated with the cell lysates determined by a scintillation counter [11,14,16,17]. However, these techniques are laborious, time consuming, and do not allow an accurate characterization of the cells and surface receptors involved with parasite uptake. Moreover, most of these studies involve human or murine monocytes-macrophages and Leishmania major. "In vitro" studies using canine macrophages and Leishmania chagasi are rare in the literature. As far as we know, Marzochi et al. (1999) were the first to report binding-assays between different species of Leishmania and canine macrophages [18].
In this study we propose a flow cytometric method to study peritoneal canine macrophage-Leishmania chagasi interaction by a binding assay using the stable intra-cytoplasmic fluorocrome, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) [19]. This methodology is less laborious than cell-cultures techniques, and is not limited by the subjective and time-consuming nature of microscopy, necessary to calculate parasitism ratios. Moreover, when associated with conventional flow cytometry analysis, this technique also allows the phenotypic characterization of the infected cells and the identification of the cell surface molecules involved in parasite uptake. Lastly, this method allows for the analysis of large populations of cells rather than the limited number that must be analyzed using microscopy.
Methods
Animals
Four mongrel dogs of unknown age were obtained from the City of Sabará (Suburban area of Belo Horizonte, City) (City hall Zoonosis Department), MG, Brazil. All dogs were negative for Leishmania by direct immunofluorescence (RIFI) and complement fixation tests (RFC). The dogs received anti-helminth and anti-ectoparasite treatment. Animals were maintained in quarantine with food and water "ad libidum". All animal studies were performed under the guidance and approval of the institutes' animal welfare committee under the supervision of a certified veterinarian.
Ethics approval
The experiment protocol using dogs, was approved by CETEA (COMITÊ DE ÉTICA EM EXPERIMENTAÇÃO ANIMAL – UFMG), by number 034/2004.
Obtaining the Peritoneal cells
For obtaining the peritoneal macrophages the dogs were sacrificed using a lethal dose (0,3 ml/Kg) of T-61® drug (Intervet). The animals were positioned in decumbency and the peritoneal cavity was disinfected using alcohol-iodine-alcohol and washed with sterile PBS (Phosphate-Buffered Saline). The peritoneal cavity was opened by a 10 cm incision along the medial line using a sterile scalpel. Next, 700 to 900 mL of sterile cold PBS was added to the cavity and mixed. The PBS was collected using a 60 mL sterile syringe and poured into several 50 mL conical centrifuge tubes on ice. Immediately the cells were centrifuged at 250 g, 4°C, 15 minutes and the supernatant was discarded. Washed peritoneal cell suspensions were adjusted to 3 × 106 cells/mL in culture medium (D-10 - DMM + 10% fetal calf serum (FCS) + L-glutamine + penicillin-streptomycin).
Obtaining the parasites
Leishmania (Leishmania) chagasi parasites (stationary-phase) were adjusted to 5 × 107 cells/mL in "phagocytosis buffer" culture medium (equal parts of Dulbecco's Modified Eagle Medium and Medium 199 supplemented with1% of BSA and 12,5 mM HEPES) [14].
The CFSE (carboxyfluorescein diacetate succinimidyl ester – Molecular Probes C-1157) dye was used as previously described [19]. Briefly, parasites were washed twice, in PBS 1,200 g, 10 minutes, 4°C. Parasites were resuspended in 1 mL of PBS (5 × 107 parasites/mL) with × nM of CFSE (using a stock of 2.8 μg/mL in DMSO) and incubated in a 37°C water bath for 10 minutes in the dark.. After this, they were washed in 10 mL of cold PBS + 10% of fetal bovine serum and centrifuged at 1,200 g. The pellet was diluted in phagocytosis buffer culture medium protected in the dark.
We have tested the viability and vitality of the CFSE-stained Leishmania, by observing their mobility under the light microscope. The fluorescence was confirmed by observing stained Leishmania using a fluorescent microscope. Dyed parasites were also used to infect mice (BALB/c) and hamsters to observe their virulence. The spleen and liver were collected after 4 weeks of infection and slides were prepared for analysis using light microscopy. The results indicated that CFSE stained parasites have an equivalent infective capacity as non-stained parasites (data not shown).
Leishmania binding assay
The interaction between CFSE-stained Leishmania-chagasi and canine peritoneal macrophages was performed in polypropylene tubes to avoid macrophage adhesion. 100 μL of macrophages (3 × 105 cells) and 100 μL of CFSE stained-Leishmania chagasi (5 × 106 cells) were combined in the tubes and maintained for 45–60 minutes at 37°C in a 5% CO2 environment.
We carried out assays in the presence of normal serum or in the presence of a final concentration of 5% of C5 deficient (serum from AKR/J mice) mouse serum [20].
Specific staining with anti-CR3 (CD11b/CD18)
Macrophages obtained as described above were stained with anti-CR3 (CD11b/CD18) rat anti-Human CD18-RPE antibody (Serotec) that shows cross reactivity with canine cells. Non-conjugated, purified Mouse anti-Canine CD11b (Serotec) conjugated using the Kit Zenon tricolor (Molecular probes – Z-25080) as described by the manufacturer with the "C" compound that represent a 647 nm emission band and can be used with CFSE and R-PE. Cells were incubated with labeled antibody solutions for 20 min at 4°C. After staining, preparations were washed with 0.1% sodium azide PBS, fixed with 200 μl of 2% formaldehyde in PBS and kept at 4°C until data were acquired using a flow cytometer (FACSVantage, Becton & Dickinson, San José, CA, USA).
Flow cytometry analysis
The cells were run on an analytical flow cytometer equipped with a laser emitting at 488 nm (FACSVantage, Becton-Dickinson, San Diego, CA, USA). Whole cells were excluded from fragments by gating based on the forward and side scatter signals. CFSE-stained promastigotes bound to macrophages were detected according to their relative fluorescence intensities using the FL1 (green) detector as compared to those of uninfected cells. Both the frequency of cells associated with Leishmania, as well as the intensity of cells associated with Leishmania was determined. Analyses were performed on 40,000 to 100,000 gated events, and numeric data were processed with WinMDI software version 2.8 (Joseph Trotter: ).
In order to confirm the intensity of macrophages binding with dyed Leishmania, we did an experimental "in vitro" assay using different macrophage/ parasite in ratios of : 1:1, 1:2, 1:5, 1:10, 1:15, 1:20 and 1:30 Macrophages:Leishmania).
Statistical analysis
Results are given as complete randomized design and the means from each group were compared using "student t test". P value less than 0.05 was considered significant.
Results
Flow cytometry was used to analyze the interaction between canine peritoneal macrophages and L. chagasi promastigotes, stained with the fluorescent dye CFSE (Fig. 1), and compared to a conventional methodology ("in vitro" binding-assays). Experiments were carried out to evaluate the frequency and intensity of Leishmania bound macrophages using gates designed to encompass the macrophage population and further identifying CFSE positive (Leishmania bound macrophages) and CFSE negative (Leishmania unbound macrophages) populations. Aliquots of CFSE-stained parasites were evaluated using the flow cytometer to analyze the efficiency of CFSE staining. More than 95% of the parasites were strongly and homogeneously stained by CFSE, and non-stained parasites were easily distinguished (Fig. 1A, 1B). To evaluate the macrophage-parasite interaction, macrophages were selected based on the forward and side scatter profile, depicted in Figure 1C (region R1), excluding lymphocytes (lower left hand population). Macrophages bound, or not, with Leishmania were easily distinguished following 1 hour of interaction using flow cytometric analysis and detection of CFSE fluorescence with the FL1 (green) detector (Fig. 1E and 1D, respectively). The Leishmania bound macrophages are identified by the M1 marker (Fig. 1E,1F), and position themselves to the right of the negative macrophages which are defined by control cultures of macrophages (Fig. 1D) in the absence of CFSE stained Leishmania. More than one population of macrophage-Leishmania conjugates are seen within the M1 marker and represent different intensities of association. The frequency and intensity of macrophages bound with Leishmania were increased when a C5 deficient mouse sera was used during the macrophage-Leishmania interaction (Fig. 1F). Free parasites present in the cultures were not detected during our analysis of infected macrophages, due to the fact that they are gated out of the analysis region based on size and granularity which puts them in the extreme lower left region of a FSC vs. SSC plot (data not shown).
Figure 1 The use of CFSE stained- L. chagasi to evaluate parasite-canine macrophage interaction. L. chagasi promastigotes were stained with the intracellular dye CFSE and the efficiency of the staining procedure evaluated trough flow cytometry (A) and conventional fluorescence microscopy (B). CFSE-stained parasites were used for in vitro infection of peritoneal dog macrophages (X to Y ratio) and the parasite-macrophage interaction was evaluated through flow cytometry. Macrophages were selected based on forward- versus side-scatter parameters (C) and the frequency and intensity of CFSE+ macrophages was evaluated (D to F – MI Marker). At least 30,000 events were collected. (D) Non-infected macrophages. (E) Macrophages infected by CFSE-stained parasites in absence of C5 deficient sera. (F) Macrophages infected by CFSE-stained parasites in presence of C5 deficient sera. Data represents the mean ± SE of the frequency of infected macrophages and the CFSE fluorescence intensity of infected macrophages, obtained for one dog, representing identical experiments of four dogs.
Simultaneously, Leishmania-binding assays were carried out using 24-well plastic plates covered with coverslips, previously described by others [13,14]. Through microscopic morphologic analysis of the cells, the populations harvested from the canine peritoneum was found to be composed of mostly macrophages, monocytes and neutrophils (Fig. 2). Using a Leishmania-binding assay (Fig. 3 and 4) it was determined that macrophages show parasite binding after 1 hour of macrophage-Leishmania interaction and that the use of C5 deficient sera (serum dependent system) markedly increased the parasite burden. Serum dependent (SD) "in vitro" binding-assays showed an average of 2.43:1 (Leishmania:macrophage) in contrast to the serum independent (SI) assays which showed a lower average of 1.7:1. In addition, the numbers of macrophages bounded to Leishmania were higher in the SD assays than in the SI assays as seen by 37.41% and 11.41% macrophages bound to Leishmania, respectively. Finally, the increased interaction of Leishmania with macrophages when using SD as compared to SI conditions was also seen by flow cytometric analysis of the mean fluorescent intensity (MFI) and % bound macrophages (Fig. 7 and 8). Both MFI and percent macrophages bound with Leishmania were higher under the SD conditions (Fig. 7 and 8).
Figure 2 Cells from peritoneal lavage adhered to coverslips. The image shows the predominance of macrophages (arrow) and neutrophils (arrow head). Giemsa stain – 400×.
Figure 3 Conventional Binding assay showing the interaction of macrophages from one dog with several Leishmaniae. Giemsa stain – 400×.
Figure 4 Conventional Binding assay showing the interaction of one macrophage with several Leishmaniae. Giemsa stain – 1000× optical microscope – 2× digitalized image.
Figure 7 Serum dependent (SD) conditions induce a higher association of Leishmania with macrophages as measured by both mean fluorescent intensity and percent macrophages associated with Leishmania. This figure shows the MFI of serum independent SI and SD. analysis. n = 4 animals. * = p < 0.05.
Figure 8 Percent of CFSE positive macrophages from SI and SD cultures. Canine macrophages were incubated for 1 hour with CFSE labeled Leishmania and analyzed using flow cytometry as described in Materials and Methods. n = 4 animals. * = p < 0.05.
Next to determine if increased numbers of CFSE-stained parasites would lead to a corresponding increase in the intensity of macrophage/parasite-CFSE pairs, a number of ratios of macrophage to parasite combinations were studied. As seen in Figure 5, an increased parasite/macrophage ratio leads to an increase in the mean fluorescent intensity (MFI) of the positive macrophage population.
Figure 5 Increased parasite/macrophage ratios leads to an increased intensity of macrophages associated with CFSE marked parasites. The intensity of fluorescence increases in accordace with the number of parasites per macrophage used. Macrophages and parasites were combined as described in Materials and Methods and analyzed for fluorescent intensity using flow cytometry. The data represent the mean flourescent intensity of posive macrophage populations from cultures at each ratio studied. These studies are representative of 4 experiments.
Evaluation of CR3 expression using flow cytometry
The expression of CR3 (CD11b/CD18) by peritoneal macrophages was determined using flow cytometry before and after interaction with Leishmania under both the SD and SI conditions (Figure 6).
Figure 6 The levels of CD11b and CD18 fall dramatically following incubation with Leishmania under serum dependent (SD) culture conditions. Peritoneal macrophages were selected based on forward- versus side-scatter parameters as described in Materials and Methods and Figure 1 using flow cytometry. A) Isotype control with macrophages showing delineation of the M1 marker based on the negative population. B and C represent expression of CD18 (B) or CD11b (C) before incubation with Leishmania. D and E represent expression of CD18 (D) or CD11b (E) after association with Leishmania in a SD assay. In all cases the percent positive cells within the M1 marker are identified. This Figure is a representative diagram of 4 experiments with similar findings.
There was no significant difference in the percent cells expressing CD11b versus those expressing CD18 amongst all the samples analyzed (Fig. 9). Interestingly, upon interaction with Leishmania, the frequency of macrophages expressing CD11b or CD18 dropped significantly in both the SI and SD conditions (Fig. 10 and 11). However, no difference in the expression of CD11b/CD18 was seen between the SI and SD conditions (data not shown).
Figure 9 Percent peritoneal cells expressing CR3 as determined by expression of CD11b and CD18. Peritoneal cells from 4 dogs were stained ex vivo and analyzed using flow cytometry as described in Materials and Methods.
Figure 10 The expression of both CD11b and CD18 is dramatically reduced following association of peritoneal macrophages with Leishmania in both assays (SI or SD). Percent of CD11b expressing macrophages before and after incubation with Leishmania (SD assay).
Figure 11 The expression of both CD11b and CD18 is dramatically reduced following association of peritoneal macrophages with Leishmania in both assays (SI or SD). Percent of CD18 expressing macrophages before and after incubation with Leishmania (SD assay). Macrophages were incubated with or without Leishmania for one hour and stained with the appropriate antibody followed by analysis using flow cytometry as described in Materials and Methods. n = 4 animals, and * p < 0.05.
Discussion
American Visceral Leishmaniasis (AVL) is a zoonosis with dogs representing the principal domestic reservoir of the disease. AVL is present in at least a dozen Latin American countries, with 90% of human cases reported from Brazil, especially the north-eastern region [1-3,18,21,22]. Canine Visceral Leishmaniasis (CVL) appears to be spreading further in Brazil, and outbreaks have occurred in major Brazilian cities in the northeast of the country, such as Teresina, (PI), São Luiz (Maranhão), Fortaleza (Ceará), Salvador (Bahia), [23,24] and also in the southeast as in Belo Horizonte, [25,26] and Rio de Janeiro (RJ) [18]. These urban or predominantly peri-urban outbreaks appear to be increasing, but historically they are cyclical and recur following several year intervals of low endemicity. Domestic dogs have been the target of intense research concerning leishmaniasis with hopes of improving control of infected animals, and due to findings that show common clinical, immunological and pathologic aspects with human visceral disease. [27-34].
Macrophages are the main cells involved in the pathogenesis of the disease and they represent an ideal target cell population for "in vitro"studies. However, data concerning the behaviour of canine macrophages from naturally or experimentally infected animals with Leishmania chagasi, "in vitro", are limited. In this work we have demonstrated a reliable and practical method to quantify the frequency and intensity of host cells bound with Leishmania. This methodology is less laborious and more quantitative than cell-cultures techniques, removing the disadvantages of subjectivity and the time-consuming nature of microscopy when establishing parasitism ratios.
Our results, using the conventional Leishmania-binding assay, have confirmed previous work in the literature [35] where it has been demonstrated that the number of parasites bound to macrophages was dramatically increased when using serum containing complement. Importantly, these same results were obtained using analysis by flow cytometry with CFSE stained Leishmania. Moreover, a strong correlation was seen between both conventional microscopy based methods and our flow cytometry based approach.
In addition, this methodology permits the simultaneous use of conventional flow cytometry analysis to quantify the expression of membrane receptors related to Leishmania-macrophage interactions. For example, we have performed assays to determine the expression of CD11b/CD18 (CR3) [14,36] integrins involved in the interaction of Leishmania promastigotes with canine macrophages. Moreover, the MFI of macrophages associated with Leishmania increased with higher parasite/macrophage ratios, indicating a direct correlation between the number of parasites and the intensity of interaction with the host macrophage. Finally, other phenotypic markers can be used to clearly characterize the infected population and to describe functional characteristics of cell subpopulations through the analysis of cytokines and activation markers.
Our results have shown that CR3 (CD11b/CD18) receptor expression is lower in the presence of promastigote forms of Leishmania. Since we have performed binding-assays (not infection assays) we can consider that these receptors are either occupied by Leishmania binding, or that the receptor complexes have been internalized. Future "in vitro" studies are being carried out in our laboratory to resolve this question.
Several studies have been designed to study the interaction of parasites and macrophages. The majority used dead microorganisms (bacteria or fungi), inert or fluorescent particles (latex/polystyrene beads or zimozan), lamb's hematia, or finally, microorganisms labelled with antibodies [37]. The use of antibody labelled-living L. amazonensis was shown by Bertho et al (1992) as a poor method, because labelling antibodies were shed too quickly [38]. We have found a staining method using living-parasites that can be used without interfering with the interaction between macrophages and parasites, and it represents a stable association of the fluorescence with the parasite. The stability of the CFSE staining has been largely employed in other studies using living cells [39-42], and was recently used for staining and tracking the association of T. cruzi with host cells as well [43].
While CFSE is typically used in proliferation assays [44], our novel studies have defined another use for this marker to analyze the interaction between a parasite and host cells.
Conclusion
In this work we propose a method for the study of the interaction between live Leishmania and macrophages using flow cytometry. Moreover, we have also demonstrated a system that can be useful for studies of the interaction of L. chagasi with canine peritoneal macrophages. These techniques open the door to many future studies designed to further investigate the interaction of Leishmania with host cell populations. Finally, CFSE stained Leishmania have been shown to be stable for many days, and could be used to study phagocytosis or Leishmania-survival.
Competing interests
The author(s) declare that they have no competing interests
Authors' contributions
RG conceived and designed the study, carried out the assay development, data analysis, and wrote the first draft of manuscript. ER participated in cytometry assay, cytometry analysis and critical review of the manuscript. MNM provided the parasite Leishmania (Leishmania) chagasi. KJG provided expert input for cytometry assay, critical review of manuscript and supervised the cytometry study. DMM provided expert critical review of manuscript. WLT carried out the Leishmania binding assay, provided expert input for writing and supervised the study. All authors have read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Doctor Alan Lane de Melo for gently providing the C5 deficient mice (AKR/J). - Laboratório de taxonomia e biologia de invertebrados do Departamento de Parasitologia da UFMG. This work was supported by Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG grant CDS2263/97) and Consellho Nacional de Desenvolvimento da Pesquisa Tecnológica e Científica - (grant CNPq 472287/01-0 – NV), and CT-INFRA-FINEP, Brazil. This investigation also received financial support from the UNICEF/UNDP/World bank/WHO Special Program for Research and Training in Tropical Diseases (TDR).
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| 15913461 | PMC1166554 | CC BY | 2021-01-04 16:28:16 | no | BMC Infect Dis. 2005 May 24; 5:39 | utf-8 | BMC Infect Dis | 2,005 | 10.1186/1471-2334-5-39 | oa_comm |
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BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-5-451594903610.1186/1471-2334-5-45Research ArticleThe therapeutic effect of the neuropeptide hormone somatostatin on Schistosoma mansoni caused liver fibrosis Chatterjee Shyama [email protected] Gunther [email protected] Inge [email protected] Theo [email protected] Marck Eric [email protected] Laboratory of Pathology, Faculty of Medicine, University of Antwerp, Universiteitsplein-1, B-2610 Antwerp, Belgium2 Laboratory of Gastrointestinal Hormones, Gasthuisberg, Leuven, Belgium2005 10 6 2005 5 45 45 30 12 2004 10 6 2005 Copyright © 2005 Chatterjee et al; licensee BioMed Central Ltd.2005Chatterjee et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The neuropeptide somatostatin is one of the major regulatory peptides in the central nervous system and the digestive tract. Our recent work has delineated an association between fibrosis and low levels of endogenous somatostatin plasma levels in Schistosoma mansoni infected subjects. Based on these results this paper explores the therapeutic potential of somatostatin in a mouse model of hepatic fibrosis associated with S. mansoni infections.
Methods
Groups of outbred Swiss mice were infected with 100 S. mansoni cercariae, infection maintained till weeks 10 or 14, and then somatostatin therapy delivered in two regimens – Either a one or a two-day treatment. All animals were sacrificed one week after therapy and controlled for liver, spleen and total body weight. Circulating somatostatin levels in mice plasma were measured at the time of sacrifice by means of a radio-immuno assay. GraphPad Prism® was used for statistical calculations.
Results
Somatostatin administration showed little toxicity, probably due to its short half-life. Total liver and spleen weights of S. mansoni infected animals increased over time, with no changes observed due to somatostatin therapy. Total body weights were decreased after infection but were not affected by somatostatin therapy. Snap frozen liver sections were stained with haematoxylin-eosin or Masson's trichrome to study parasite count, hepatocyte status, granuloma size and cellularity. After somatostatin treatment mean egg counts per liver section (43.76 ± 3.56) were significantly reduced as compared to the egg counts in untreated mice after 10 weeks of infection (56.01 ± 3.34) (P = 0.03). Similar significant reduction in parasite egg counts were also observed after somatostatin treatment at 14 weeks of infection (56.62 ± 3.02) as compared to untreated animals (69.82 ± 2.77)(P = 0.006). Fibrosis was assessed from the spectrophotometric determination of tissue hydroxyproline. Infection with S. mansoni caused increased hydroxyproline levels (9.37 ± 0.63 μmol at wk10; 9.65 ± 0.96 μmol at wk14) as compared to uninfected animals (1.06 ± 0.10 μmol). This significant increase in collagen content (P = 0.01; 0.007 respectively) marks the fibrosis observed at these time points. Treatment with somatostatin resulted in a significant decrease in hydroxyproline levels both at wk10 (4.76 ± 0.58 μmol) and at wk14 (5.8 ± 1.13 μmol) (P = 0.01; 0.03 respectively). Endogenous somatostatin levels were increased at wk10 (297 ± 37.24 pg/ml) and wk14 (206 ± 13.30 pg/ml) of infection as compared to uninfected mice (119 ± 11.99 pg/ml) (P = 0.01; 0.008 respectively). Circulating somatostatin levels in infected animals were not significantly affected by somatostatin treatment. Hepatocyte status remained unaltered and granulomas were not remarkably changed in size or cellularity.
Conclusion
Our experiments reveal an antifibrotic effect of somatostatin in schistosomiasis. We have previously shown that the somatostatin receptors SSTR2 and SSTR3 are present on the parasite egg and worms. We therefore hypothesize that somatostatin reduces either the number of parasite eggs or the secretion of fibrosis inducing-mediators. Our data suggest somatostatin may have therapeutic potential in S. mansoni mediated liver pathology.
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Background
Somatostatin (SOM) or somatostatin-14, a 14 amino acid peptide hormone was originally isolated from the hypothalamus. It was soon after reported in the central nervous system (CNS), the stomach, gastrointestinal (GI) tract and pancreas. By 1980, the gene coding for prosomatostatin had been identified and two molecular forms (SOM-14 & SOM-28) were reported. As the five specific receptors for somatostatin (SSTR1-SSTR5) belonging to the G protein-coupled receptor family were identified, it became clear that the different molecular forms and various receptors are part of a complicated control system that regulate a variety of body functions. Somatostatin exerts profound inhibitory functions not only on growth hormone secretion but also on many GI functions. These different aspects form the basis for the therapeutic potential of somatostatin in various diseases [1].
Somatostatin alone or in combination with endotherapy has been successfully used in the management of bleeding esophageal varices (BOV) [2]. Following an acute bleeding episode somatostatin is administered over a period of 5 days and acts by reducing the splanchnic blood flow. Somatostatin is useful in reducing peptide ulcer bleeding and the lack of recurrence [3]. More recently, somatostatin has also shown proven benefits in the management of pancreatic disorders, such as pancreatic fistulae, acute pancreatitis and the prevention of complications following pancreatic surgery [4-6]. Somatostatin is well tolerated, with very few side effects.
Complications resulting from hepatic fibrosis are the principal cause of death in Schistosoma mansoni infected patients [7]. In such patients portal hypertension leads to the formation of gastro-esophageal collaterals (varices). Fatal bleeding of these esophageal varices can occur depending on the severity of fibrosis. We have previously shown an association between severe hepatic fibrosis and low levels of endogenous somatostatin [1]. Taking these reports into consideration, our present study was aimed to test the therapeutic capacity of exogenously administered somatostatin on S. mansoni caused liver pathology.
In schistosomiasis the principal inflammatory response is directed against the parasite eggs some of which enter the portal circulation, become lodged in hepatic portal venules initiating a granulomatous response. During periovular granuloma formation fibronectin produced by macrophages is deposited around the inflammatory cells [8,9] composing a large part of the extracellular matrix. This is followed by the deposition of the proteoglycans dermatan sulphate, to a lesser extent heparan sulphate [10], and the interstitial collagen types I & III that all together form the fibrotic tissue.
Collagen is composed of three chains, wound together in a tight triple helix (Fig. 1.). A repeated sequence of three amino acids forms this sturdy structure. Every third amino acid is glycine, a small amino acid that fits perfectly inside the helix. Many of the remaining positions in the chain are filled by two unexpected amino acids: proline and a modified version of proline, hydroxyproline. The glycine forms a tiny elbow packed inside the helix, and the proline and hydroxyproline smoothly bend the chain back around the helix.
Figure 1 Structure of collagen triple helix.
Our recent studies have elucidated the presence of somatostatin receptors (SSTR2A, mostly associated with inflammatory cells, and receptor SSTR3 expressed in the liver) on S. mansoni egg and worm stages (manuscript submitted). Soluble egg antigen (SEA) secreted by the egg stage parasite in the liver triggers inflammation and fibrosis [11]. Circulating host somatostatin levels might inhibit SEA production via interaction with the SSTR receptors on the parasite surface, thereby regulating the level of liver fibrosis and as a result portal hypertension, variceal bleeding and fatality due to this disease. In related studies the group of Reynaert [12] have shown that the presence of SSTRs 1, 2 and 3 on hepatic stellate cells could be used as therapeutic targets for reducing fibrosis in chronic liver disease because treatment with somatostatin decreased collagen I and III and alpha-SMA synthesis.
Based on these reports we presented the hypothesis that exogenous administration of somatostatin could alleviate the pathology caused by schistosomiasis. To prove this we tested the effect of somatostatin treatment on collagen deposition in the liver of Schistosoma mansoni infected Swiss mice as compared to control uninfected or untreated mice. We have on several occasions stressed the therapeutic potential of somatostatin on S. mansoni caused liver fibrosis [13]. Our results may indicate an endogenous function of the somatostatin receptors on the parasite surface and point to their role in host-parasite interactions.
Methods
Pharmacokinetic profile of somatostatin
Separate groups of male Swiss mice were injected with 25 μg of somatostatin (somatostatin-ucb®, UCB Pharma, Brussels) intraperitoneally (in the abdomen) or intravenously (in the caudal vein in the tail). At defined moments after injection (10, 20, 40, 60 minutes) the animals were anaesthetized with Nembutal® (60 mg/kg). The thoracic cavity was cut open and blood collected from the right ventricle of the heart into chilled syringes containing EDTA (1 mg/ml) and Aprotinin (500 KIU/ml blood). The collected blood was centrifuged at 3000 rpm for 15 minutes at 0°C. The plasma was immediately frozen at -80°C freezer. Untreated naïve mice were also bled to ascertain background levels of somatostatin.
Schistosoma mansoni infection
The maintenance of the S. mansoni life cycle and the transcutaneous infection of mice with S. mansoni have been previously described [14]. Male Swiss mice (age 7 weeks) were anaesthetized with Nembutal® (60 mg/kg) and the abdomen was shaved. A metal ring was placed on the abdomen and then filled with treated water containing infectious cercariae of a Puerto Rican strain of S. mansoni. The cercariae were allowed to penetrate during 20 minutes after which the water was removed and checked for remaining cercariae.
Experimental setup
Groups of Swiss mice were infected with 100 S. mansoni cercariae each as mentioned above. Age matched mice were maintained as uninfected control animals. Groups of 10 mice composed Group 1 that were maintained till 10 weeks following infection, other 10 mice composed Group 2 that were maintained till 14 weeks of infection.
At such times animals of Groups 1 and 2 were treated with somatostatin (Somatostatine-ucb®), that was kindly gifted by UCB Pharma, Brussels. Somatostatin was administered in two regimens – a one-day treatment or a two-day treatment. One day treatment consisted of 3 doses of 30 μg somatostatin each, two day treatment was the double of this dose, that is 6 doses of 30 μg somatostatin to each animal. Somatostatin was administered intravenously via the caudal vein in the tail and intraperitoneally at regular intervals during the day. One week after the last somatostatin administration, mice were killed, the weight of the animal, liver and spleen were noted, plasma was extracted and stored as mentioned above, and the liver frozen immediately in liquid nitrogen and then at -80°C. The overall planning is depicted in Table 1. Untreated animals of the acute and chronic stage were also sacrificed at the respective times together with their treated counterparts. Research protocols involving rodents received ethical clearance by the University of Antwerp ethical committee.
Table 1 The experimental planning of our study is depicted.
Criteria Gp 1 Gp 1 Gp 1 Gp 2 Gp 2 Gp 2
Time of SOM treatment Acute (wk. 10) 1 day treatment Acute (wk. 10) 2 days treatment Acute (wk. 10) Untreated Chronic (wk. 14) 1 day treatment Chronic (wk. 14) 2 days treatment Chronic (wk. 14) Untreated
No. doses 3 × 30 μg (2 × IP; 1 × IV) 6 × 30 μg (4 × IP; 2 × IV) NA 3 × 30 μg (2 × IP; 1 × IV) 6 × 30 μg (4 × IP; 2 × IV) NA
Total SOM 90 μg 180 μg NA 90 μg 180 μg NA
NA: not applicable; SOM: somatostatin®; Wk.: week; IP: Intraperitoneal; IV: Intravenous; No.: number.
Parasite egg count and hepatocyte status after somatostatin treatment
All animals of Groups I and II were controlled for liver, spleen and total body weight, before sacrifice. The adult worms were recovered from the hepatic portal system and the liver by perfusion with citrate saline (0.85% sodium chloride; 1.5% sodium citrate). The livers were cut out and snap frozen in liquid nitrogen. For cyosectioning, liver fragments were embedded in Tissue-Tek OCT compound, 4 μm thick transverse sections were cut on a cryostat, mounted on slides coated with 0.1% ploy-L-lysine and stored at -20°C until use. To study parasite egg count, hepatocyte status, granuloma size and cellularity, series of sections were stained with Haematoxylin-eosin stain.
Hydroxyproline determination
The collagen concentration was determined by assessing hydroxyproline amount. Herein is described the protocol of Technique B for the biochemical assessment of fibrosis used by Bergman and Loxley [15]. Just as was done by Cheever [16] we neutralized our samples for the color reaction.
Reagents used
1. Dowex/Norit: Twenty mg of Dowex (Sigma, St. Louis, Missouri, US) and 10 mg Norit A (Merck, Darmstadt, Germany) were mixed together in 200 ml of 6N HCl. The Dowex/Norit mixture was recovered with the help of a vacuum pump and thereafter washed thrice more with 6N HCl. The residue was rewashed with 95% ethanol and 100 % ethanol, and the powder dried over several days.
2. Solution A: One part 7% chloramine T-solution (Sigma) was mixed with 4 parts of citrate/acetate buffer (pH 6.0: 57 g sodium acetate.3H2O (Merck) + 37.5 g Na3citrate.2H2O (Sigma) + 5.03 g Citrate + 385 ml isopropanol made up to 1 liter with distilled water).
3. Solution B: Composed of 3 parts of Ehrlichs reagent (25 g p-dimethyl amino benzal dehyde (Sigma) dissolved in 37.5 ml 60% perchloric acid) and 13 parts isopropanol.
4. Hydroxyproline standard solution: 6.56 mg hydroxyproline standard (Merck) dissolved in 500 ml gave a 0.1 mM hydroxyproline-solution. This solution remains stable at 4°C thereby enabling us to set up a standard curve of known hydroxyproline values.
Hydrolysis of liver
For the measurement of the hydroxyproline content in the liver, about 200 mg of liver was treated with 5 ml of 6N HCl for 18 hours at 110°C. This acidic hydrolysis breaks down the collagen to individual amino acids. Remaining undissolved matter was removed by adding 40 mg Dowex/Norit in 5 ml of distilled water. After centrifugation for 15 minutes at 2000 rpm, the supernatant was filtered with the aid of 0.22 μm millipore filters (Millipore S.A., Molsheim, France).
Neutralization
Two ml of hydrolysate was pipetted out to which was added 1 drop (40 μl) of 1% phenolphthalein. When the solution became colorless, 10 N NaOH was added drop wise till the color changed to purple red. Return titration was done with 5 μl drops of a 3N HCl solution, till all red color was lost. The total volume was next restored to 4 ml with distilled water and the solution kept stable at 4°C.
Color reaction
Starting from this step we used a series of standard hydroxyproline concentrations made from 0-25-50-75-100 μmol/l. (200 μl/test tube). From the test sample above 200 μl was placed in a separate test tube. After vortexing 200 μl test sample/200 μl standard mixed together with 400 μl of isopropanol, 200 μl of solution A (chloramine T/citrate-acetate buffer) was added that provided an optimal binding between color and tissue. This reaction needed at least 4 minutes to work after which 2.5 ml of solution B was added and the contents well mixed. The tubes were covered with aluminum foil and incubated for 25 minutes in a warm water bath maintained at 60°C. To stop the reaction the test tubes were cooled in cold water for 3 minutes.
Measurement
Within 30 minutes, the absorbance for each sample was measured in an Ultrospec 3000 UV/Visible Spectrofotometer at a wavelength of 558 nm.
Measurement of somatostatin levels in plasma
The measurement of somatostatin concentrations in the Swiss mice plasma was carried out in the laboratory of Gastro-intestinal Hormones, at Gasthuisberg, K.U. Leuven, by means of a radio-immuno assay (RIA). The RIA was performed by incubating the samples with 1.7 pM 3-[125I] iodotyrosyl11 somatostatin-14 (specific activity 2000 Ci/mmol, Amersham Pharmacia Biotech, Buckinghamshire, UK) and a rabbit antibody against human SOM [1-14] in a 50 mM sodium phosphate buffer (pH 7.4, 0.25% EDTA, 0.5% charcoal-BSA, 500 U/ml Trasylol) for at least two days at 4°C. At the end of the incubation period the SOM bound to the antibody was separated from the free SOM by adding 500 μl dextran-charcoal followed by centrifugation for 15 min at 3000 rpm. Both fractions were counted in a gamma counter and the results were read from a standard curve (0–250 pg/ml) included in the RIA. The minimal detectable dose was 2.5 pg/ml.
Results
Toxicity
Somatostatin is a highly purified compound reflected by the fact that it has very little toxicity. Studies in mice have shown that the LD50 is comparable to 10,000 times the acute therapeutic dose used in humans. The favorable metabolic profile of somatostatin is further supported by the fact that this compound has a very short half-life, and thus any undesirable effects may be rapidly reversed. In our experiment we have used 90 μg/24 hours in the Swiss mice that weighs about 40 g. In humans the therapeutic dose is 3.5 μg/kg/hour or 6 mg/24 h for a 75 kg man. A comparison of these values tells us that we are working with about 100 times higher values in mice as compared to that used in humans.
Pharmacokinetic profile of somatostatin in outbred mice
Following an intravenous infusion of a therapeutic dose of somatostatin (250 μg/hour or 6 mg/24 hours) to healthy volunteers, the plasma profile demonstrated that the drug reaches a plateau of 300–3000 pg/ml within 15 minutes. Somatostatin has a very short half-life of 1–3 minutes in man. In animal studies the same profile has been observed in dogs, whereas in rats somatostatin is stable for up to 30 minutes in whole blood, indicating that it is broken down in tissues [17]. To determine the pharmacokinetic profile in outbred Swiss mice, 25 μg of somatostatin was administered per mouse via the intraperitoneal or intravenous route. To assess the break down in these mice, plasma was collected at regular time intervals and circulating somatostatin levels assayed (Figure 2). At 10 minutes post injection somatostatin levels fall to 30233 pg/ml (IP) and 17413 pg/ml (IV). After intravenous injection thus somatostatin appears to be more rapidly degraded than after intraperitoneal injection, however after one hour of injection using either means the levels of circulating somatostatin in blood reaches baseline control levels. Thus in our experimental set up of somatostatin treatment in S. mansoni infected mice the schedule of 3 treatments per day (morning, noon and evening), assured that for at least 3 hours per day there were significantly high levels of circulating somatostatin in vivo.
Figure 2 Pharmacokinetic profile of somatostatin in Swiss mice. Figure depicts the breakdown of somatostatin in vivo after intraperitoneal and intravenous administrations. Values were noted after 10, 20, 40 and 60 minutes and compared to a control situation where no somatostatin was administered.
Body and Organ weight after somatostatin treatment
The weight of the liver increased with infection, with a significant rise at the acute (p = 0.01) and chronic stage (p = 0.01) as compared to the uninfected animals (Fig. 3.). Acute and chronic infected animals after 2 days of somatostatin treatment showed no significant change in their liver weights (p = 0.19; 0.90 respectively).
Figure 3 Variations in liver weight after infection and somatostatin treatment. Figure shows the variations in mice liver weights after S. mansoni infection and/or somatostatin treatment. Results are depicted as a box and whiskers plot starting from the left – uninfected mice, uninfected mice with 2 days treatment, 10 weeks infected mice, 10 weeks infected mice with 1 day treatment, 10 weeks infected mice with 2 days treatment, 14 weeks infected mice, 14 weeks infected mice with 1 day treatment, 14 weeks infected mice with 2 days treatment. Values represent the 5th, 25th, 50th, 75th and 95th percentiles.
Mean spleen weights were significantly increased in acute (p = 0.01) and chronic (p = 0.01) infected animals as compared to uninfected controls (Fig. 4.). Acute and chronic infected animals after 2 days somatostatin treatment showed no significant changes in their spleen weights (p = 0.28; 0.73 respectively).
Figure 4 Variations in spleen weight after infection and somatostatin treatment. Figure shows the variations in spleen weight after S. mansoni infection and/or somatostatin treatment. Results are depicted as a box and whiskers plot as mentioned in Fig. 3.
The total body weight of the acute infected animals showed a decrease as compared to uninfected animals however this difference was borderline significant (p = 0.06) (Fig. 5). There were no significant changes in body weight in acute or chronic infected animals after somatostatin treatment.
Figure 5 Variations in total body weight after infection and somatostatin treatment. Figure shows the variations in total body weight after S. mansoni infection and/or somatostatin treatment. Results are depicted as a box and whiskers plot as mentioned in Fig. 3.
Parasite egg count after somatostatin treatment, hepatocyte, granuloma and fibrosis status
S. mansoni egg stage parasites were counted in liver sections before and after somatostatin treatment. A series of 10 slides were examined from each sub group, the sections obtained at intervals of at least 40 μm so as to avoid counting repetitive eggs. Two days of somatostatin treatment in acute infected animals significantly reduced the mean parasite egg counts form 56.01 ± 3.34 to 43.76 ± 3.56 (p = 0.03). At the chronic stage of infection, mean egg counts were only significantly (p = 0.006) reduced after 1 day of somatostatin treatment from69.82 ± 2.77 to 56.62 ± 3.02 (Fig. 6.).
Figure 6 Variations in parasite egg count after somatostatin treatment. Figure shows the variations in parasite egg count after somatostatin treatment. Box and whiskers plot depicting the 5th, 25th, 50th, 75th and 95th percentiles represent egg counts at 10 weeks of infection, variations after 1 day of treatment or 2 days of treatment, egg counts at 14 weeks of infection, variations after 1 day or 2 days of treatment.
Haematoxylin-eosin staining of liver sections displayed the evolution of granuloma formation in infected animals as compared to treated animals. After treatment with somatostatin hepatocyte status remained unaltered, granulomas were not remarkably changed in size or cellularity (results not shown).
Hydroxyproline determination
Hepatic fibrosis is the process of excessive deposition of collagen in the liver. Collagen generation involves 3 polypeptide chains, each chain composed of 19 amino acids consisting of glycine (30%), proline (12%) and two other rather uncommon amino acids – hydroxyproline (10%) and to an even lesser extent hydroxylysine. Proline and hydroxyproline are responsible for the angle (kink) in the polypeptide backbone furthermore hydroxyproline stabilizes the collagen via intramolecular hydroxyl bridges. Normal hydroxylated collagen is equivalent to 10% hydroxyproline in weight. Thus 1 μmol hydroxyproline (MW = 131.13) is equivalent to 1.3113 mg collagen. Hydroxyproline concentrations in the livers (controls versus acute, chronic, treated and untreated) of the Swiss mice are depicted in Table 2 and figure 7. The differences in hydroxyproline concentrations between uninfected and infected mice with or without treatment are illustrated by non-parametric (Mann-Whitney U) or parametric (unpaired Student's t) test as shown in Table 2.
Table 2 The hydroxyproline values calculated in the different groups.
Group 1 Group 2 Group 1 vs. Group 2
No SOM treatment Uninf.: 1.06 ± 0.10
Acute: 9.37 ± 0.63
p-value: 0.01** Chronic: 9.65 ± 0.96 Uninf.: 1.06 ± 0.10
Chronic: 9.65 ± 0.96
p-value: 0.007**
Acute: 9.37 ± 0.63
Chronic: 9.65 ± 0.96
p-value: 0.90 (NS)
1 day SOM treatment Acute: 9.37 ± 1.24 Uninf.: 0.85 ± 0.08
Chronic: 8.99 ± 0.61
p-value: <0.0001** Acute: 9.37 ± 1.24
Chronic: 8.99 ± 0.61
p-value: 0.90 (NS)
2 day SOM treatment Uninf.: 0.81 ± 0.09
Acute: 4.76 ± 0.58
p-value: 0.007**
Acute (untreated): 9.37 ± 0.63
Acute (2d SOM): 4.76 ± 0.58
p-value: 0.01** Uninf.: 0.93 ± 0.09
Chronic: 5.88 ± 1.13
p-value: 0.02*
Chronic (untreated): 9.65 ± 0.96
Chronic (2d SOM): 5.8 ± 1.13
p-value: 0.03* Uninf. (17 wks.): 0.81 ± 0.09
Uninf. (21 wks.): 0.93 ± 0.09
p-value: 0.41 (NS)
Acute: 4.76 ± 0.58
Chronic: 5.88 ± 1.13
p-value: 0.41 (NS)
SOM: somatostatin; Uninf.: uninfected; Inf.: infected; Wks.: weeks;
*Significant difference (p < 0.05); ** significant difference (p < 0.01);
NS: not significant difference
Figure 7 Hydroxyproline levels after somatostatin treatment. Figure depicts the hydroxyproline levels after S. mansoni infection and/or somatostatin treatment. Values are represented as a box and whiskers plot as described in Fig. 3.
At 10 weeks post infection with 100 S. mansoni cercariae, levels of hydroxyproline were significantly higher as compared to that assayed in uninfected animals (p = 0.01) (Fig. 7.). After treatment with somatostatin for 2 days, circulating levels of this collagen at one-week post treatment time were significantly lower as compared to infected animals that were not treated (p = 0.01).
Progression of the infection from week 10 to week 14 did not result in a significant rise in hydroxyproline levels. Treatment of chronically infected animals with a two-day regimen of exogenous somatostatin resulted in a drop in hydroxyproline levels at one-week post treatment. This fall was significant (p = 0.03) as compared to chronic untreated animals.
Somatostatin levels
Somatostatin concentrations in the plasma (controls versus acute, chronic, treated and untreated) of the Swiss mice are depicted in Table 3 and figure 8. The differences in somatostatin concentrations between uninfected and infected mice with or without treatment were illustrated by a non-parametric (Mann-Whitney U) test as shown in Table 3. The distribution of somatostatin levels in all groups is depicted in Figure 8. At 10 weeks post infection with 100 S. mansoni cercariae, levels of circulating somatostatin were significantly higher as compared to that assayed in uninfected animals (p = 0.01). After treatment with somatostatin for 1 or 2 days, circulating levels of this neuropeptide at one week post treatment time were not significantly different as compared to infected animals that were not treated.
Table 3 The somatostatin levels calculated in the different groups.
Group 1 Group 2 Group 1 vs. Group 2
No SOM treatment Uninf.: 119.4 ± 11.99
Acute: 297.3 ± 37.24
p-value: 0.01** Chronic: 206.1 ± 13.30 Uninf.: 119.4 ± 11.99
Chronic: 206.1 ± 13.30
p-value: 0.008**
Acute: 297.3 ± 37.24
Chronic: 206.1 ± 13.30
p-value: 0.06 (BL)
1 day SOM treatment Acute: 240.3 ± 21.30 Uninf.: 131.6 ± 19.34
Chronic: 226.3 ± 5.41
p-value: 0.01** Acute: 240.3 ± 21.30
Chronic: 226.3 ± 5.41
p-value: 0.71 (NS)
2 day SOM treatment Uninf.: 136.9 ± 11.69
Acute: 234.2 ± 23.65
p-value: 0.008**
Acute (untreated): 297.3 ± 37.24
Acute (2d SOM): 234.2 ± 23.65
p-value: 0.28 Uninf.: 151.6 ± 41.45
Chronic: 247.9 ± 13.11
p-value: 0.11 (NS)
Chronic (untreated): 206.1 ± 13.30
Chronic (2d SOM): 247.9 ± 13.11
p-value: 0.06 (BL) Uninf. (17 wks.): 136.9 ± 11.69
Uninf. (21 wks.): 151.6 ± 41.45
p-value: 0.9 (NS)
Acute: 234.2 ± 23.65
Chronic: 247.9 ± 13.11
p-value: 0.73 (NS)
SOM: somatostatin; Uninf.: uninfected; Inf.: infected; Wks.: weeks;
* Significant difference (p < 0.05); ** significant difference (p < 0.01); BL: borderline significant difference; NS: not significant difference
Figure 8 Somatostatin levels after infection and therapy. Figure shows a box and whiskers plot depicting somatostatin values after S. mansoni infection and/or somatostatin therapy. Results are described as in Fig. 3.
Progression of the infection from acute (week 10) to chronic (week 14) stages resulted in a drop in the somatostatin levels. Comparison of somatostatin levels between these two time points depicted borderline significant differences (p = 0.06). Treatment of chronically but not of acute infected animals with a two-day regimen of exogenous somatostatin resulted in an elevated level of circulating hormone at one-week post treatment. This rise in circulating somatostatin level was borderline significant (p = 0.06) as compared to chronic untreated animals.
Discussion and conclusions
The neuropeptide somatostatin is one of the major regulatory hormones in the central nervous system and the digestive tract. Improved knowledge of the patho-physiological processes of schistosomiasis can be obtained by studying the regulatory mechanisms of somatostatin in the human body. The activity of somatostatin is mediated via binding to specific cell surface receptors. To understand the expression, regulation, true role and distribution of these receptors, animal models come in helpful in defining the physiological and pathological conditions set up by schistosomiasis. Somatostatin, produced by neuroendocrine, inflammatory and immune cells inhibits various cellular functions including secretions, motility, and proliferation [18,13]. Preprosomatostatin (pp-SOM), a precursor of somatostatin is synthesized in the pancreas, the gastrointestinal network, and the brain. Two products of this pp-SOM are somatostatin-14, a 14 amino acid peptide, and somatostatin-28 (SS-28) that contain the SS-14 but is prolonged at the N-terminus. Its action is mediated by five specific somatostatin receptors (SSTR1 – SSTR5), which belong to the G protein-coupled receptor family.
The last years we have been studying the potential of somatostatin in modulating the pathology caused by schistosomiasis, in particular hepatic fibrosis. The trematode, Schistosoma mansoni is phylogenetically the oldest class of invertebrate in which we have identified immunopositivity to the somatostatin receptors SSTR2 and SSTR3 (manuscript submitted). The presence of these receptors on the parasite stages indicate that circulating somatostatin levels might interact with the pathogenic parasitic stages and hence influence hepatic fibrosis caused by this parasite.
The helminth parasite Schistosoma mansoni and its related species cause the fibrotic hepatic disease, schistosomiasis. Fibrosis is the process of excessive deposition of collagen in a tissue together with other extracellular matrix (ECM) components like fibronectin, proteoglycans, laminin and elastin. To some extent ECM deposition is necessary for wound healing since it provides strength and temporary structure to damaged tissues. However, if not limited this process is pathogenic. Liver fibrosis can be particularly detrimental leading to portal hypertension and its attendant sequelae that include splenomegaly and rupture-prone gastroesophageal varices.
The collagen peptide-synthesis in the liver of infected mice attains a peak activity at 8 weeks post infection. The triple polypeptide chains, assembled to form procollagen, once leaving the cell come together to form polymeric collagen fibers. The hydroxyproline residues take care of the stability of the three dimensional helix via the formation of hydroxyl bridges between the polypeptide chains. The important points in collagen biosynthesis are thus the positioning of the hydroxylproline residues in the triple backbone structure, the knipping off of the propeptides from procollagen to form collagen as it leaves the cell and finally the cross-linking to form fibers. The deposition of fibronectin and heparansulphate round the egg rises rapidly till week 11 post infection after which levels plateau off. All these extracellular matrix components interact with different cells and modulate various cellular activities like migration, proliferation, differentiation, chemotaxis etc. [19].
Somatostatin that has earned itself the nickname 'endocrine cyanide' due to its slowing down a number of biological processes in the body, might add another feature to its ubiquitous nature – that of reducing fibrosis in Schistosoma mansoni infections.
We have quantified fibrosis (amount of collagen) generated by S. mansoni infections and the reductions after somatostatin treatments by measuring the levels of hydroxyproline at each time point.
In acute and chronic infected animals that were untreated, the increased collagen levels were associated with increased somatostatin levels respectively. Two days treatment of acute and chronic infected animals with somatostatin significantly reduced hydroxyproline levels. Endogenous somatostatin levels were not affected during the acute phase after somatostatin treatment although in chronic infected mice endogenous somatostatin levels tended to increase. However, at both time points, a significant reduction in parasite egg counts was observed, suggesting that therapeutic doses of somatostatin might, by binding to somatostatin receptors on the parasite surface, inhibit the production of the parasite stage that induces the inflammatory granulomatous response that can lead to fibrosis.
Reports by Reynaert et al. [12] have identified the somatostatin receptors SSTR1, 2 and 3 on the hepatic stellate cells that are responsible for collagen synthesis. In our experiments exogenous somatostatin might also have bound to these receptors thereby inhibiting collagen synthesis.
Taken together we have elucidated the therapeutic capacity of somatostatin in reducing hepatic fibrosis. This report in fact confirms a previous report by Mansy [20], where using a somatostatin synthetic analog octreotide in S. mansoni infected animals, a reduction in hepatic fibrosis was noted. Since octreotide is limited in binding to somatostatin receptors, our results with natural somatostatin that binds to all 5 somatostatin receptors equally well assures us of the anti fibrotic capacities of this neuropeptide hormone, and points to its potential use in human conditions.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SC designed this study, performed the parasite egg counting, and statistical analysis. GV carried out the animal infection, somatostatin treatment, and hydroxyproline determinations. IDP and TP performed the somatostatin detection in the mice plasma. EVM participated in the design of the study and coordination. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors wish to thank Mr. Michel Segers, UCB Pharma, Brussels, for the kind gift of somatostatin® that was used in all experiments. This work was supported by the Inter-University Attraction Program (Grants P4/16 and P5/20) Services of the Prime Minister Federal Agency for Scientific, Technical and Cultural Affairs. The contribution of Kristel Kuypers and Koen Van de Vijver in the standardization of the hydroxyprolene procedure is much appreciated. Many thanks to Frank Rylant and Liliane Moeneclaey for the cryo-sectioning and staining of liver samples.
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| 15949036 | PMC1166555 | CC BY | 2021-01-04 16:28:15 | no | BMC Infect Dis. 2005 Jun 10; 5:45 | utf-8 | BMC Infect Dis | 2,005 | 10.1186/1471-2334-5-45 | oa_comm |
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BMC Med EducBMC Medical Education1472-6920BioMed Central London 1472-6920-5-181594387610.1186/1472-6920-5-18Research ArticleMedical students who decompress during the M-1 year outperform those who fail and repeat it: A study of M-1 students at the University of Illinois College of Medicine at Urbana-Champaign 1988–2000 Kies Susan M [email protected] Gregory G [email protected] College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA2005 19 5 2005 5 18 18 4 1 2005 19 5 2005 Copyright © 2005 Kies and Freund; licensee BioMed Central Ltd.2005Kies and Freund; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
All medical schools must counsel poor-performing students, address their problems and assist them in developing into competent physicians. The objective of this study was to determine whether students with academic deficiencies in their M-1 year graduate more often, spend less time to complete the curriculum, and need fewer attempts at passing USMLE Step 1 and Step 2 by entering the Decompressed Program prior to failure of the M-1 year than those students who fail the M-1 year and then repeat it.
Method
The authors reviewed the performance of M-1 students in the Decompressed Program and compared their outcomes to M-1 students who failed and fully repeated the M-1 year. To compare the groups upon admission, t-Tests comparing the Cognitive Index of students and MCAT scores from both groups were performed. Performance of the two groups after matriculation was also analyzed.
Results
Decompressed students were 2.1 times more likely to graduate. Decompressed students were 2.5 times more likely to pass USMLE Step 1 on the first attempt than the repeat students. In addition, 46% of those in the decompressed group completed the program in five years compared to 18% of the repeat group.
Conclusion
Medical students who decompress their M-1 year prior to M-1 year failure outperform those who fail their first year and then repeat it. These findings indicate the need for careful monitoring of M-1 student performance and early intervention and counseling of struggling students.
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Background
All medical schools are faced with poor performing M-1 students. The challenge is to encourage these students to take remedial programs that address their academic problems and assist them in becoming high performing physicians. During Academic Year 2001–2002, the LCME reported that 47 schools employed an Extended Time or Decompressed Program to assist poor performers [1]. Despite this widespread use of decompressed programs, little outcome information is reported in the literature.
In reviewing the literature, few outcome studies regarding remedial programs were found. Most articles discuss predictive measures of academic success in medical school, including both cognitive and non-cognitive variables that can assist admission committees to choose those students most likely to achieve well in medical school, while at the same time, steering committees away from admitting students likely to fail. Study results have provided medical school admission committees with data to apply to their admission policies and procedures [2-11]. Prominent among medical school admission policy and procedure changes are the development of 'prematriculation' programs and post baccalaureate programs, aimed at assisting the academically at risk students to find success after matriculation [12]. However, these policies and programs are not infallible and do not reflect the struggle failing students face, their rates of graduation, time spent in the program, number of attempts at USMLE Step 1 and Step 2. Importantly, curriculum track options were not discussed.
Once a student is admitted to medical school, it is important that academic progress be monitored. Typically, Student Affairs Offices and academic advisers are charged with this responsibility. Unfortunately, the literature has little information regarding the formulation and assessment of remedial programs [13]. Kassebaum and Szenas' study of all medical students matriculating from 1976 to 1988 revealed a decline in four year graduation rates, from 91.4% to 81.2%. During the same period, students requiring five years to graduate increased from 5.5% to 10.6%, with some students taking an additional sixth or seventh year to complete medical school [14]. The Alternative Curriculum developed at Boston University School of Medicine while not designed as a remedial program has become an entity where students experiencing academic difficulty enroll. McCahan [15] reported that half of those enrolled in the Alternative Curriculum either dropped out or were dismissed from medical school. Finally, in order to increase the retention of first year students at Indiana University School of Medicine, the Reduced Load Program was created in 1973. This remedial option allowed students to take two academic years to complete the first-year course requirements. In a study of this program, Ficklin, et al found it successful in retention of students, as 49% of this population, subject to dismissal for poor performance, graduated [16]. This research was conducted to provide students at risk for failure with the best possible guidance in choosing their curricular path.
At the University of Illinois College of Medicine in Urbana-Champaign approximately 125 M-1 students enroll each year. Since 1988, two options exist for M-1 students with performance deficiencies who wish to continue in medical school: the decompressed program or the repeat year. The decompressed program allows students to spread out their M-1 courses over two years. Students may opt into this program at any time between matriculation and one week after the M-1 second exam results are recorded. The repeat year is a full repeat of the entire curriculum. Repeat years are given to students, who have passed at least half of the M-1 curriculum after makeup examinations.
Research question/hypothesis
We sought to determine the best remedial program for our students with academic problems in their first year of medical school. Based on working with students over several years, our hypothesis was: For failing M-1 students, those who entered the decompressed program would, as a group, perform better than those who repeated based on graduation rate, length of time to complete the curriculum, and the number of attempts at USMLE Step 1 and USMLE Step 2.
Methods
Records of all medical students admitted to the M-1 track at the University of Illinois College of Medicine at Urbana-Champaign between 1988 and 2000 were examined (n = 1625). In this cohort, 107 students were found to have entered a remedial program to address M-1 deficiencies. The performance of these academically at risk M-1 students was analyzed comparing those in the decompressed program to those who fully repeated the M-1 year. After Institutional Review Board approval, 63 decompressed M-1 students were compared to 44 repeating M-1 students. Please note the students involved in the study are in the traditional curriculum. Medical Scholar Program Students (M.D./Ph.D.) were excluded from the study. Further, the cohorts are comparable as analysis was adjusted for differences in Cognitive Index and MCAT score. Subjects have comparable Cognitive Index scores and MCAT scores. 57% of the decompressed group students have underrepresented minority status while those in the repeating group 41% have underrepresented minority status.
Sample sizes of 63 in the decompressed group and 44 in repeating group are sufficient for having substantial power to detect differences between groups that are of practical importance. For instance, when using a two-sample t-statistic to test for a difference between the mean values of variables such as USMLE Step 1 scores, with a significance level of 0.05, these sample sizes result in 71% and 91% power, when the true means differ by 0.5 and 0.67 standard deviations, respectively.
To compare the decompressed and repeating groups, pre- and post- admission metrics were examined. The pre-admission metrics analyzed were: Cognitive Index (a proprietary formula that includes undergraduate GPA, rating of undergraduate institution and MCAT score); and MCAT score. The post-admission metrics analyzed were: graduation rates; length of time to graduation; and attempts to pass USMLE Step 1 and Step 2. For each comparison statistical analysis employed the Inman and Conover t Test (JMP Statistical Discovery Software, SAS Institute, Cary, NC). Possible confounding variables include potential personal pressures such as financial, health, and family issues. Any of these issues could have potentially affected subjects in this study, in both groups, but were unobtainable to the authors.
Results
Comparison of entering metrics
Predictive measures of success in medical school have been well studied [2-11]. However, dealing with medical students experiencing academic problems has not been well studied as outlined in the literature review above.
To compare our two groups upon admission, Inman and Conover t-Tests comparing the Cognitive Index of students and MCAT scores from both groups were performed. Results showed that there was no significant difference between the decompressed group and the repeaters relative to Cognitive Index (Table 1) or MCAT score (Table 2.).
Table 1 Comparison of Cognitive Index Between the Decompressed and Repeating Groups
Oneway Analysis of CI By Program t-Test
Difference t-Test DF Prob>/t/
Estimate -0.0648 -0.063 101 0.9501
Std Error 1.0331
Lower 95% -2.1141
Upper 95% 1.9847
Means and Standard Deviations Level Number Mean Std Dev Std Err Mean Lower 95% Upper 95%
Decompress 59 59.0586 5.34525 0.69589 57.666 60.452
Repeat 44 59.1234 4.9646 0.74849 57.614 60.633
This table shows there was no significant difference between the decompressed group and the repeating group relative to Cognitive Index scores. (Cognitive Index data not available for 4 students in the decompressed group.)
Table 2 Comparison of MCAT Between the Decompressed and Repeating Groups
Oneway Analysis of MCAT By Program t-Test
Difference t-Test DF Prob>/t/
Estimate -0.38724 -1.411 102 0.1613
Std Error 0.27448
Lower 95% -0.93167
Upper 95% 0.15719
Means and Standard Deviations Level Number Mean Std Dev Std Err Mean Lower 95% Upper 95%
Decompress 60 7.61253 1.33963 0.17295 7.2665 7.9586
Repeat 44 7.99977 1.4402 0.21712 7.5619 8.4376
Table Two shows there was no significant difference between decompressed group and the repeating group relative to MCAT score. (MCAT data not available for 3 students in the decompressed group.)
Comparison of medical school performance
Table 3 shows that of the 63 students who enrolled in the decompressed program 58.7% (37 students) graduated compared to those 44 students in the repeat program where only 27.2% (12 students) graduated. Further, of those students in the decompressed program 46% (29 students) graduated in five years compared to the 12 students in the repeat program in which 18% (8 students), finished in five years. Of the decompressed group, 46% (29 students) passed USMLE Step 1 on the first attempt while 16% (7 students) of the repeating group passed on the first sitting of USMLE Step 1. Decompressed students passed USMLE Step 2 on the first attempt at the rate of 51% (n = 32), while Repeating students passed USMLE Step 2 on the first attempt at a rate of 21% (n = 9). Analysis of USMLE performance by groups also showed that there was a significant difference between groups on number of attempts at USMLE Step 1 (Table 4.) but no significant difference in the number of attempts on USMLE Step 2 (Table 5.).
Table 3 1988–2000 Performance Data on Students in decompressed program and repeating program
Decompressed Repeated
Raw % Raw %
Number Enrolled 88-00 63 44
Number Graduated 37 58.7 12 27.2
For those who completed:
Five Years to Graduate 29 46 8 18
Six Years to Graduate 4 6 3 7
Seven Years to Graduate 3 5 0 0
Eight Years to Graduate 1 2 0 0
Twelve Years to Graduate 0 0 1 2
1 Attempt to Pass Step 1 29 46 7 15.9
2 Attempts to Pass Step 1 7 11 1 2
3 Attempts to Pass Step 1 1 2 4 9
1 Attempt to Pass Step 2 32 50.8 9 20.5
2 Attempts to Pass Step 2 3 4.8 2 4.5
3 Attempts to Pass Step 2 2 3 1 2.3
Table Three contains Raw and Percentage data comparing students who either entered the decompressed program or repeated the M-1 year. Percentages are calculated using the number enrolled in each program during the M-1 year.
Table 4 Comparison of USMLE Step 1 Performance Between the Decompressed and Repeating Groups
Oneway Analysis of Step One By Program t-Test
Difference t-Test DF Prob>/t/
Estimate -0.52616 -2.685 57 0.0095
Std Error 0.19594
Lower 95% -0.91852
Upper 95% -0.13381
Means and Standard Deviations Level Number Mean Std Dev Std Err Mean Lower 95% Upper 95%
Decompress 43 1.34884 0.612711 0.09344 1.1603 1.5374
Repeat 16 1.875 0.806226 0.20156 1.4454 2.3046
Table Four shows there was a significant difference between decompressed group and repeating group on the number of attempts at USMLE Step 1. (Note of the 63 students in the decompressed group, 43 students took USMLE Step 1, but 6 did not graduate. Of the 44 students in the repeating group, 16 took USMLE Step 1, of those 4 did not graduate.
Table 5 Comparison of USMLE Step 2 Between the Decompressed and Repeating Groups
Oneway Analysis of Step 2 By Program t-Test
Difference t-Test DF Prob>/t/
Estimate -0.11712 -0.587 47 0.5598
Std Error 0.19942
Lower 95% -0.5183
Upper 95% 0.28407
Means and Standard Deviations Level Number Mean Std Dev Std Err Mean Lower 95% Upper 95%
Decompress 37 1.21622 0.583816 0.09598 1.0216 1.4109
Repeat 12 1.33333 0.651339 0.18803 0.9195 1.7472
Table Five shows there was no significant difference on the number of attempts on USMLE Step 2 between the decompressed group and the repeating group.
Discussion
These data demonstrate that upon admission to the College of Medicine at Urbana-Champaign student performance could not be predicted based on either MCAT score, nor on Cognitive Index score. Further, the selection of the decompressed program to aid poor performing M-1 students is the best predictor of success later in the medical school curriculum. Decompressed students graduated at a rate of 59% compared to repeating students who graduated at a rate of 27%. Decompressed students took less time to complete the curriculum and required fewer attempts at USMLE Step 1.
This research would be greatly enhanced if an additional comparison were made of those students who matriculated with the same admissions record data, but did not experience academic difficulty. This would allow for broader conclusions. Further, if the study analyzed additional data related to the relationship of variables such as financial stress, personal hardships, health etc., broader conclusions could be drawn. Unfortunately those data are unknown and unobtainable at this time.
Conclusion
The results of this study demonstrate that the Decompressed Program is the best option for failing M-1 students, at the University of Illinois College of Medicine at Urbana-Champaign, as they performed better in the subsequent years of the curriculum. This study is a first step in understanding the remedial process with failing medical students. Further investigation is needed to develop criteria with which Student Affairs Offices and Promotions Committees can advise students on the most efficient/effective of handling M-1 remediation. In addition, identification of pre-enrollment metrics that would identify to medical school admissions committees those students likely to require participation in a decompressed program would be beneficial.
List of abbreviations
Liaison Committee on Medical Education (LCME)
United States Medical Licensing Examination (USMLE)
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SK conceived of the study, designed the study, performed the statistical analysis and helped to draft the manuscript.
GF helped to refine the design of the study, has been involved in drafting the article and revising it critically for intellectual content.
Both authors read and approved the final manuscript.
Funding
This research was supported by grants from the National Institutes of Health (DDK064862 to G.G.F.).
Pre-publication history
The pre-publication history for this paper can be accessed here:
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| 15943876 | PMC1166556 | CC BY | 2021-01-04 16:30:57 | no | BMC Med Educ. 2005 May 19; 5:18 | utf-8 | BMC Med Educ | 2,005 | 10.1186/1472-6920-5-18 | oa_comm |
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BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-5-361596323310.1186/1471-2180-5-36Research ArticleCarvacrol and p-cymene inactivate Escherichia coli O157:H7 in apple juice Kiskó Gabriella [email protected] Sibel [email protected] Corvinus University of Budapest, 1118 Budapest, Hungary2 Microbiology Research Group, Faculty of Health and Human Sciences, Thames Valley University, 18–22 Bond Street, LondonW5 5AA, UK2005 17 6 2005 5 36 36 7 2 2005 17 6 2005 Copyright © 2005 Kiskó and Roller; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Outbreaks of food poisoning associated with drinking un-pasteurised apple juice contaminated with enterohaemorrhagic Escherichia coli O157:H7 are a cause of serious illness and occasionally death. Whilst a well-established heat process (pasteurisation) will readily eliminate the pathogen, some consumers are demanding more fresh-like foods that have not been subjected to processing methods that are perceived as severe and may lead to loss of flavour and vitamins. Therefore, alternative methods are being investigated to replace pasteurisation and improve the safety of minimally-processed juices. The addition of natural antimicrobial substances such as the phenolic substances carvacrol and p-cymene (derived from the essential oils of herbs and spices) provides a potential new route to assure safety and extend the shelf-life of raw fruit juices.
The aim of this study was to evaluate the addition of very low concentrations (0.25–1.25 mM) of carvacrol and p-cymene both individually and in combination as a novel means of controlling Escherichia coli O157:H7 in un-pasteurised apple juice.
Results
When inoculated at a level of 4 log CFU/ml into un-pasteurised apple juice (pH 3.20 ± 0.06), Escherichia coli O157:H7 survived for up to 3 and 19 days at 25° and 4°C, respectively. Treatment of the juice with 1.25 mM carvacrol or p-cymene reduced the numbers of E. coli O157:H7 to undetectable levels within 1–2 days at both storage temperatures. The effective concentrations of carvacrol could be reduced even further by combining it at 0.5 mM with cymene at 0.25 mM. The phenolic compounds were biocidal against both spoilage yeasts and E. coli O157:H7 thereby increasing the shelf-life and improving the safety of un-pasteurised apple juice, particularly when stored at chill temperatures.
Conclusion
The results showed that the natural antimicrobial compounds carvacrol and p-cymene could potentially be used to extend the shelf life and improve the safety margins in un-pasteurised chilled fruit juices.
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Background
Conventional fruit juice processing involves a heating step to inactivate the vegetative forms of pathogenic and spoilage microorganisms. Any remaining bacterial spores are generally unable to germinate due to the acidic nature of the juices [1]. This ensures acceptable safety margins and extends the shelf-life of the juice. However, heat treatment causes vitamin losses and changes in flavour of the juices and some consumers regard heat-treated, shelf-stable products as low in quality. In the last 15 years, there have been several outbreaks of food poisoning associated with drinking un-pasteurised apple juice contaminated with enterohaemorrhagic Escherichia coli O157:H7 and several children have died in the USA [2]. Warning labels are now required in the USA for all fruit juices unless a 5-log pathogen reduction treatment has been applied to the product [3-5]. Therefore, there is a need for new methods of fruit juice preservation that rely less heavily on severe heat treatment or the addition of synthetic preservatives. It has been suggested that many natural antimicrobial compounds from plant, animal and microbial sources might fulfil this need [6-8].
Carvacrol (2-methyl-5-(1-methylethyl)-phenol) is a major component of the essential oils of oregano and thyme [9,10]. Generally recognized as a safe food additive, carvacrol is used as a flavouring agent in baked goods, sweets, beverages and chewing gum [11]. Carvacrol-containing essential oils are biostatic and/or biocidal against many bacteria, yeast and fungi in laboratory media and consequently have attracted considerable research attention as potential food preservatives [reviewed in [12]]. Carvacrol has also been shown to inactivate microorganisms in biofilms on stainless steel surfaces [13,14]. The biocidal mode of action of carvacrol on bacteria is similar to that of other phenolic compounds and occurs via membrane damage resulting in an increase in membrane permeability to protons and potassium ions, depletion of the intracellular ATP pool and disruption of the proton-motive force [15,16].
The biological precursor of carvacrol, p-cymene (1-methyl-4-(1-methylethyl)-benzene), is less antimicrobial than carvacrol when used alone. p-Cymene lacks a hydroxyl group, which is thought to play an important role in antimicrobial activity [17]. Synergism between carvacrol and p-cymene against B. cereus in vitro and in rice has been reported [18].
The minimum inhibitory concentrations (MICs) of carvacrol, thymol, cinnamic acid and other phenolic compounds from herbs and spices against some food-borne bacteria in vitro have been reported at around 1 mM [12,18,19]. However, a much higher concentration is usually needed to achieve the same biocidal or biostatic effects in foods. For example, the MIC of carvacrol in mushroom soup inoculated with B. cereus was 50 times higher than that found in laboratory media [20]. Similarly, the concentrations of carvacrol and cinnamic acid required to delay spoilage of mango, kiwifruit and melon slices was >5 mM [21,22]. A number of intrinsic (fat and protein content, pH, salt, etc.) and extrinsic (temperature, oxygen availability, etc.) factors are thought to play a role in protecting microorganisms from the biocidal effects of carvacrol in foods. Furthermore, the potent aromatic and antioxidant properties of phenolic compounds at these high concentrations have been reported to lead to undesirable colour, odour and flavour changes in food products [8,22].
The aim of this study was to evaluate the addition of very low concentrations of carvacrol and p-cymene both individually and in combination as a novel means of controlling Escherichia coli O157:H7 in un-pasteurised apple juice.
Results
Inactivation/inhibition of microorganisms with 1.25 mM carvacrol and p-cymene used individually
At 25°C, the total viable counts in un-pasteurized, untreated apple juice increased in the first 4 days to a level of 8 log CFU/ml, where they remained up to Day 7 (Figure 1). Yeast counts reflected the total counts. When inoculated into raw apple juice, E. coli O157:H7 survived for up to 3 days at 25°C but by Day 4, numbers decreased to below the detection limit of the plate-counting method (0.5 log CFU/ml). Enrichment methods allowed the detection of E coli up to 3 days after inoculation at 25°C but by Day 4 (and beyond to Day 20) none were detected (Table 1).
In the presence of 1.25 mM carvacrol, there was an initial reduction (within the first two days) in total and yeast counts of about 2 log CFU/ml (Figure 1). This was followed by re-growth of total viable numbers to about 6 log CFU/ml within 7 days where they remained steady until Day 20. Yeast numbers continued to fall in the presence of 1.25 mM carvacrol and by Day 4 reached levels below the detection limit of the assay (0.5 log CFU/ml). In the presence of 1.25 mM p-cymene, total plate counts increased more slowly than in the untreated controls but by Day 12, numbers were similar to those in the controls. Yeasts were initially inactivated by p-cymene and remained at low levels (1–2 log CFU/ml) from Day 1 to 4 but then increased to about 6–7 log CFU/ml by Day 8 where they remained up to Day 20. Notably, numbers of E. coli decreased to below the limit of detection of the plate-counting method within less than one day of exposure to both carvacrol and p-cymene. E. coli were no longer detectable by enrichment methods after 2 days in the presence of carvacrol and after 3 days in the presence of p-cymene (Table 1).
At 4°C, total viable numbers and yeasts in the untreated juice remained at about 4–5 log CFU/ml for the first 12 days of incubation, followed by very slow growth to about 7 log CFU/ml by Day 20 (Figure 1). In the presence of 1.25 mM carvacrol and p-cymene, there was a gradual decline in total and yeast numbers with carvacrol affecting a slightly steeper reduction in total viable numbers than p-cymene. By Day 20, total numbers were about 1 and 4 log CFU/ml in the presence of 1.25 mM carvacrol and p-cymene, respectively. Notably, E. coli O157:H7 survived at a level of 3–4 log CFU/ml for up to 14 days at 4°C, was still countable on Day 18 (1 log CFU/ml) and was detectable (by enrichment) on Day 19 of incubation. In contrast, E coli was not countable (above the detection limit of 0.5 log CFU/ml) or detectable (by enrichment) after 1 day of exposure to either carvacrol or p-cymene (Figure 1). The results for E. coli shown in Figure 1 were obtained using thin TSA overlaid on selective CT-SMAC agar (see Methods section) in an attempt to resuscitate injured organisms. The results obtained on plain CT-SMAC agar and on Chromagar (with and without a thin agar layer on top) were very similar and are consequently not illustrated.
It was noted that the addition of either carvacrol or cymene at 1.25 mM imparted an intense "spicy" aroma to the treated apple juices.
Inactivation of microorganisms with 0.5 mM carvacrol and/or 0.25 mM p-cymene at 4°C
Since the results above indicated that survival of E. coli O157:H7 was substantially extended at chill temperatures and therefore represented a greater cause for concern than survival at ambient temperatures, further work was undertaken at 4°C only. In order to study the effect of combinations of carvacrol and cymene, both compounds were used at concentrations that had no or very little inhibitory effect on microorganisms. As shown in Figure 2, addition of 0.5 mM carvacrol or 0.25 mM p-cymene individually to apple juice had a very slight inhibitory effect on total microbial counts at 4°C. Like in Figure 1, survival of E. coli O157:H7 persisted for two weeks at this temperature (Figure 2 and Table 2). Using the plate-counting technique, it is shown in Figure 2 that survival of E. coli was curtailed by both 0.5 mM carvacrol and 0.25 mM p-cymene to about 5 days. Enrichment techniques showed that E. coli was detectable up to 7 and 14 days in the presence of 0.5 and 0.25 mM carvacrol and p-cymene, respectively. However, when the two compounds were added to the juice together, E. coli could not be counted or detected after 1 day of exposure to the treatment (Figure 2 and Table 2). Plating out on selective agars with or without a thin layer of TSA gave very similar results to those shown in Figure 2 and so these counts are not illustrated.
It was noted that the addition of 0.5 mM carvacrol and 0.25 mM p-cymene, alone or in combination, imparted a very slight "spicy" aroma to the treated juices.
Throughout this study, the pH of the juices remained essentially unchanged at around 3.20 ± 0.06, irrespective of the type of treatment or storage temperature.
Discussion
Freshly-pressed apple juice prepared from healthy fruit can be expected to contain around 3–5 log CFU/ml of viable microorganisms, of which the majority are yeasts [23]. The results in this study were consistent with this expectation, as shown in Figures 1 and 2. The pH of the juices used in this study was at the lower end (3.20 ± 0.06) of the expected pH range (2.9–4.5) for fresh apple juice [23].
Despite the low pH, E. coli O157:H7 survived in raw apple juice for 3 days at 25°C and nearly 3 weeks (19 d) at 4°C. These findings agree with those reported elsewhere [23-28]. The acidic nature of apple juice does not ensure its safety as E. coli O157:H7 may survive for extended periods of time, especially at chill temperatures.
The antimicrobial properties of carvacrol and similar phenolic compounds from the essential oils of herbs and spices have been reported previously against individual microorganisms, tested in vitro [12,20,29-32]. The application of carvacrol in the preservation of some foods such as rice and fresh-cut fruit has also been reported [18,22]. However, this is the first report of the successful application of relatively low doses (0.25–1.25 mM) of carvacrol and p-cymene against E. coli O157:H7 in the presence of the mixed microflora of fruit juice. The results demonstrate that the addition of 1.25 mM carvacrol or p-cymene to apple juice inactivated E. coli O157:H7 within less than 1 day from about 4 log CFU/ml to levels that were undetectable using conventional microbiological techniques (Figure 1). Furthermore, once inactivated, E. coli remained undetectable for the duration of the trial (20 days; Table 1). In addition, the results obtained using the thin agar layer method for resuscitating injured organisms were no different from those using selective media alone, suggesting that the cellular injury caused by the phenolic compounds was irreversible. The substantial reduction in the number of days (from 19 to 1) that E. coli O157:H7 was able to survive in apple juice when treated with 1.25 mM carvacrol or p-cymene and stored at chill temperatures represents an opportunity to improve the safety of un-pasteurised fruit juices.
In addition to their antibacterial activity, carvacrol and p-cymene were also biocidal against the yeast flora naturally present in the apple juice (Figure 1). However, 1.25 mM p-cymene was not as effective as the same concentration of carvacrol in eliminating the yeast population at ambient temperature. Those yeasts surviving the treatment with p-cymene recovered and eventually reached the same viable numbers as in the untreated control. By contrast, the presence of either phenolic compound at 1.25 mM resulted in similar, gradual die-off of the yeast population at chill temperatures. The results confirm the broad antimicrobial spectrum of carvacrol but also suggest that yeasts may be somewhat less sensitive to phenolic compounds than bacteria. The reduction in numbers of spoilage yeasts would clearly benefit both the manufacturer and consumer by extending the shelf-life of the product.
When carvacrol and p-cymene were added to apple juice at 0.5 and 0.25 mM, respectively, E. coli O157:H7 and the yeast population were reduced but to a different extent than was observed at the higher concentrations of 1.25 mM. The shapes of the inactivation curves for the total viable numbers (Figure 2) would suggest that there may have been some synergism between carvacrol and p-cymene but the results for E. coli (Figure 2 and Table 2) suggest that the effect may have been additive. In the presence of 0.5 mM carvacrol and 0.25 mM p-cymene used alone, E. coli was not detectable for 13 and 6 d longer than in the control but in the presence of both compounds used in combination, the organism was not detectable for 18 d longer. Therefore, it is not possible to conclude unequivocally whether the effect of adding the two substances was synergistic or merely additive.
It has been reported previously that carvacrol was more effective in reducing the viable count of the natural microflora on kiwifruit (pH 3.2–3.6) than on honeydew melon (pH 5.4–5.5) [22]. At low pH, the hydrophobicity of essential oil components increases, enabling them to partition more easily into the lipids of the cell membrane of bacteria.
It is known that direct plating on selective media following exposure to physical or chemical stresses can lead to gross underestimation of viable counts by as much as 3–4 log CFU ml-1 [33,34]. Although a thin agar layer method [35] and enrichment techniques [36] were used in this study to allow some resuscitation of the inoculated pathogen, it is possible that the results shown in Figures 1 and 2 represent an underestimate of numbers. Furthermore, it is possible that a larger sample volume (25 ml instead of 2.5 ml) in the pre-enrichment step would have allowed the resuscitation and recovery of an even greater number of E. coli from the control juices.
In the present study, ethanol was used as a solvent for the preparation of stock solutions of carvacrol and cymene. Whilst the final concentrations of ethanol in the juices were below the tolerance limit previously reported for E. coli [37], it is possible that the low levels of ethanol present (0.95, 1.9 and 4.75% in the presence of 0.25, 0.5 and 1.25 mM carvacrol/cymene, respectively) may have potentiated the biocidal action of the phenolic compounds. This possibility would need experimental confirmation in future work.
Conclusion
When inoculated into un-pasteurised apple juice, E. coli O157:H7 survived for up to 3 and 19 days at 4° and 25°C, respectively. At 1.25 mM and at both storage temperatures, carvacrol and p-cymene reduced the numbers of E. coli O157:H7 to undetectable levels within 1–2 days. The effective concentrations of carvacrol could be reduced even further by combining it at 0.5 mM with p-cymene at 0.25 mM. Carvacrol and p-cymene were biocidal against both spoilage yeasts and E. coli O157:H7 thereby increasing the shelf-life and improving the safety of un-pasteurised apple juice, particularly when stored at chill temperatures.
Methods
Materials
Freshly pressed, unclarified, raw juice from a mixture of Bramley and Cox apples was obtained directly from a manufacturer in Suffolk, England. The pH of the juice was 3.20 ± 0.06 on arrival at the laboratory. All microbiological media and diluents were from Oxoid (Basingstoke, UK) and all chemicals from Sigma Chemicals Co. Ltd. (Poole, Dorset, UK) unless otherwise indicated.
Un-pasteurised apple juice is often referred to as "cider" in the USA. This should not be confused with UK cider, which is a fermented alcoholic beverage. In this paper, the term "apple juice" refers strictly to the raw, unprocessed, unfermented juice of the apple.
Microorganisms and their cultivation
Bacteria, yeasts and moulds were routinely cultivated on Plate Count Agar (PCA) and Malt Extract Agar (MEA). PCA plates were incubated at 30°C for 2–3 days whilst MEA plates were incubated at 25°C for 3–5 days. The attenuated strain of Escherichia coli O157:H7 (Dr. P. McClure, Unilever Research, Colworth House, Bedford, UK) was maintained in Brain Heart Infusion (BHI) broth and agar and incubated overnight at 37°C. For challenge experiments, fresh overnight cultures of E. coli O157:H7 in BHI broth were washed three times and re-suspended in sterile saline solution. Viable counts of the washed cell suspensions were determined by serial dilution (1:10) in Maximal Recovery Diluent (MRD) and spread-plating 0.1 ml of the suspensions (in duplicate) on Tryptone Soya Agar (TSA) and incubating the plates at 37°C for 24 h.
Treatment of apple juice with carvacrol and cymene
Containers (200 ml capacity) of apple juice (100 ml) were prepared in duplicate for each treatment and stored at 4°C and 25°C. Stock solutions (25 mM) of carvacrol and cymene were prepared in 95% ethanol and added to the apple juice to give final concentrations of 0.25, 0.5 and/or 1.25 mM. The juice was inoculated with the washed suspension (prepared as described above) of E. coli O157:H7 to give an approximate count of 104 CFU/ml. Control juices inoculated with E. coli O157:H7 but containing no antimicrobials, as well as absolute controls containing no antimicrobials or E. coli O157:H7 were also prepared.
Samples (10 ml) were taken periodically for up to 20 days from each container for microbiological analysis and pH determination. Serial (1:10) dilutions were prepared in sterile MRD. Total viable numbers were determined by pour-plating (1.0 ml) on PCA. Yeasts and moulds were enumerated by spread-plating (0.1 ml) on Tryptone Glucose Yeast Extract Agar (TGYEA) supplemented with chloramphenicol (0.1 mg/ml). PCA and TGYEA plates were incubated at 30°C for 2–3 days. Single samples were removed from each duplicate batch of juice periodically during incubation for viable counting and plating out in triplicate; therefore, mean counts for each time point were calculated from six replicate determinations.
Pre-enrichment and enrichment steps were used to detect low levels of E. coli O157:H7 in the apple juice following treatment with carvacrol and/or cymene. An aliquot of 2.5 ml of juice was added to 22.5 ml BPW and incubated at 37°C for 24 h (in the case of the un-inoculated control, 25 ml apple juice was added to 225 ml BPW). Following incubation, an aliquot (1 ml) was dispensed into 10 ml EC broth (reduced bile salts supplemented with novobiocin) and incubated at 37°C for 24 h. A loopful of the EC broth was streaked onto Sorbitol MacConkey (SMAC) Agar or onto CHROMagar (M-Tech Diagnostic Ltd., EE-220-Trial; both agars supplemented with cefixime and potassium-tellurite) and incubated at 37°C for 24 h.
For enumeration of injured cells of E. coli O157:H7, the Thin Agar Layer method (TAL) was used [35]. Spread plates were prepared on selective agars (CT-SMAC agar or CHROMagar supplemented with cefixime and potassium-tellurite) and on selective agars overlaid with TSA agar. The number of injured cells was calculated by subtracting the viable count on selective agar alone from the viable count obtained on the overlaid agar.
Authors' contributions
GK participated in the design of the study and carried out all the experimental work.
SR conceived the study and participated in its design and coordination.
GK and SR drafted the manuscript jointly.
Acknowledgements
The financial support of the European Commission in the form of a Marie Curie Individual Fellowship awarded to author Kiskó (QLK1-CT-2000-51126) to undertake this work in author Roller's laboratory in London is gratefully acknowledged.
Figures and Tables
Figure 1 Survival/growth of microorganisms in apple juice treated with 1.25 mM carvacrol or p-cymene at 25° and 4°C. Data points represent: control with no additions (●); control inoculated with E. coli O157:H7 (○); juice inoculated with E. coli O157:H7 and treated with 1.25 mM carvacrol (▲); juice inoculated with E. coli O157:H7 and treated with 1.25 mM p-cymene (■). Sampling of control batches at 25°C was discontinued after 7 d due tovisible spoilage. Dotted line represents the lower detection limit of the plating technique. The pH of the juice was 3.17 on Day 0 and ranged between 3.30 and 3.44 on the final day of sampling (Days 7 and 20 at 4° and 25°C, respectively).
Figure 2 Survival of microorganisms in apple juice treated with 0.5 mM carvacrol and/or 0.25 mM p-cymene at 4°C. Data points represent: control with no additions (●); control inoculated with E. coli O157:H7 (○); juice inoculated with E. coli O157:H7 and treated with 0.5 mM carvacrol (▲); juice inoculated with E. coli O157:H7 and treated with 0.25 mM cymene (■); and juice inoculated with E. coli O157:H7 and treated with the combination of 0.5 mM carvacrol plus 0.25 mM cymene (◆). The dotted line represents the lower detection limit of the plating technique. The pH of the juice was 3.21 on Day 0 and ranged between 3.16 and 3.32 on the final day of sampling (Day 20).
Table 1 Presence/Absence of E. coli in apple juice stored at 25°C (A) and 4°C (B). Results (in duplicate) were obtained by enrichment of samples from the experiment illustrated in Figure 1.
A
Antimicrobial treatment Time (days)
0 1 2 3 4 7 12 15 19 20
Control, no additions - - - - - - - - - -
Control, E. coli O157:H7 inoculated + + + + - - - - - -
Carvacrol, 1.25 mM + ± - - - - - - - -
Cymene, 1.25 mM + ± ± - - - - - - -
B
Antimicrobial treatment Time (days)
0 1 2 3 4 7 12 15 19 20
Control, no additions - - - - - - - - - -
Control, E. coli O157:H7 inoculated + + + + + + + + + -
Carvacrol 1.25 mM + - - - - - - - - -
Cymene 1.25 mM + - - - - - - - - -
Table 2 Presence/Absence of E. coli in apple juice stored at 4°C. Results (in duplicate) were obtained by enrichment of samples from the experiment illustrated in Figure 2.
Antimicrobial treatment Time (days)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Control, no additions - - - - - - - - - - - - - - - - - - - - -
Control, E. coli O157:H7 inoculated + + + + + + + + + + + + + + + + + + + + -
Carvacrol, 0.5 mM + + + + + + + ± - - - - - - - - - - - - -
Cymene, 0.25 mM + + + + + + + + + + ± + ± ± + - - - - - -
Carvacrol (0.5 mM) plus cymene (0.25 mM) + + - - - - - - - - - - - - - - - - - - -
==== Refs
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| 15963233 | PMC1166557 | CC BY | 2021-01-04 16:03:41 | no | BMC Microbiol. 2005 Jun 17; 5:36 | utf-8 | BMC Microbiol | 2,005 | 10.1186/1471-2180-5-36 | oa_comm |
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BMC Med Inform Decis MakBMC Medical Informatics and Decision Making1472-6947BioMed Central London 1472-6947-5-171596704010.1186/1472-6947-5-17Research ArticleA Severe Acute Respiratory Syndrome extranet: supporting local communication and information dissemination Valaitis Ruta K [email protected] Noori [email protected] Cathy M [email protected] Glenn M [email protected] Helen [email protected] Public Health Research and Development Program, Public Health and Community Services Department, 1 Hughson St N, Hamilton, L8R 3L5, Canada2 Public Health and Community Services Department, Hamilton, L8R 3L5, Canada3 School of Nursing, McMaster University, 1200 Main Street West, Hamilton, L8N 3Z5, Canada2005 20 6 2005 5 17 17 15 12 2004 20 6 2005 Copyright © 2005 Valaitis et al; licensee BioMed Central Ltd.2005Valaitis et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The objective of this study was to explore the use and perceptions of a local Severe Acute Respiratory Syndrome (SARS) Extranet and its potential to support future information and communication applications. The SARS Extranet was a single, managed electronic and limited access system to manage local, provincial and other SARS control information.
Methods
During July, 2003, a web-based and paper-based survey was conducted with 53 SARS Steering Committee members in Hamilton. It assessed the use and perceptions of the Extranet that had been built to support the committee during the SARS outbreak. Before distribution, the survey was user-tested based on a think-aloud protocol, and revisions were made. Quantitative and qualitative questions were asked related to frequency of use of the Extranet, perceived overall usefulness of the resource, rationale for use, potential barriers, strengths and limitations, and potential future uses of the Extranet.
Results
The response rate was 69.4% (n = 34). Of all respondents, 30 (88.2%) reported that they had visited the site, and rated it highly overall (mean = 4.0; 1 = low to 5 = high). However, the site was rated 3.4 compared with other communications strategies used during the outbreak. Almost half of all respondents (44.1%) visited the site at least once every few days. The two most common reasons the 30 respondents visited the Extranet were to access SARS Steering Committee minutes (63.3%) and to access Hamilton medical advisories (53.3%). The most commonly cited potential future uses for the Extranet were the sending of private emails to public health experts (63.3%), and surveillance (63.3%). No one encountered personal barriers in his or her use of the site, but several mentioned that time and duplication of email information were challenges.
Conclusion
Despite higher rankings of various communication strategies during the SARS outbreak, such as email, meetings, teleconferences, and other web sites, users generally perceived a local Extranet as a useful support for the dissemination of local information during public health emergencies.
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Background
An Extranet is a private network that uses the Internet to securely share information or operations with selected partners; it is accessible from any web browser. The literature describes the use and potential benefits of local area Extranets in hospitals,[1] physician offices,[2,3] and small managed health care organizations [4]. A current U.S. National Emergency Medical Extranet (NEME) is being constructed to support emergency medicine and public health [5]. Building on the Centre for Disease Control's (CDC) national communication infrastructure, the US has begun to develop their Extranet to manage local public health emergencies, and plans to expand it for communicable disease surveillance and control activities [6].
A systematic review of the use of information technology in the event of bioterrorism evaluated web-based communication systems that link public health officials with clinicians and the public [7]. Although such systems have not been tested in crisis situations, the review indicates that they can securely manage disease-reporting needs of local and state officials. The systems evaluated most were those communicating abnormal findings in electronic medical records between institutions and clinicians. Three communication systems had the capacity to support rapid reporting and dissemination of information related to naturally occurring and bioterrorist-related infectious disease, although they required the use of electronic medical records.
A provincial Severe Acute Respiratory Syndrome (SARS) emergency was declared in Ontario on March 26, 2003. Two days later, a SARS Steering Committee was convened in Hamilton, Ontario, linking Public Health and Community Services (PHCS) with other partners (Table 1). Hamilton is located about 70 kilometers west of Toronto, which was the centre of the outbreak. Information management was critical during the crisis. Communication from the Ontario Provincial Operations Centre proceeded through email and fax to hospitals, long-term care facilities, health units, and the Ontario Medical Association. Simultaneously, information came from the World Health Organization (WHO), Health Canada, other health units and health care partners. The multi-source nature of the information made it difficult to manage and synthesize the most current, accurate information in a timely fashion. In addition, information impacting health units and Emergency Medical Services (EMS) might have reached only hospital staff. Directives changed often, necessitating version control measures to ensure that everyone was working from the most recent documents. This situation was difficult to manage with emails as updates were often sent daily and document file sizes were too large to send. A decision was made to develop a single, limited-access web-based system to manage constantly changing local and provincial SARS control information.
Table 1 Respondents by Agency or Institution (n = 34)
Agency or institution Frequency Percentage
Hospitals 11 32.4
Public Health and Community Service 10 29.4
Ontario Ministry of Health and Long-term Care (MOHLTC) 5 14.7
Emergency services 4 11.8
Community Care Access Centre 3 8.8
Other 1 2.9
Total 34 100.0
In response to demands from the steering committee for up-to-date information, the City of Hamilton's Public Health and Community Services (PHCS) and Information Technology (IT) Division developed and launched an Extranet on April 3, 2003. The Extranet was developed to support the SARS steering committee in disseminating to partners comprehensive information that was timely, current, accurate and relevant. The Extranet also supported the implementation of local strategies using a secure accessible and managed centralized access point. SARS steering committee members (N = 53) received a common ID and password to access the site. The site did not require users to connect through a Virtual Private Network (VPN), nor was there any validation of IP addresses to validate users. The Extranet, however, was viewed as a private network using the Internet to securely share information with partners. The security aspects made it an Extranet.
The secure site provided document distribution, version control of documents such as clinical guidelines and directives, and control of links to information for schools and workplaces. An email link was provided to access the health unit's web master. Because of the speed at which this site had to be developed, there was insufficient time for developing other interactive communication tools or tracking IP addresses. Ideally, participatory design principles, [8,9] would have been used to design the site, but there was no time to actively involve end users or track the costs to set up and maintain the site. Set-up and maintenance of the site was estimated to require one programmer (100 hours @ $40/hr), one Information Architect and Business/Technical Liaison (95 hours @ $50/hr), and one communicable disease expert (10 hours @ $75/hr) over the twelve weeks the Extranet was in use (total = $9,500).
The objectives of this research were to examine a) the use and perceptions of a local SARS Extranet, and b) its potential to support future public health information dissemination and communication with the local community.
Methods
The local university ethics review board approved the study. Participants included all 53 SARS Steering Committee members. The authors designed and tested a survey containing 13 forced-choice and 8 open-ended questions, which focused on perceptions on the Extranet and on other SARS information and communication sources. Both web-based and paper-based surveys were used to ease completion and simplify data collection as well as to increase the response rate.
User testing was conducted with the online version of the survey to identify problems with use, ensure survey comprehension, calculate average completion time (10–15 minutes), and identify if dynamic or technical functionality posed challenges. A three-member observation team tested the web survey with four PHCS employees who were familiar with the SARS Extranet and comfortable using the web. Instructions were given to each participant based on a think-aloud protocol, [10-14]. Participants were asked to complete the web survey, while observers took notes. Participants were instructed to think out loud, make statements, or ask questions as they made their way through the survey. They were informed that observers would not respond, but that observers would benefit from hearing their comments and watching their actions. Observers noted problems spoken and/or observed, which were later discussed in a debriefing session after each test. They participated in a final debriefing session to identify problems that occurred with more than one participant; this process provided direction for survey improvements. Several minor survey modifications were made prior to the launch. The online survey form was dynamic and, depending on how a respondent replied to one question, determined the next series of questions. Thus, changes identified through the think-aloud technique related to branching of questions (If yes, go here; If no, go there). No other significant issues were identified. The paper-based survey was also modified slightly to match the web version.
On July 3rd 2003, all steering committee members were sent an information email along with a letter of introduction, an invitation to participate, and a direct link to the online survey. One week later, a reminder email was sent to non-respondents; two weeks later, the paper-based survey and self-addressed stamped envelope was mailed. Quantitative data were entered into SPSS for analysis. Two researchers independently coded the open-ended questions. Consensus was reached with the entire research team whenever differences in coding existed.
Results
Of the 53 SARS Steering Committee members, four were ineligible as follows: one person moved, one was a research team member, and two were not active on the committee. Of the 49 eligible participants, three refused to complete the survey, three of them were disqualified because their responses were inconsistent, and nine of them did not respond. Respondents were representative of the steering committee, which included members from various agencies (Table 1). The response rate was 69.4% (n = 34).
Hamilton SARS Extranet compared with other SARS communication strategies
Thirty-four respondents rated the usefulness (1-lowest to 5-highest) of communication strategies used during the outbreak (Table 2). Email, teleconferences, and face-to-face meetings were rated the highest. Among SARS web-based resources, the Ontario Ministry of Health and Long-Term Care (MOHLTC) (mean = 4.1), the Center for Disease Control (CDC) (mean = 4.0), and the World Health Organization (WHO) (mean = 4.0) sites were the most valued. The Hamilton SARS Extranet site was rated fifth among the web resources (mean = 3.4).
Table 2 Usefulness of Various Communication Strategies during the SARS outbreak to respondents (N = 34) (1= not at all useful to 5 = extremely useful)
Communication strategy N Mean SD
E-mail 34 4.4 0.8
Teleconference 33 4.3 0.8
Face-to-face meeting 31 4.2 1.0
Ontario MOHLTC SARS private website 28 4.1 0.9
Centre for Disease Control website 24 4.0 1.0
WHO SARS website 24 4.0 1.2
Health Canada 28 3.9 1.2
City of Hamilton's SARS Extranet 28 3.4 1.2
Fax 23 3.3 1.1
City of Toronto Public Health Department's website 20 3.3 1.3
Use and perceived utility of the Hamilton SARS extranet
The site had 3478 hits, and 30 respondents (88.2%) visited the Extranet (Table 3). The home page received the average number of 37.4 hits per day, while clinical guidelines and directives (10.3) and SARS Steering Committee minutes (7.3) received the next-highest average number of hits per day. Twenty-eight of them rated the quality of the Extranet and reported frequency of use. Even though the Extranet was rated lower than some other SARS web sites, when participants were asked specifically about the Hamilton Extranet, they rated it high overall (Mean = 4.0; SD = 0.8) (1 – lowest to 5 – highest). Criteria included relevance (Mean = 4.4; SD = 0.7), comprehensiveness (Mean = 4.0; SD = 0.8) accuracy (Mean = 4.3; SD = 0.6), and timeliness (Mean = 3.9; SD = 0.9). Most respondents (63.3%; n = 19) who used the Extranet visited the site at least once every few days. There was a difference in visiting habit between those who rated the Extranet most favourably (mean value ≥ 4 out of five points) and others. Those rating it most favourably visited the site more often (not statistically significant using two-sided Fisher's exact test, P > 0.50) than the others.
Table 3 Analysis of Hits to Pages on Site
Page Total hits Average hits per day
Clinical Guidelines and Directives 954 10.3
SARS Steering Committee minutes 683 7.3
Directives and Advisories 316 3.4
Resources for other professionals 187 2.0
Epidemiology 184 2.0
Schools/Workplace 171 1.8
Health Services 57 0.6
Hamilton Medical Advisories 42 0.5
Thirty participants were asked a forced-choice question about whether they visited the site and why they visited. Most commonly, users visited to obtain SARS Steering Committee minutes (63.3%), access "Hamilton medical advisories" (53.3%), and obtain the SARS Steering Committee contact list (53.3%) (Table 4). These results corroborate the number of actual hits per page, Table 3. Other reasons for visiting included getting access to provincial directives and links to other websites. Although the difference was only marginally significant (P = 0.063), those who rated the website most favorably cited more reasons for using it than did others.
Table 4 Extranet Resources Used by Number of Extranet Users (N = 30)
Reason Frequency Percentage
Steering committee minutes 19 63.3
Hamilton medical advisories 16 53.3
Steering committee contact list 16 53.3
Provincial directives 14 46.7
Links to other websites 12 40.0
Emergency telephone numbers 10 33.3
Clinical guidelines 8 26.7
SARS signs 5 16.7
SARS assessment clinic information 5 16.7
Resources for other health professionals 5 16.7
Link SARS private website: Ontario Ministry of Health and Long-Term Care 2 6.7
Other 3 10.0
Eighteen participants revealed that they shared information from the Extranet with others outside of the committee. The information they shared most often included provincial directives and local information such as Hamilton medical advisories and Steering Committee minutes (Table 5). The average number of hits per page corroborates this finding, as these resources were used the most (Table 4). Eight (22.8%) respondents reported sharing the website address, username and password with middle management and staff.
Table 5 Type of Information Shared by those who Shared Information with People Outside the SARS Steering Committee (N = 18)
Information Frequency Percentage
Provincial directives clinical guidelines 10 55.6
Hamilton medical advisories 9 50.0
Steering committee minutes 8 44.4
Resources for other health professionals 7 38.9
SARS signs 6 33.3
Steering committee contact list 4 22.2
SARS assessment clinic information 3 16.7
Website/ Web address 3 16.7
Emergency telephone numbers 3 16.7
Open-ended questions were included to gain a better understanding of the barriers, strengths, limitations, and other features of a local Extranet. Most respondents indicated that they experienced no personal barriers to accessing the Extranet. However, a few respondents indicated that time to access the site and duplication of information sent through email and the Extranet were barriers. A concern was raised related to Extranet access: "Some folks had access/password who should not have access, and were following information not yet implemented or disseminated internally." One person indicated that access was expanded to others as the outbreak was prolonged.
Regarding the strengths of the system, many respondents indicated that they appreciated the ability to access current, timely and comprehensive information in one location. One respondent noted " [It was] very useful for staff to have info in one place, in early days of SARS where no other provincial web sites [were] set up to access information." Another participant noted that the Extranet was able to "dispel rumours [and] contained a broad scope of information in one locale."
Respondents identified no weaknesses of the Extranet; however, a few commented on the manpower and time needed to keep the site current as well as the reliance on IT staff to upload information in a timely fashion. A few also noted the redundancy of the site; for example, "Once the provincial site [was] up [there was] no need for the Extranet." One participant highlighted a problem with Internet access: " [The] web network went down on one occasion during [the] crisis and delayed [the] info getting out."
Respondents mentioned that using an electronic system for communication during an emergency was a good learning experience. Some noted areas for improvement: They wanted technical support to be available (help-desk) for participants who might have difficulties accessing the secure web site. They also identified a need for a system that can be activated immediately in future public health emergencies.
Potential future use of a local Extranet
Thirty respondents identified features they would like to see on a future Extranet so as to enhance communication with the public health department. Of these, 19 (63.3%) wanted to use the Extranet as a mechanism to send private email to public health experts; 19 (63.3%) wanted to use it to share a database for surveillance of infectious diseases; 15 (50%) wanted to use it as a bulletin board communication system; 9 (30.0%) wanted online certificate courses on public health matters; and 1 (3.3%) wanted to use it as a mechanism for physicians to send immunization data. From the open-ended questions, eleven respondents mentioned that the Extranet could be used for communicable disease communication, surveillance and/or reporting, and two respondents believed that the Extranet could support preparedness for future public health emergencies.
Discussion
Our results showed that participants preferred email, face-to-face meetings, or phone communication to any Internet communication strategy. This is not surprising, considering these strategies are more interactive than visiting a typical web site. Respondents indicated that they wanted future Extranets to support private email to experts as well as have a bulletin board for feature use, validating our conjecture that they would prefer more interactivity. Two-way communication features may be important features to consider in the creation of future Extranet solutions so as to enhance interactivity. Further research is needed to measure the perceived value and impact of interactive technologies. In addition to having synchronous communication modes, such as a discussion board or email link, web-conferencing and real-time online communication using text chat may provide more effective interactive web-based solutions.
We can speculate on the various reasons why email was a preferred method of communication compared with web sites. An administrator likely views the use of email as part of his or her daily routine activity, but does not view searching web sites as a routine. Information is automatically 'pushed' to users in email, whereas users need to actively seek information from an Internet site. Furthermore, a web site is typically less interactive than email, with which users can easily reply to senders. Although the Hamilton Extranet had an email link, it was not used. The Extranet was likely not perceived by users as a two-way communication tool but, rather, a place to find information.
The Hamilton Extranet was rated low relative to other SARS web sites. Perhaps this was because the newly developed Hamilton Extranet had not had a chance to establish its credibility compared with long-established sites such as those from the CDC, WHO and the provincial MoHLTC. Despite this finding, the majority rated the Hamilton Extranet high overall, considering its own merits. Possible reasons for this positive opinion may be extrapolated from the findings: Given the most common reasons for visiting the site, the Extranet was perceived as useful to support local information needs such as meeting minutes, and Hamilton directives. In addition, until the provincial SARS site was launched, the Hamilton Extranet was the only source of provincial information. Once the MOHLTC website was launched, redundancy with Extranet information was inevitable; our findings supported this view. Also, provincial MoHLTC staff may not have been interested in specific local issues.
Despite the Extranet's shortcomings, the findings generally support the view that access to both local and provincial information is needed; however, better planning, coordination and alignment of local and provincial web communication strategies are required. It is essential to clarify without delay where the responsibility for communication begins and ends among local, provincial and federal public health agencies during emergencies.
The development of a local Extranet must be viewed as an evolutionary process. SARS Extranet users were moving away from email and fax communication to rely more on the Internet. The Extranet was built quickly to meet immediate local needs during the SARS emergency without taking time to fully engage end-users in design. Governance structures are needed to set guidelines about the practice of sharing passwords. Health units are thus urged to work with their communities before the next emergency to create a secure password-protected Internet communication system. Through such partnerships, local communities will feel more ownership and familiarity with the communication infrastructure.
Two areas of concern for a future Extranet are the security of the site and privacy of information. Many users shared with others both the information and the user IDs and passwords. Had there been sensitive information on the Extranet, a risk of improper access or release of information would have existed. These issues can be dealt with by more stringent security policies and technological improvements to the Extranet.
Looking beyond SARS, respondents were asked to respond to potential future Extranet applications for public health: Communication on communicable disease was the most common response. Not surprisingly, this response reflects the needs of the SARS Steering Committee, which was responsible for infection control during the SARS emergency. An Extranet also has the potential to include activities such as surveillance and public health education. In addition, an Extranet for use during non-emergency situations may create a useful infrastructure to support local communication in future emergencies.
This study has some important limitations. The survey was conducted three months after the initial SARS outbreak, so participants may not have accurately recalled their use of the site. This uncertainty is further complicated by the wide variety of SARS sites. The lack of statistical significance may be a result of the small size of the respondents. Finally, because the Extranet was developed quickly, it was not possible to plan for a rigorous evaluation. Qualitative evaluation methods would have been useful to answer questions about why participants rated the Hamilton site lower than other sites. Given more time for planning, set-up, and design of the Extranet, long-and short-term objectives would have been developed from which to frame a program evaluation. A more accurate system for tracking individual use would also have been incorporated. Better assessment of the human and financial costs of running the Extranet would have been a useful measure against the perceived value of the Extranet.
Conclusion
In conclusion, our study showed that interactive communication strategies – email, face-to-face meetings and teleconferences – were preferred over static web sites during the SARS outbreak. This finding has important implications for the future development and evaluation of web-based communication solutions. It is likely that more interactivity in web-based communication solutions would enhance their use and value. Although the CDC, WHO, and federal and provincial sources of SARS health information on the Internet were rated higher than that on the Hamilton Extranet, a local area Extranet appears to have an important role in supporting local information sharing and communication in an emergency situation. There is a need, however, for anticipatory planning, coordination and development of an Extranet communication system to ensure we are prepared for the next public health emergency. In addition, the findings indicate that users envisioned an expanded role of a local Extranet to support public health communication and education beyond the management of emergencies. Lessons learned from this study provide a foundation on which to build for future emergencies.
Competing interests
The authors have no competing financial interests with regard to any aspect of this work. Two of the authors (CK and GB) were involved in the development of the site and participated in various aspects of the research work (see authors contributions for details).
Authors' contributions
RV and HT conceived of the study and the design; all the authors, led by RV and HT, participated in the creation of the data collection tool. Data collection and entry was led by RV and NA. Data analysis of open-ended questions was conducted primarily by RV, HT and NA; however, CK and GB assisted in the interpretation of the results because they had a better understanding of the context of some participants' comments, having been involved in the development and maintenance of the site. Statistical analysis was conducted by NA. All the authors assisted in drafting the article and making edits, and they all contributed to the discussion and conclusion.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We would like to acknowledge support for this project from the City of Hamilton, Public Health and Community Services Department, Public Health Research and Development Program. We would also like to acknowledge the assistance of two McMaster nursing students, Liz Pawlowski and Debbie Woods, for their help with tracking the surveys and distributing them.
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Van Waes L Thinking aloud as a method for testing the useability of websites: The influence of task variation on the evaluation of hypeertext IEEE Transactions on Professional Communication 2000 43 279 291 10.1109/47.867944
| 15967040 | PMC1166558 | CC BY | 2021-01-04 23:52:11 | no | BMC Med Inform Decis Mak. 2005 Jun 20; 5:17 | utf-8 | BMC Med Inform Decis Mak | 2,005 | 10.1186/1472-6947-5-17 | oa_comm |
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BMC Med Res MethodolBMC Medical Research Methodology1471-2288BioMed Central London 1471-2288-5-181590449910.1186/1471-2288-5-18Research ArticleRecruitment of ethnic minority patients to a cardiac rehabilitation trial: The Birmingham Rehabilitation Uptake Maximisation (BRUM) study [ISRCTN72884263] Jolly Kate [email protected] Gregory Y [email protected] Rod S [email protected] Jonathan W [email protected] Deirdre A [email protected] Kaeng W [email protected] Andrew J [email protected] BRUM Steering Committee 1 Department of Public Health and Epidemiology, University of Birmingham, Edgbaston, Birmingham B15 2TT, England2 University Department of Medicine, City Hospital, Birmingham B18 7QH, England3 Department of Primary Care & General Practice, University of Birmingham, Edgbaston, Birmingham B15 2TT, England2005 17 5 2005 5 18 18 21 1 2005 17 5 2005 Copyright © 2005 Jolly et al; licensee BioMed Central Ltd.2005Jolly et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Concerns have been raised about low participation rates of people from minority ethnic groups in clinical trials. However, the evidence is unclear as many studies do not report the ethnicity of participants and there is insufficient information about the reasons for ineligibility by ethnic group. Where there are data, there remains the key question as to whether ethnic minorities more likely to be ineligible (e.g. due to language) or decline to participate. We have addressed these questions in relation to the Birmingham Rehabilitation Uptake Maximisation (BRUM) study, a randomized controlled trial (RCT) comparing a home-based with a hospital-based cardiac rehabilitation programme in a multi-ethnic population in the UK.
Methods
Analysis of the ethnicity, age and sex of presenting and recruited subjects for a trial of cardiac rehabilitation in the West-Midlands, UK.
Participants: 1997 patients presenting post-myocardial infarction, percutaneous transluminal coronary angioplasty or coronary artery bypass graft surgery.
Data collected: exclusion rates, reasons for exclusion and reasons for declining to participate in the trial by ethnic group.
Results
Significantly more patients of South Asian ethnicity were excluded (52% of 'South Asian' v 36% 'White European' and 36% 'Other', p < 0.001). This difference in eligibility was primarily due to exclusion on the basis of language (i.e. the inability to speak English or Punjabi). Of those eligible, similar proportions were recruited from the different ethnic groups (white, South Asian and other). There was a marked difference in eligibility between people of Indian, Pakistani or Bangladeshi origin.
Conclusion
Once eligible for this trial, people from different ethnic groups were recruited in similar proportions. The reason for ineligibility in the BRUM study was the inability to support the range of minority languages.
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Background
Proportional representation of research participants by race is a requirement of The National Institutes of Health in the USA [1]. However, no such requirement exists in the UK, but concern has been expressed about the low representation of patients from ethnic minority groups in clinical trials [2,3]. There are some examples of good practice with a representative proportion of trial participants from minority ethnic groups recruited from areas of the UK with a diverse ethnic population [4,5]. In addition, there is some evidence that once patients have met the inclusion criteria for a clinical trial, then equal recruitment of patients from different ethnic groups can be achieved [6-8].
Much research on ethnicity has focused on testing hypotheses about differences in outcome by race or ethnicity whilst the issue that participants of trials should represent the population that will be receiving an intervention has been relatively neglected. This is essential for the generalisability of the results [8,9], and to achieve this, trial participants need to mirror the demographic profile of the disease group being studied. Whether participants in trials of chronic disease management reflect the population who are affected by such disease is an issue of growing importance in the UK as the black and minority ethnic population ages.
There are some unanswered questions with respect to the low representation of patients from minority ethnic groups in clinical trials. Firstly, compared to the majority white population, are patients from ethnic minority groups less likely to meet the eligibility criteria for a trial (e.g. because of language)? Secondly, are patients from ethnic minority groups more likely to refuse to participate in randomized trials [10]? Birmingham is an excellent place to explore this issue – some 30% of the population are from black and minority ethnic groups [11]. We have the opportunity to address these questions in relation to the Birmingham Rehabilitation Uptake Maximisation (BRUM) study, a randomized controlled trial (RCT) comparing a home-based with a hospital-based cardiac rehabilitation programme in a multi-ethnic population in the UK [12].
Methods
The BRUM study recruited 525 (26.3%) participants from the 1997 patients who presented following a myocardial infarction, percutaneous transluminal coronary angioplasty (PTCA) or coronary artery bypass graft (CABG) surgery to one of four West Midland hospitals from February 2002–January 2004 [12]. The hospitals are located in an area of the UK with a high proportion of people of South Asian origin. The BRUM study sought to include patients who spoke either English or Punjabi (which is the most frequently spoken minority language locally), but was unable to include patients with other minority languages because of the lack of validated outcome measures in these languages.
Basic demographic data including self-defined ethnic group was sought on all presenting patients. Ethnic status was recorded using the same categories as the 2001 UK Census [13]. Data on ethnicity was obtained for 1933 (96.8%) of the patients presenting following a heart attack, PTCA or CABG, following which the ethnic categories were then combined into three broad groups: White, South Asian (Pakistani, Indian and Bangladeshi) and Other (Caribbean, African, Chinese). No-one described themselves as being of mixed ethnicity. Data on individual social class was not collected on all presenting patients, so postcodes were used to assign the Index of Multiple Deprivation 2004 (IMD) [14]. Details about the eligibility of patients, reasons for exclusion from the trial, and reason for declining to participate were collected. Data were entered into a Microsoft Windows ACCESS database (Microsoft Inc, USA) and analysed using SPSS version 12 (SPSS Inc, Chicago USA). Chi-squared tests were used to compare proportions across ethnic groups. For normally distributed data the one-way ANOVA was used to compare means, and the Kruskal-Wallis test to compare means for non-parametric data. Logistic regression was used to determine relative risks of eligibility and recruitment adjusted for sex, age, deprivation index and initial diagnosis.
Results
Table 1 details the proportion of patients excluded and the reasons given. Reasons for non-recruitment are divided into exclusion criteria (e.g. language, severe disease, co-morbidity) and refusal. Significantly more South Asian patients were excluded (51.9%), but mainly for the reason of language (24.3%). Exclusions for co-morbidity were lowest in the South Asian patients (6.8% v 12.1% of white ethnic group), probably as a result of their significantly lower mean age (see table 1).
Table 1 Recruitment to and exclusion from the BRUM trial, by ethnic group
White South Asian Other fp-value
All presenting, n 1452 412 69
MI n (%) 811 (56.4) 230 (56.5) 42 (61.8) 0.6
PTCA n (%) 514 (35.7) 149 (36.6) 19 (27.9)
CABG n (%) 113 (7.9) 28 (6.9) 7 (10.3)
Mean (SD) age 66.3 (12.0) 61.0 (12.4) 66.5 (12.2) <0.001
Males n (%) 998 (68.9) 311 (75.5) 47 (69.1) 0.04
Mean (SD) Index of Multiple Deprivation (IMD) [14] 32.9 (17.1) 43.7 (16.6) 48.3 (16.8) <0.001
Excluded n (%) 525 (36.1) 214 (51.9) 25 (36.2) <0.001
Reason for exclusion (more than 1 may apply) n (%)
aLanguage 4 (0.30) 120 (29.4) 3 (4.5) <0.001
bHigh cardiac risk 191 (13.2) 50 (12.1) 6 (8.7) 0.7
cComorbidity 175 (12.1) 28 (6.8) 6 (8.7) 0.01
In other trial 18 (1.2) 6 (1.5) 2 (2.9) 0.7
Out of area 76 (5.2) 18 (4.4) 2 (2.9) 0.4
dSensory deficit 37 (2.5) 8 (1.9) 1 (1.4) 0.6
eMental health 41 (2.8) 6 (1.5) 5 (7.2) 0.06
Deceased 19 (1.3) 5 (1.2) 0 (0) 0.6
Excluded for language only n (%) 2 (0.1) 100 (24.3) 3 (4.35) <0.001
Eligible n (% of all presenting) 927 (63.8) 198 (48.1) 44 (63.8) <0.001
Males (% of presenting males) 659 (65.7) 163 (52.4) 32 (68.0)
Female (% presenting females) 268 (59.0) 35 (34.7) 12 (54.6)
Mean (SD) IMD of eligible patients 32.3 (16.6) 41.2 (16.7) 47.1 (17.3) <0.001
Refused (% of eligible) 514 (55.4) 109 (55.1) 27 (61.4) 0.2
Reason for refusal: n (%)
Did not want rehabilitation 85 (16.5) 10 (9.2) 3 (11.1)
Did not want to take part in research 272 (52.9) 38 (34.9) 11 (40.7)
Preference for hospital programme 62 (12.1) 15 (13.8) 3 (11.1)
No reason given 95 (18.5) 46 (42.2) 10 (37.0) <0.001
Total recruited (% of eligible) 418 (45.1) 88 (44.4) 19 (43.2) 0.7
(% of all) (28.8) (21.4) (21.3)
Males (% of eligible males) 312 (47.3) 76 (46.6) 14 (43.8)
Female (% of eligible females) 106 (39.6) 12 (34.3) 5 (41.7)
Mean (SD) IMD of recruited patients 31.6 (15.7) 40.0 (16.9) 47.0 (15.2) <0.001
aLanguage: unable to speak English or Punjabi, or Punjabi speaking nurse not available to recruit the patient.
bHigh risk: patients with risk factors excluding a home-based cardiac rehabilitation programme, e.g. severe angina, hart failure or documented arrhythmias.
cComorbidity: physical conditions which would interfere with the ability to undertake a cardiac rehabilitation programme, e.g. severe arthritis, malignancy.
dSensory deficit: severe hearing or visual loss such that patients would not be able to read written materials or communicate with research nurses on the telephone.
eMental health: patients with case-note documented dementia or current mental illness.
fChi square test (except for age comparison, oneway ANOVA; index of deprivation comparison, Kruskal-Wallis test)
Compared with the white ethnic group, the relative risk (95% CI) for eligibility was 0.42 (0.33, 30.55) for South Asians (table 2). However, there were no significant ethnic differences in the proportion of eligible patients recruited to the trial (45.1% of whites, 44.6% of South Asians, see Table 1). Compared with the British white ethnic group, the relative risk of recruitment of eligible patients was 0.59 (0.44, 0.78) for South Asians, when adjusted for age, gender, deprivation index and initial diagnosis.
Table 2 Eligibility and recruitment BRUM by ethnicity, adjusted for age, sex, deprivation and initial diagnosis
Ethnic group Adjusted RRa of eligibility 95% CI Adjusted RR of recruitment 95% CI
White British 1.00 (base) 1.00 (base)
Other White 1.52 0.81 to 2.84 01.10 0.60 to 2.02
South Asian 0.42* 0.33 to 0.55 0.59* 0.44 to 0.78
Afro-Caribbean 1.28 0.70 to 2.35 1.03 0.54 to 2.00
Other 0.86 0.16 to 4.19 0.97 0.23 to 4.21
White British used as the reference group (RR = 1.0)
aRR: relative risk
*P < 0.001
In each ethnic group (white, South Asian and other) women were less likely to be eligible for the study than men (table 1). This was statistically significant for the white and South Asian groups. Overall the women presenting with ischaemic heart disease were significantly older than the men (mean age 69.2 versus 63.6 years for men, p = 0.002) and were more likely to be excluded because of co-morbidity (16.8% v 8.6% of men, p < 0.001). Women of white and South Asian ethnicity were significantly less likely than the men of these ethnic groups to be recruited once eligible (p < 0.01) (table 1). There was no difference given in the reasons for declining to participate in the study by men and women.
The reasons for and rates of exclusion varied markedly between the three main South Asian groups, with exclusion rates of 29%, 63% and 70% among people of Indian, Pakistani and Bangladeshi origin respectively (Table 3). Figures for exclusions due to language barriers ranged from 5.1% (Indian) to 55% (Bangladeshi). In each South Asian sub-group the exclusion rate for language barriers was higher in people aged 65 years or more with 8.2% of people of Indian ethnicity, 43.6% of Pakistani and 87.5% of people of Bangladeshi ethnicity aged 65 years or more excluded because of language. This was approximately double the exclusion rates in people aged less than 65 years (see Table 3). A similar picture emerges for women, with higher exclusion rates for women in each of the South Asian sub-groups. Compared to people of white British origin, people of Pakistani and Bangladeshi origin were at a 4-fold increased risk of ineligibility to the trial, whilst people of Indian ethnicity were no more likely to be ineligible (see Table 3).
Table 3 Exclusions by ethnic group within the South Asian population presenting with a cardiovascular event
Indian
n (%)
n = 136 Pakistani
n (%)
n = 238 Bangladeshi
n (%)
n = 20
Mean (SD) age, years 61.0 (12.6) 61.0 (12.4) 63.1 (7.3)
Males n, (%) 96 (70.6) 184 (77.3) 15 (75)
Mean (SD) IMD score 35.95 (15.8) 47.8 (15.7) 48.9 (14.4)
Any exclusion 40 (29.4) 151 (63.4) 14 (70)
Reason for exclusion (more than one may apply) n (%)
aLanguage 10 (7.5) 94 (39.8) 11 (55)
bHigh cardiac risk 15 (11) 33 (13.9) 1 (5)
cComorbidity 8 (5.9) 19 (8.0) 0 (0)
Excluded for language only: 1
All n (%) 7 (5.1) 78 (32.8) 1 (55)
Aged <65 years 3 (3.4) 34 (24.8) 4 (40)
65+ 4 (8.2) p = 0.2 44 (43.6) p = 0.002 7 (87.5) p = 0.2
Gender males 4 (4.2) 51 (27.7) 6 (40)
females 3 (7.5) p = 0.4 27 (50) p = 0.003 5 (100) p = 0.04
Total recruited n 50 31 4
(% of eligible) (52.1) (35.6)
(% of all presenting) (36.8) (13) (20)
RR of ineligibility (95%CI)* 0.89 (0.6, 1.3) 4.0 (2.9, 5.5) 4.4 (1.6, 11.8)
aLanguage: unable to speak English or Punjabi, or Punjabi speaking nurse not available to recruit the patient.
bHigh risk: patients with risk factors excluding a home-based cardiac rehabilitation programme, e.g. severe angina, heart failure or documented arrhythmias.
cComorbidity: physical conditions which would interfere with the ability to undertake a cardiac rehabilitation programme, e.g. severe arthritis, malignancy.
SD: standard deviation
*adjusted for age, sex and deprivation index using white British as the reference group (RR = 1.0)
Discussion
This report has detailed the process of recruitment to a randomized controlled trial for patients from different ethnic groups from presentation with a cardiac event, through the eligibility criteria and consent process.
We have demonstrated that the point of inequality in recruitment between ethnic groups in this study occurred because of an inability to support the range of minority languages. This was despite additional measures employed to recruit Punjabi speaking patients, including a Punjabi speaking research nurse, translated (and recorded) patient information and the translation and validation of the Hospital Anxiety and Depression Scale into Punjabi. It is possible that those excluded on the grounds of language would have refused to participate in the trial, but we have no evidence to support or refute this possibility. Of those excluded as a result of language, 74% described themselves as of Pakistani origin and thus would have been catered for by the provision of Urdu and Mirapuri (an oral only language used by people of rural Pakistani origin). The socio-economic status of the presenting patients this is clearly a potential confounding factor, with people from minority ethnic groups who do not speak English being more likely to be of lower socio-economic status [15]. Presenting patients from minority ethnic groups had significantly higher deprivation indices than the white ethnic group, using the IMD as a measure of socio-economic status, in this study. Whilst the level of deprivation was a significant factor in the multiple regression analysis, ethnicity was still a significant factor in the likelihood of eligibility and recruitment to the trial when deprivation was included in the regression model.
We found no significant differences in the proportions of people from the three main ethnic groups who gave the reason for their refusing to participate as 'not wishing to take part in research.' This result differs from the evidence from the USA, which suggests that people from minority ethnic groups are less likely to agree to participate in clinical research because of a lack of trust [16]. The UK does not have the past history of the Tuskegee Syphilis study which appears to be the cause of this mistrust of research in black and minority ethnic groups in the US [17,18]. It is true that in this study a higher proportion of the minority ethnic groups who declined to participate in the trial gave no reason for their decision, so it is possible that there were differences in the reasons for not-participating that we failed to identify.
In addition, our findings provide further support for the limited evidence base that patients from ethnic minority groups can be recruited in a similar proportion to the majority population, once they have fulfilled the trial's eligibility criteria [7,8]. Other studies have tried to retrospectively estimate the proportion of patients from different ethnic groups, who would have been potential trial participants [19]. However, we believe that this is the first published report that has detailed the whole recruitment process of cardiac patients presenting with the index conditions, reasons for ineligibility and rates and reasons for refusal to participate. In the BRUM trial, the eligible patients of South Asian ethnicity were no more likely to decline to participate on the grounds of not wishing to take part in a research study than other ethnic groups.
Conclusion
This research raises the issue of feasibility versus inclusivity. In this study, even with considerable effort, time and resources to try to recruit a representative population to the trial, we were unable to meet the wide language requirements. This must be considered in the context that this was a trial of a behavioural intervention with a psychological questionnaire as a main outcome measure. In other trials, with only objective clinical measurements as endpoints, it might be possible to exclude less patients due to language restrictions. In a geographical area of ethnic diversity, a number of languages would need to be supported to achieve equivalent recruitment rates for different ethnic groups, which has considerable cost and methodological implications. The importance of achieving a sample that is ethnically representative will depend on the research question and the context.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All the authors were involved in the design and conduct of the trial. KJ undertook the analysis and drafted the paper. All the authors read and commented on the paper.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The BRUM study is funded by the NHS HTA Programme. The views and opinions expressed in this paper do not necessarily reflect those of the Department of Health.
The BRUM Steering Committee consists of Jolly K (Principal investigator), Lip GYH, Sandercock J, Greenfield SM, Raftery JP, Mant JW, Taylor RS, Lee KW, Lane D, Stevens AJ.
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| 15904499 | PMC1166559 | CC BY | 2021-01-04 16:32:51 | no | BMC Med Res Methodol. 2005 May 17; 5:18 | utf-8 | BMC Med Res Methodol | 2,005 | 10.1186/1471-2288-5-18 | oa_comm |
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BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central London 1471-2474-6-231590451510.1186/1471-2474-6-23Research ArticleInfluence of species and anatomical location on chondrocyte expansion Akens Margarete K [email protected] Mark B [email protected] Comparative Orthopaedic Research Laboratory, Dept. of Clinical Studies, University of Guelph, Guelph, Ontario, N1G 2W1, Canada2005 17 5 2005 6 23 23 20 10 2004 17 5 2005 Copyright © 2005 Akens and Hurtig; licensee BioMed Central Ltd.2005Akens and Hurtig; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Bovine articular cartilage is often used to study chondrocytes in vitro. It is difficult to correlate in vitro studies using bovine chondrocytes with in vivo studies using other species such as rabbits and sheep. The aim of this investigation was to study the effect of species, anatomical location and exogenous growth factors on chondrocyte proliferation in vitro.
Methods
Equine (EQ), bovine (BO) and ovine (OV) articular chondrocytes from metacarpophalangeal (fetlock (F)), shoulder (S) and knee (K) joints were cultured in tissue culture flasks. Growth factors (rh-FGFb: 10 ng/ml; rh-TGFβ: 5 ng/ml) were added to the cultures at days 2 and 4. On day 6, cells were counted and flow cytometry analysis was performed to determine cell size and granularity. A three factor ANOVA with paired Tukey's correction was used for statistical analysis.
Results
After 6 days in culture, cell numbers had increased in control groups of EQ-F, OV-S, OV-F and BO-F chondrocytes. The addition of rh-FGFb led to the highest increase in cell numbers in the BO-F, followed by EQ-F and OV-S chondrocytes. The addition of rh-TGFβ increased cell numbers in EQ-S and EQ-F chondrocytes, but showed nearly no effect on EQ-K, OV-K, OV-S, OV-F and BO-F chondrocytes. There was an overall difference with the addition of growth factors between the different species and joints.
Conclusion
Different proliferation profiles of chondrocytes from the various joints were found. Therefore, we recommend performing in vitro studies using the species and site where subsequent in vivo studies are planned.
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Background
In vitro studies regarding chondrocyte metabolism and expansion are often performed using bovine chondrocytes [1-3]. These chondrocytes are harvested from the metacarpophalangeal (fetlock) joint of slaughter-age cattle (18 months old or younger), since the distal limb is not used for meat production. However, in vivo animal studies not only tend to be performed in other animals, such as rabbits [4-9] and sheep [10-14], but also tend to use a different joint. Rather than the fetlock joint used in in vitro studies, the knee joint is used for in vivo animal studies [11,12], since it is frequently affected by osteoarthritis in humans [15,16].
Animal models of osteoarthritis are used as a bridge between mechanistic cell biology studies and phase 1 trials in human patients [17,18]. In most cases, laboratory animals such as the rabbit are used for initial studies because of their small size, low cost and faster progression of osteoarthritis. However, lapine (rabbit) cartilage is very thin, the tissue available for analysis is limited and this species retains intrinsic repair abilities at maturity [19,18,20]. Furthermore, smaller laboratory animals maintain a markedly flexed knee joint position at rest, whereas larger species have knee joint angles that are closer to that of the human knee [21]. It is therefore common that larger animals such as sheep, goats or horses are used to establish efficacy in models where serial synovial fluid analysis, topographical analysis of joint cartilage, and semi-invasive surgery are possible. It thus can be seen that in vitro and in vivo animal studies seldom use cartilage from the same species and anatomic location.
There are numerous cartilage repair treatments, including cell-based strategies such as the implantation of autologous chondrocytes (ACI) or engineered tissues [22]. ACI is performed with chondrocytes taken from a small biopsy, which are expanded in vitro [23]. Growth factors are added to chondrocyte cultures to prevent de-differentiation and to increase cell numbers [24,8,26]. It has been found that chondrocytes in the ankle and knee joints react differently to cytokine stimulation (IL-1) in rats and humans [27,28]. However, not much is known about the effect of growth factors on chondrocytes from different anatomical locations. In this study, two different growth factors were used in chondrocyte cultures: transforming growth factor β (TGFβ) and basic fibroblast growth factor (FGFb).
Transforming growth factor β (TGF-β1) is a pleiotropic cytokine that has many effects on chondrocytes. TGF-β1 can control cell proliferation, differentiation, and extracellular matrix (ECM) synthesis, as well as the biological activities of other growth factors [29]. Its effects on articular chondrocyte proliferation can be either stimulatory or inhibitory, depending on culture conditions, time of TGF-β1 addition to the culture, and state of cellular differentiation. Vivien et al. [30] and Fortier et al. [31] showed that TGF-β1 inhibited the growth of cells with 2% foetal calf serum (FCS), whereas TGF-β1 in media with 10% FCS caused a growth increase. Furthermore, the number, type and specificity of cytokine receptors, and their reaction to stimuli, may vary between joints. It has been shown that the size of the type II TGF-β receptor differs between freshly isolated and cultured bovine chondrocytes by 15 kD [32]. Also, Glansbeek et al. [2] found a species specific difference in chondrocyte expression of type II TGF-β receptor isoforms between murine, human and bovine cartilage. The murine cartilage taken from the patella expressed almost equal amounts of TGF-βbRII1 and TGF-βbRII2 mRNA, while human cartilage from femoral condyles expressed about three times more TGF-βbRII1 than TGF-βbRII2. In bovine articular cartilage from the metacarpophalangeal joint, only mRNA of TGF-βbRII1 was found. Studies have shown that in lapine chondrocytes, expression of TGF-β1 receptor systems is dependent on the stage of the cell cycle [30].
Basic fibroblast growth factor (FGFb) has been shown to prevent chondrocyte de-differentiation in culture [3] and decrease the doubling time of chondrocytes in monolayer cultures [33,25]. Chondrocytes, which have become fibroblast-like cells, are elongated and contain a distinct F-actin fibrillar structure. Martin et al. [3] concluded that chondrocytes treated with FGFb did not develop thick F-actin fibers, but instead had F-actin configurations resembling those of differentiated chondrocytes. The F-actin configuration is important since it influences the shape of the chondrocytes. FGFb was found to have a positive effect on lapine chondrocytes embedded in agarose grafts. These grafts produced better results in a modified histological O'Driscoll score compared to TGFβ or BMP treated agarose grafts [8].
This investigation studied the effect of species, anatomical location, and exogenous growth factors on chondrocyte proliferation in vitro. The large animal species and sites commonly used for harvesting chondrocytes chosen for this study were: the equine and ovine knee, shoulder and fetlock joints, as well as bovine fetlock joints. Equine cartilage was included in the study because the equine knee joint can be affected by naturally occurring disease. Furthermore, its thickness, which is similar to that of the human knee, allows for surgical manipulations [34,35]. The ovine knee is a common animal model used for cartilage repair studies [36,12]. The ovine shoulder joint provides more cartilage for in vitro studies than the fetlock joint and was also used as a third joint for comparison. The equine and ovine fetlock joints were compared to the bovine fetlock joint since it is commonly used for in vitro studies.
Methods
Chondrocytes and culture
Articular cartilage was harvested under sterile conditions from equine (EQ) and ovine (OV) shoulder (S), knee (K) and fetlock (F) joints, as well as from bovine (BO) fetlock joints within 24 h of death (Table 1). Animals between 1 – 8 years of age were available from the necropsy room or slaughterhouse, respectively. The animals had no joint or systemic disease (such as endotoxemia), which could affect chondrocyte metabolism. Cartilage samples from donors of different ages were not pooled.
Table 1 Distribution of sample collection
Sheep (OV) Horse (EQ) Cattle (BO)
Knee (K) 2 5 0
Shoulder (S) 4 3 0
Fetlock (F) 3 3 3
The chondrocyte isolation protocol was the same in all species. Cartilage was removed from articular surfaces, washed with DMEM/F-12 (Invitrogen™, Burlington, Canada) containing 100 U penicillin, 100 μg streptomycin and 0.25 μg amphotericin B (Invitrogen™, Burlington, Canada), then minced into 1 × 2 mm pieces. The cartilage pieces were incubated with pronase E (Sigma, Oakville, Canada) (25 U/ml DMEM/F-12, and 8 ml/g cartilage) for 1.5 h at 37°C and 5% CO2, washed, and digested with bacterial collagenase type II (Sigma, Oakville, Canada)(400 U/ml DMEM/F-12 + 10 % foetal bovine serum (FBS)(Sigma, Oakville, Canada) and 8 ml/ g cartilage) for 3 h at 37°C and 5 % CO2. The cells were separated from undigested material through a sieve, centrifuged at 200 × g for 10 minutes to form a pellet, washed twice and resuspended with DMEM/F-12 + 10 % FBS. Cells were seeded at a density of 0.5 × 106/ml (± 0.025 × 106/ml) and 4 ml DMEM/F-12 + 10 % FBS per 25 cm2 flask.
Growth factors
After 2 days in culture, the media was changed and either 10 μg/ml recombinant human basic fibroblast growth factor (rhFGF-b) (R & D Systems, Inc., Minneapolis, USA), 5 μg/ml recombinant transforming growth factor β (rhTGF-β) (R & D Systems, Inc., Minneapolis, USA) or both factors were added twice every other day. Chondrocytes without added growth factors served as a control. The media in the control groups was changed at the same time as that in the growth factor treated groups. In each experiment, each group consisted of two flasks, except for chondrocytes from the ovine fetlocks. Due to the size of the ovine fetlock joint, fewer cells were collected, thus producing only one flask.
Cell count
On day 6, the culture was 80–90% confluent and the chondrocytes were removed from all culture flasks by trypsinization, centrifuged at 200 × g for 10 minutes and washed twice with DMEM/F-12 medium. Day 6 was chosen for this procedure because the de-differentiation of chondrocytes, which has been observed in monolayer cultures, is still not very advanced at this stage [33,37]. The collagen type II to collagen type I ratio as an indicator for de-differentiation experienced the biggest changes in monolayer cultures between weeks 1 and 2 [37]. Before proceeding with further experiments, the influence of growth factors and cell culture conditions on the first passage was assessed. Cell count and cell viability were performed with the trypan blue exclusion test.
Flow cytometry
Flow cytometry analysis of fluorescent dye labelled chondrocytes (data not shown) stimulated with FGFb and TGF-β1 revealed an influence of the growth factors on cell size and granularity. The morphological profile of a cell can be observed by combining forward light scatter (FSC) and orthogonal or side light scatter (SSC). The FSC measurement is related to the cell size and can change through cell cycle progression and activation. Measurement of light scattered at a 90° angle, SSC, is related to internal granularity. Changes in these parameters were also found in the non-labelled cells and therefore flow cytometry analysis was performed in this investigation to study influence of growth factors on cell activation (Figure 1). Changes in cell morphology were confirmed with a cytospin slide preparation of the chondrocytes stained with Wright's stain (Figure 2). The cell size and granularity were analysed by flow cytometry with 0.5 × 106/ml cells in serum-free DMEM/F-12. The analyses were performed with a FACScan (Becton Dickinson, Oakville, Canada) by gating 10,000 events. The settings were as follows: excitation: 488 nm; emission: 585 nm; forward side scatter (x - axis) – photodiode voltage set to E00, AmpGain set to 1.00, Lin; side scatter (y - axis) – photo multiplier voltage set to 410, AmpGain 1.12, Lin. The same settings were used throughout the whole experiment for all species and joints. The results of FSC and SSC measurements are given in the mean value of channels. A channel is a measured value of a parameter, representing the signal intensity of an event after amplification. To appear on a plot (FSC: x- axis; SSC: y-axis), data for an event must fall into one of 1024 channels. The mean value is calculated from 10,000-gated events. A conversion from the channel value to cell size in μm was not performed.
Figure 1 Density plots (FACS) of equine chondrocytes after 6 days in culture and/or two stimulations with growth factors (FSC: forward scatter (size); SSC: sideward scatter (granularity))
Figure 2 Cytospin slide preparation of equine chondrocytes stained with Wright's stain (Mag. 100×)
Statistical analysis
A random block design was used in this study. The cell proliferation rate (increase in cell numbers), cell size and cell granularity of growth factor treated chondrocytes were compared to chondrocytes free of any treatment (control group). The results of the equine and ovine shoulder and knee joints were analysed separately from bovine, equine and ovine fetlock joints. A three factor ANOVA with paired Tukey's Correction was used for statistical analysis. The level of significance was p ≤ 0.05.
Results
Cell number
After 6 days in culture, cell numbers had increased slightly in control groups for EQ-F (equine fetlock), OV-S (ovine shoulder), OV-F (ovine fetlock) and BO-F (bovine fetlock) chondrocytes in contrast to EQ-K (equine knee), EQ-S (equine shoulder), and OV-K (ovine knee), where no increase in cell numbers was found. The cell viability was over 95 % similar to chondrocyte viability immediately after isolation. Chondrocyte monolayer cultures had variable proliferation profiles after the addition of FGFb and/or TGF- β. The addition of FGFb led to a significant increase in cell numbers for all species and joints (Table 2). The highest increase in cell count was found in BO-F, followed by EQ-F and OV-S chondrocytes cultures, as compared to the control groups. The addition of TGF-β increased cell numbers significantly in EQ-F (p = 0.0004) and in EQ-S (p = 0.0002) chondrocyte cultures, but showed nearly no mitogenic effect on EQ-K, OV-K, OV-S, OV-F and BO-F chondrocytes. Addition of both growth factors (FGFb and TGF-β1) to equine chondrocytes from all tested joints resulted in a higher increase in cell numbers compared to that from the addition of FGFb alone. In contrast to these findings, increases in cell numbers were lower in ovine and bovine chondrocyte cultures when both growth factors were added, compared to the addition of FGFb alone.
Table 2 Comparison of cell number, cell size and granularity of growth factors treated chondrocytes to control groups
Cell number FGFb TGF-β1 FGFb/TGF-β1
EQ-F 0.003 ↑ 0.0004 ↑ < 0.0001 ↑
EQ-S 0.0031 ↑ 0.0002 ↑ < 0.0001 ↑
EQ-K 0.1500 0.7494 0.0077 ↑
OV-F 0.016 ↑ 0.667 0.153
OV-S < 0.0001 ↑ 0.8037 0.0204 ↑
OV-K 0.1223 0.4245 0.1249
BO-F < 0.0001 ↑ 0.964 < 0.0001 ↑
Cell size
EQ-F 0.4983 0.4709 0.2509
EQ-S 0.8786 0.0061 ↓ 0.2194
EQ-K 0.0033 ↑ 0.4283 0.0008 ↑
OV-F 0.0343 ↓ 0.4044 0.9033
OV-S 0.6132 < 0.0001 ↑ < 0.0001 ↑
OV-K 0.4310 0.0041 ↑ 0.0041 ↑
BO-F 0.036 ↓ 0.001 ↓ < 0.0001 ↓
Cell granularity
EQ-F 0.0015 ↓ 0.0514 0.0009 ↓
EQ-S 0.0074 ↓ 0.2470 0.0020 ↓
EQ-K 0.0995 0.0098 ↑ 0.0273 ↓
OV-F < 0.0001 ↓ 0.0996 < 0.0001 ↓
OV-S 0.0092 ↓ 0.0005 ↑ 0.1356
OV-K 0.0008 ↓ 0.3767 0.0009 ↓
BO-F < 0.0001 ↓ 0.6367 < 0.0001 ↓
↑ : significant increase
↓ : significant decrease
Equine fetlock chondrocytes responded with the largest cell expansion to the addition of FGFb and FGFb/TGF-β whereas the knee chondrocytes expressed the lowest reaction to the growth factors. In the ovine species, the growth factors had the greatest effect on shoulder chondrocytes and fetlock chondrocytes, whereas knee chondrocytes responded with a lower proliferation rate. The bovine species showed the highest proliferation rate of all the tested species with respect to stimulation of fetlock chondrocytes with FGFb.
Cell proliferation rate of chondrocytes is age dependent. In this study, specimens from animals over 1 year of age were used and a trend of higher proliferation rates in younger animals was present, when compared to the older animals (up to 8 years of age). The influence of age on cell proliferation was significant, when data from animals < 1 year old were included (data not shown). Age did not influence the species and anatomical location differences or the chondrocyte reaction to growth factor treatment.
Cell size
Chondrocyte cell size was given in mean value of channels and was affected by the addition of growth factors (Table 2). FGFb (p = 0.0033), and the combination of both factors (p = 0.0008) increased the cell size significantly in EQ-K chondrocytes. Equine shoulder chondrocytes were significantly smaller after treatment with TGF-β1, compared to control cells. In contrast to the equine species, the cell size in ovine shoulder and knee chondrocytes increased significantly after the addition of TGF-β1 to the culture, as compared to the control groups. The average ovine fetlock chondrocyte was significantly smaller after the treatment with FGFb (p = 0.0343). Combined treatment with both growth factors led to larger shoulder (p < 0.0001) and knee (p = 0.0041) chondrocytes in the ovine species. All treatments in bovine cultures resulted in significantly smaller chondrocytes (FGFb: p = 0.036; TGF-β1: p = 0.001; FGFb/TGF-β1: p < 0.0001). An interesting additional observation, without further investigation, was that the ovine chondrocytes were generally larger and more granular than the equine chondrocytes.
Cell granularity
After the treatment with FGFb, all chondrocytes, except EQ-K, showed a significant decrease in cell granularity (Table 2). The same results were found for the combined treatment trials, except for the OV-S chondrocytes. The addition of TGF-β1 to the EQ-K and OV-S chondrocyte cultures resulted in an increase of granularity. Correlation between cell number, size and granularity was analysed using the Pearson Correlation Test and no relevant correlation was found.
Discussion
Distinctions in cartilage, related to anatomical location (i.e. from knee and ankle joints), in regard to catabolism have been found [38]. Furthermore, the frequency of human osteoarthritis (OA) differs between joints. It is most common in the hands, knees and hips, while occurring less frequently in ankle and shoulder joints [16,38]. Dieppe and Kirwan [16] listed several factors that influence susceptibility and severity of osteoarthritis. Several authors have reported differences between joints [27,38], but this factor is not always considered when observations from in vitro studies are transferred to in vivo studies. This investigation details the influence of species and anatomical location on chondrocyte proliferation, as well as the influence of growth factors on chondrocytes from different joints and animals.
Isolated chondrocytes are used for cartilage repair in human patients [23] and in tissue engineering. These techniques rely on in vitro expansion of chondrocytes from small biopsies. The goal is to expand the chondrocytes quickly and to maintain their chondrogenic potential and their ability to re-differentiate. To this end, different growth factors and growth factor combinations have been tested [33]. However, the differences related to anatomical location of the knee and hip chondrocytes were not accounted for [33]. In tissue engineering, cells are placed in a three dimensional matrix to form new cartilage [39,40,25]. However, the newly formed tissue can vary from species to species, despite the fact that the chondrocytes were taken from the same anatomical location [39]. These studies involved the encapsulation of bovine and ovine chondrocytes into a photo-polymerising hydrogel system and it was found that bovine chondrocytes produced more glycosaminoglycans, whereas the cell number increased in the ovine constructs.
It was evident in our study that chondrocytes from the fetlock have a higher proliferation rate than chondrocytes from the knee joint in horses, as well as in sheep. This result indicates that the chondrocyte cell cycle varies according to species and location. The doubling time of human chondrocytes in monolayer cultures ranges between 1.7 to 3.5 days [23,33], while in equine and bovine chondrocytes it is 5 – 6 days [41,1]. In our study, doubling of the chondrocytes could not be achieved within the 6-day culture time for the control groups. It would be of interest to study several time points to evaluate changes over time and find the optimal time point for the addition of growth factors and their combination for chondrocyte expansion. When several time points are studied, the differentiation state of the cells has to be considered, because the de-differentiation progress in monolayer culture changes dramatically between the first and second week [37].
Age and culture conditions are also factors to consider when working with isolated chondrocytes. Age is an important factor, since young animals and young humans have chondrocytes that proliferate much faster than chondrocytes from older animals and humans [22,26]. The influence of age on the proliferation rate was observed to be significant when analysing data obtained from young animals (data not shown). In animals over 1 year of age, a trend of higher proliferation rates in younger animals was present, when compared to the older animals (up to 8 years), but age did not influence the species and anatomical location differences or the chondrocyte reaction to growth factor treatment. Therefore, in this study, only specimens from animals over 1 year of age were taken, because at this age their growth is no longer exponential [42]. Sheep, horses and cattle reach skeletal maturity at the age of 3–4 years [43,44]. Preliminary experiments showed that cell number could not be maintained at a steady state in the control group without the addition of FBS. Thus 10% FBS was added to the chondrocyte cultures. When chondrocytes are expanded for later in vivo use, they are cultured with homologous serum. This was not an option in this study because the specimens were retrieved from the necropsy room or slaughterhouse.
Chondrocytes modify their cell shape from round to flat in monolayer cultures and increase their DNA synthesis [45]. Lee et al. [1] suggest that these cell shape changes affect responsiveness to IGF-1 due to receptor level changes or affinity of the receptors in flattened cells. A change of cell size and granularity was found after cells were twice treated with growth factors FGFb and TGF-β1, which are indicators for different cell activation stages. Further investigations are necessary to study the influence of cell size and granularity changes on intracellular signalling. Changes in granularity can be caused by an engorged endoplasmatic reticulum, as a consequence of increased intracellular protein production.
It should be noted that human recombinant growth factors were also used in this study, since only bovine FGFb is available. The amino acid homology between human growth factors FGFb and TGF-β1 and the tested animals is above the 60 % threshold, which is necessary for cross-reaction between species [46]. Furthermore, FGFb and TGF- β1 belong to the β-sheet based folds family, which makes cross-reaction between species more likely, since these are highly conserved cytokines. The amino acid homology to human growth factors is 98% for equine, 97% for ovine and 98% for bovine FGFb and 84% for equine, 87% for ovine and 86% for bovine TGFβ-1. Increased biological activity may be more influenced by the receptor binding capacity and efficiency [46]. If the differences in cell proliferation were related to species-specific cytokine – receptor-binding interaction, the same proliferation pattern within the tested species would be found. It is very unlikely that the same growth factor interacts with cell surface receptors differently in different joints within one species. Rather, the inconsistent results between joints of the same species indicate the presence of different numbers of receptors on the cell surface.
The varying response between joints can also be due to different types or number of receptors per cell. Bovine fetlock chondrocytes express only one type of TGF-β receptor (TGF-βbRII1) in contrast to murine and human knee chondrocytes, which express two types of receptors (TGF-βbRII1 and TGF-βbRII2) [2]. The expression rate of the TGF-βbRII receptor is higher in freshly isolated bovine metacarpophalangeal chondrocytes as compared to cultured chondrocytes [47]. These findings indicate that time of treatment may influence receptor expressions. It would be of interest to study whether the changes in receptor density could be avoided by three dimensional chondrocyte cultures.
A synergistic effect of TGF-β1 and FGFb is reported in the proliferation rate of human hip and ankle [33], as well as, in lapine knee chondrocytes [48]. In our study, the same effect was found on equine chondrocytes from all tested joints. In contrast, ovine chondrocytes from the various joints and the bovine fetlock chondrocytes did not demonstrate this effect. The proliferation rate in ovine and bovine chondrocyte cultures with the addition of both growth factors was lower than the cultures with FGFb alone. This synergistic effect is species specific, but is not related to the anatomical location of cartilage origin.
The signal pathways activated by growth factors are complex. TGF-β1 activates two independent signal pathways, which lead to the activation of different transcription factors [49]. It could be speculated that the smaller cell size is a result of freshly divided cells, but a correlation between cell number and size was not found in our study. To investigate the cause of the cell size and granularity changes after growth factor treatment, further studies such as cell cycle studies and/or intracellular matrix protein production studies would be necessary. It would also be of interest to analyse the type and number of FGFb and TGF-β receptors on the cell surface of the chondrocytes from different joints and their changes over culture time. A different number of cell surface receptors on chondrocytes may suggest that chondrocytes from one joint produce more matrix than chondrocytes from another joint. This factor would be an important consideration when harvesting chondrocytes for tissue engineering. For example, a smaller cartilage biopsy from the ankle joint would be necessary to produce the same amount of new tissue than a bigger biopsy from the knee joint [38].
Healthy chondrocytes from various joints within the same species proliferate differently, depending on the addition or absence of growth factors. This indicates varying capabilities of articular chondrocytes to react to stimuli, which is further supported by the catabolic differences found between human ankle and knee cartilage explants [38]. Therefore, one could expect that an in vitro study performed with fetlock chondrocytes regarding cell proliferation and tissue engineering gives different results when knee chondrocytes are used. Furthermore, the use of a different species could add another variable. The use of animal models to establish treatment strategies for osteoarthritis before clinical studies is very important since technical application difficulties and the development of associated joint pathology would be revealed in these models [22,20].
Conclusion
The animal model, site and the size of the lesion require careful consideration in making decisions about treatment strategy [22]. In vitro studies are important to adequately preparing for in vivo testing. Thus, in vitro studies should be performed using the species and site where subsequent in vivo studies are planned.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MKA made substantial contributions to conception and design as well as acquisition and interpretation of data.
MBH revised the article critically and gave final approval of the version to be published.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We would like to thank Mrs Michelle Beaudoin-Kimble and Mrs Nicole Kudo for technical assistance and Mrs Gabrielle Monteith for statistical analysis.
A Canadian Arthritis Network Fellowship and the Morris-Animal Foundation supported this work financially.
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| 15904515 | PMC1166560 | CC BY | 2021-01-04 16:32:03 | no | BMC Musculoskelet Disord. 2005 May 17; 6:23 | utf-8 | BMC Musculoskelet Disord | 2,005 | 10.1186/1471-2474-6-23 | oa_comm |
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BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-6-431596975210.1186/1471-2202-6-43Research ArticleAmplitude-dependency of response of SI cortex to flutter stimulation Simons Stephen B [email protected] Vinay [email protected] Joannellyn [email protected] Oleg V [email protected] Barry L [email protected] Mark [email protected] Departments of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA2 Cellular and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA2005 21 6 2005 6 43 43 7 2 2005 21 6 2005 Copyright © 2005 Simons et al; licensee BioMed Central Ltd.2005Simons et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
It is established that increasing the amplitude of a flutter stimulus increases its perceived intensity. Although many studies have examined this phenomenon with regard to the responding afferent population, the way in which the intensity of a stimulus is coded in primary somatosensory cortex (SI) remains unclear.
Results
Optical intrinsic signal (OIS) imaging was used to study the evoked responses in SI of anesthetized squirrel monkeys by 25 Hz sinusoidal vertical skin displacement stimulation. Stimuli were 10 sec duration with a 50 sec inter-stimulus interval. Stimulus amplitude ranged from 50 to 400 microns and different amplitudes were interleaved. Control levels of activity were measured in the absence of stimulation, and used to compare with activation levels evoked by the different stimulus amplitudes. Stimulation of a discrete skin site on the forelimb evoked a prominent increase in absorbance within the forelimb representational region in cytoarchitectonic areas 3b and 1 of the contralateral hemisphere. An increase in stimulus amplitude led to a proportional increase in the magnitude of the absorbance increase in this region of areas 3b and 1 while surrounding cortex underwent a decrease in absorbance. Correlation maps revealed that as stimulus amplitude is increased, the spatial extent of the activated region in SI remains relatively constant, and the activity within this region increases progressively. Additionally, as stimulus amplitude is increased to suprathreshold levels, activity in the surround of the activated SI territory decreases, suggesting an increase in inhibition of neuronal activity within these regions.
Conclusion
Increasing the amplitude of a flutter stimulus leads to a proportional increase in absorbance within the forelimb representational region of SI. This most likely reflects an increase in the firing rate of neurons in this region of SI. The relatively constant spatial extent of this stimulus-evoked increase in absorbance suggests that an increase in the amplitude of a 25 Hz skin stimulus does not evoke a larger area of SI neuronal activation due to an amplitude-dependent lateral inhibitory effect that spatially funnels the responding SI neuronal population.
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Background
The way in which stimulus intensity is represented in primary somatosensory (SI) cortex has remained an intriguing question in the study of the cortical correlates of perception. Although there have been numerous studies of the SI response to changes in stimulus intensity, few have focused on the response at the population level of analysis. Thus, a dearth of information about the global SI response to changes in stimulus intensity exists – current knowledge of the subject depends almost exclusively on reconstruction of predictions of the SI neuronal population response from afferent recordings [1-5] and single unit cortical recordings [6,7].
A number of studies have examined the global SI response using imaging techniques such as fMRI (functional Magnetic Resonance Imaging) [8-10] and MEG (MagnetoEncephaloGraphy)[11,12]. In general, results from these studies indicate that increases in stimulus intensity are accompanied by increases in the intensity of the evoked signal as well as increases in the activated volume of cortex. As a result these studies predicted that amplitude might be coded not only by the average firing rates of individual SI neurons, but also by the total aggregate of responding neurons. However, although each of these imaging techniques provide measures which are indirectly related to neuronal activity, their resolution is limited in two important ways. First, it is difficult to determine the nature of the neuronal activity being imaged (whether or not it is excitatory or inhibitory); and second, both fMRI and MEG studies have limited spatial resolution, which is typically on the order of ~1 mm2 [8,11].
Recently, Chen and colleagues used the optical intrinsic signal (OIS) to demonstrate that a proportionally greater (larger magnitude) response is evoked in SI of squirrel monkeys as the amplitude (as measured by force) of a skin stimulus is increased[13]. However, the primary focus of their report was that the response to simultaneous stimulation of multiple adjacent sites on the skin produced a smaller, more intense region of SI activation than would be normally predicted by summation of the two, and their findings did not detail the effects of amplitude on the dynamics of the response to a flutter stimulus at a single site on the skin. In this report, we extend the aforementioned work by imaging the OIS evoked in SI cortex of squirrel monkeys by a range of amplitudes of skin flutter stimulation. The results suggest that increasing the amplitude of a skin flutter stimulus evokes a proportionally larger absorbance increase in SI that remains confined to the same SI territory. In addition, it was found that increasing the amplitude of flutter evokes a large decrease in absorbance in the territory that borders the activated region of SI. Neurons in the SI region that demonstrate decreased absorbance in response to flutter stimulation are proposed to undergo stimulus-evoked inhibition and to contribute importantly to the SI processing of high-amplitude skin flutter stimuli.
Results
Figure 1(A–C) illustrates typical examples of the OIS response in SI of three different subjects in the absence of stimulation (control), and during low (50 μm) versus high (400 μm) amplitude stimulation. Each image shown in Figure 1 represents the sum of frames taken from the time of stimulus onset to 5 seconds after stimulus offset (frames 1–16). Areas of high absorbance are indicated by dark patches within each image; regions of high absorbance in each case correspond to the SI locus that represents the stimulated site on the skin. SI in each experiment underwent a larger increase in absorbance within the region of interest (ROI) in response to the 400 μm stimulus than evoked in the same region by the 50 μm stimulus. Moreover, in each subject the increase in absorbance appears more evenly distributed and less diffuse throughout the ROI under the 400 μm condition.
Figure 1 OIS response to low vs. high amplitude stimulation. OIS images taken from three subjects (A, B, C). All images are anatomically oriented as shown in the top left image. Images were obtained by averaging across 10 experimental trials and then summing frames taken from the time of stimulus onset to 5 seconds after stimulus offset to better show regions of high absorbance indicated by dark pixels. The left column shows responses acquired in the absence of stimulation (control), while the middle and right columns show the stimulus-evoked response to 50 and 400 μm respectively in the same subjects.
To identify the boundaries of the regions of increased absorbance spatial histograms were constructed. Figure 2 illustrates the average results obtained from all experiments (n = 5) as well as the methodology used to evaluate the spatial extent of the stimulus-evoked activation. In each experiment, the image was segmented along a line 6 mm long and roughly centered on the area of increased absorbance, as shown in the top panel. Pixels along the line were binned (bin size 40 × 200 μm) and absorbance values averaged and plotted as a function of distance along the line. The plots demonstrate that at all amplitudes of stimulation, the spatial extent of the region of above-background absorbance (ie. absorbance values larger than control) is similar and at every stimulus amplitude occupies a circular-shaped territory in SI between 1.8 – 2.24 mm in diameter. The ROI (to be used for further analysis) was therefore defined as the region displaying above background levels of absorbance within the activated region of SI.
Figure 2 Spatial histograms of activity at different amplitudes. Absorbances were measured at each amplitude along the red line shown in the OIS image at top left. Each plotted value represents an average of pixels spanning 100 μm above and below the line and a distance of 40 μm along the line (bin size was 40 × 200 μm). The control condition is plotted on each graph to indicate "background" levels absorbance. Dashed lines on plots indicate where stimulus-evoked activity crosses background absorbance levels (indicating the boundaries of above background absorbance). Histograms indicate no significant change in cortical territory displaying above background absorbance with respect to stimulus amplitude. Higher amplitude stimulation produces regions of below-background absorbance directly outside of the regions of above-background absorbance.
Figure 3 demonstrates (for one exemplary subject) the method used to evaluate the time course of stimulus-evoked SI absorbance. Panel A shows a green filter image of the cortical surface, which highlights the vasculature. Panels B&C are the OIS responses (dark regions) evoked by the low (50 μm) and high (400 μm) stimulus amplitudes respectively. The ROI is the circular territory enclosed by the dashed white lines. Absorbance values within the ROI were averaged for each amplitude of stimulation and plotted as a function of time. The time course of the absorbance values measured between 1 and 22 sec after stimulus onset is plotted for each of the stimulus conditions, and in the absence of stimulation ("control"). Arrows along x-axis of plots at bottom left of Figure 3 indicate stimulus onset (1 sec) and stimulus offset (11 sec), and reveal how absorbance increased with increasing amplitude of stimulation. For each stimulus amplitude absorbance is maximal near to stimulus offset.
Figure 3 Absorbance time course and anatomical registration in SI. A) Green filter image of SI cortex in vivo, used for anatomical registration of OIS images. B&C) Resulting OIS image obtained from averaging across 10 experimental trials and then summing frames taken from the time of stimulus onset to 5 seconds after stimulus offset: B) at stimulus amplitude 50 μm, C) at 400 μm. Dashed circles enclose the ROI within SI. Bottom left) Plot of the averaged absorbance. Arrows indicate time of stimulus onset (1s) and stimulus offset (11s). Absorbance values within the enclosed ROI were averaged and plotted at each amplitude as a function of time.
The analysis approach illustrated in Figure 3 was performed on all experiments (n = 5) and the resulting absorbance plots were averaged (Figure 4). Similar to Figure 3, the plots in Figure 4 demonstrate that absorbance increases with increasing stimulus amplitude. To quantify this relationship a measure of ΔAbsorbanceevoked was used. ΔAbsorbanceevoked was defined as the difference between the absorbance measured at 1 sec (prior to stimulus onset) and 11 sec (point of stimulus offset), and is shown in the plot at the bottom of Figure 4. The plot of ΔAbsorbanceevoked vs. amplitude is well described (coefficient of determination R2 = 0.9921) by the linear function (solid line) ΔAbsorbanceevoked = (4 × 10-6)*d + 0.0005. This type of analysis, however, gives little or no information about the spatial properties of the response.
Figure 4 A) Plots of absorbance and standard deviation averaged across all experiments (n = 5). All data was normalized prior to being averaged. Plotted absorbances were measured within the ROI which was defined as all pixels within a 1 mm radius of the center of activation (as defined from spatial histogram analysis). Controls in which no stimulus was administered are plotted simultaneously for comparison to test conditions. B) Plot of ΔAbsorbanceevoked which was defined as the change in absorbance measured from frame 1 (prior to stimulus onset) to frame 11 (point of stimulus offset). The plot is fit with a linear function (solid line) described by the equation A = (4 × 10-6)d + 0.0005 where A is absorbance and d is stimulus amplitude (displacement). Coefficient of determination for the linear regression (R2) is shown below.
Radial histograms were constructed to better visualize the spatiotemporal relationship of the OIS response at different amplitudes. The radial histograms shown in Figure 5 are representative time-space plots at each amplitude. From the ROI center (as determined by spatial histogram analysis), average absorbance values were determined for the pixels within concentric rings located at progressively larger distances from the center at each frame acquired. Absorbance values are color coded (red indicating areas of high absorbance, blue indicating areas of low absorbance) and plotted as a function of time and radial distance from the center of the ROI. Figure 5 demonstrates that the major differences that exist in the SI global responses to different amplitudes of stimulation are differences in the magnitude of absorbance and not the spatial properties of the absorbance pattern (this also is apparent in the spatial histogram analysis of Figure 2). As would be expected based on the absorbance curves shown in Figures 3 &4, higher stimulus amplitudes evoked a more intense and discrete region of increased absorbance than did the lower amplitudes. Interestingly, one of the more robust differences between low- and high-amplitude stimulation, is the magnitude of decreasing absorbance detected in the territory that surrounds the region in which absorbance increases. This response is most pronounced under the 400 μm condition where it can be seen to occur much sooner after stimulus onset at radial distances as small as 1.5 mm from the ROI center. Spatially, the regions of above- and below-background absorbance are nearly the same at each stimulus amplitude, with the above-background portion extending nearly 1 mm away in all directions from the center of activity, whereas the below-background portion of the response comprises a ring beginning at a radial distance of 1.5 mm from the ROI center and extending out beyond the area that was analyzed.
Figure 5 Radial time space plots. Radial histograms were performed on OIS data at all stimulus amplitudes. Radii were measured from the center of activation as demonstrated in the image at top-left. The dashed blue line is the maximum radial distance used in the maps shown. The schematic at bottom left indicates the anatomical orientation of the cortex in the image above. Absorbance values in maps shown represent an average at each radial distance at each frame after stimulus onset. Green arrows indicate stimulus onset (1s) while red arrows indicate stimulus offset (11s). Absorbance values are color coded as per the color bar shown at the right.
Using similar techniques to those we used to analyze above-background activity in the ROI (as in Figures 2 &3), regions outside the designated ROI were examined to determine whether a similar amplitude-dependency could be established for the time courses of the below-background absorbance observed in the surround. Figure 6 shows plots constructed from averaging the absorbance values in pixels lying 1.5 – 2 mm away from the center of the ROI. Data were normalized and then averaged across experiments (n = 5). It is apparent that the time courses at different amplitudes of stimulation are different with respect to the stimulus timing, (compared with above background levels of activity, which all show maximum absorbance at the point of stimulus offset). Accordingly, a different measure was adopted to quantify this relationship: In this case ΔAbsorbancemax was defined as the difference between the minimum absorbance and the maximum absorbance value obtained at any point during the recording. Interestingly, the relationship between stimulus amplitude and ΔAbsorbancemax in the surround is not linear (Figure 6b). Instead, each of the higher stimulus amplitudes employed (100–400 μm) evoked a very similar level of below-background absorbance. The sole difference between the different curves (Figure 6a) is the time required to reach the peak of the decrease in absorbance. That is, as amplitude is increased from 100 to 400 μm the point of minimum absorbance was attained earlier in time. This is also apparent in Figure 4.
Figure 6 Absorbance trends surrounding the ROI across five experiments. A) Plots of absorbance and standard deviation averaged across all experiments (n = 5). Plotted absorbances were measured at radial distances between 1 and 1.5 mm away from the center of the ROI (defined earlier). B) Plot of the maximal change in absorbance (solid line) as a function of stimulus amplitude. The linear trend, obtained from averaged absorbance measured within the ROI (dashed line from figure 3) has been simultaneously plotted on the axes to demonstrate the significant differences between the two regions.
Correlation maps were constructed to further characterize the spatial properties of the SI response to 25 Hz flutter. A correlation map compares every pixel in the image with the signal referenced from the ROI, and assigns a correlation coefficient (r) to the location of the pixel being compared. This gives a fairly good approximation of the signal at all locations in the image. Since there is no significant difference in the spatial properties between stimuli at intermediate amplitudes (as demonstrated by radial histograms) only the 50 and 400 μm amplitudes will be compared with this technique. Figure 7 shows correlation maps of the OIS responses to stimulus amplitudes of 50 and 400 μm (Top panels). The bottom panels of the figure show the input signal (solid dark red line) used for correlation of each pixel in the map, and the negative (opposite) of the input signal (dotted blue line). A coefficient of +1 (although it never appears in the map) indicates that a pixel's time course perfectly matches the input signal while a coefficient of -1 indicates that pixel's time course perfectly matches the opposite of the input signal (dotted blue line). At 50 μm the correlation map (color-coded image) shows that the correlation is weak and more dispersed within the ROI in area 3b. At the highest amplitude, however, there is a pronounced and well-defined positive correlation within the ROI that is more evenly distributed throughout the ROI. A large region of negatively correlated activity (corresponding to strong below background activity) surrounds the ROI in the high-amplitude map.
Figure 7 Correlation maps for stimulus amplitudes of 50 (left) and 400 (right) microns. The schematic at top-right indicates the anatomical orientation of the OIS image as well as all maps: A-anterior, M-medial, P-posterior, L-lateral. Color bars show coefficient of determination values for each map. The correlated signal, obtained from averaging of absorbance values within the ROI (encircled in the image at top-left) at each frame, is shown under each map (time course in solid red). Only the portion of the signal enclosed within the dashed lines (corresponding to the stimulus duration, green-on, red-off) is correlated. r values describe the degree of similarity between a given pixel's time course and the corrselated signal. Negative r values indicate that absorbance decreases to below-background levels. Thus, the signal in solid red represents the time course that would be observed in a pixel with an r value of +1 and the dotted blue line indicates the time course that would be observed in a pixel with an r value of -1.
To examine the spatial dynamics of the SI response in more detail we examined the patterns of activity generated by low- and high-amplitude stimulation in a 16 mm2 (4 × 4) area centered around the ROI. Figure 8 demonstrates the patterns of activity evoked at three time intervals during the delivery of the stimulus. The 3D surface plots show activity measured within the boxel indicated by the dashed box in the image at the top. In each 3D plot absorbance is plotted in two-dimensional space and is indicated by two measures: height of the peak along the z-axis (as shown in the schematic at the top right), and the color (indicated by the color bar to the right of each row of 3D plots). These data make it apparent that after a short period of stimulation (1 sec) the activity pattern is very similar for the different amplitudes. That is, at this early time interval both patterns are diffuse and occupy much of the ROI. However as stimulus duration increases, the pattern of increased absorbance evoked by high-amplitude stimulation tends to become restricted to the center of the ROI and within this region becomes homogeneous. Standard deviation was used to measure the variability within the ROI at low and high amplitudes. At 10s after stimulus onset, standard deviations for low- and high-amplitude surface plots were 0.1415 and 0.1166 respectively. Average standard deviation across all sets of maps (n = 5) differs very little from these values (0.1448 at low amplitude vs. 0.1201 at high amplitude). These differences suggest that within the ROI evoked absorbance levels are more homogeneous in response to high- vs low-amplitude stimulation. In addition, at 5 and 10s after onset of high-amplitude (but not low-amplitude) stimulation the territory surrounding the ROI becomes dominated by below-background changes in absorbance.
Figure 8 Spatial plots of activity evoked by low- (50 μm) and high-amplitude (400 μm) stimulation. The schematic at the top-left indicates the anatomical orientation of the cortex: A-anterior, M-medial, P-posterior, L-lateral. Absorbances were measured within the boxel shown in the top-middle panel. The schematic (top-right) shows how each frame is spatially represented with respect to the boxel of interest. Both stimulus amplitudes are mapped 1, 5 and 10 seconds after stimulus onset. Absorbance values at each pixel (x, y location) are represented two fold: both by their height along the z-axis, and by their color.
Discussion
This study evaluated the global SI response to different amplitudes of flutter stimulation by imaging the optical intrinsic signal (OIS). The OIS indirectly reflects both cortical neuronal spike discharge activity and the local, subthreshold changes in neuronal membrane potential evoked by sensory stimulation [14-16]. As a result, the observed tendency for absorbance in the same localized region of area 3b to increase with increasing stimulus amplitude (Figures 3 &4) most likely is due to the amplitude-dependence of the average firing rate of neurons in the same region [17]. An increase in SI absorbance in response to an increase in stimulus intensity has been reported previously by Chen et al [13]. The observed increase we observed in the stimulus-evoked SI OIS that accompanies increases in stimulus amplitude is well described by a linear function.
One important distinction between previous work done using the OIS and the present study is our use of near-infrared wavelengths for illumination during acquisition of the OIS. The OIS obtained using infrared light has been shown to be highly correlated with light scattering effects that accompany astrocyte swelling subsequent to the clearance of extracellular K+ and neurotransmitter [14,15,18] and local increases in blood volume [19,20]. Although the OIS at this wavelength may be influenced by changes in hemoglobin concentration and oxygenation, it is likely that contributions from these factors are small when compared with light scattering effects[20]. Additionally, OIS imaging using near-infrared illumination not only minimizes the contributions of artifacts introduced by changes in the vasculature (which can dominate the OIS at lower wavelengths) [19] but the time course of the OIS detected at shorter wavelengths (600 nm) is markedly different (shorter) than the protracted OIS observed in this study [13,15,19].
Previous studies conducted by this laboratory have reported that the SI optical response evoked by an extended period (>1 sec) of flutter stimulation not only consists of an increase in absorbance in the region that receives its input from the skin site that was stimulated, but also decreases in absorbance (frequently to levels well below-background) that occur in the surrounding cortex [21]. The present study demonstrates that the below-background component of the SI optical response to flutter stimulation is particularly evident at large stimulus amplitudes (figures 5, 7 &8). However, unlike the increase in absorbance evoked by flutter, the relationship between the magnitude of the stimulus-evoked decrease in absorbance and stimulus amplitude is not satisfactorily described by a linear function. Indeed, the results shown in figure 6 suggest that this component of the optical response to skin flutter is either absent or extremely small at small stimulus amplitudes, and remains maximal or near-maximal across a wide range of suprathreshold stimulus amplitudes. Interestingly, this stimulus-evoked decrease in absorbance (in the surround) appears later in time than the increases in absorbance (within the ROI) and as amplitude is increased it tends to develop earlier after stimulus onset.
The correlation maps shown in figure 7 provide a comprehensive overview of the time course of absorbance at each location in the image. The optical signal at each pixel is cross-correlated with a known input signal. In this case, it is the average absorbance measured within the ROI. The assigned coefficient of determination indicates the degree of similarity between a pixel's time course of absorbance and the input signal. Therefore pixels with a large positive correlation undergo increases in absorbance very similar to the input signal, while pixels with a large negative correlation undergo a decrease in absorbance which more closely resembles the opposite (negative) of the input signal. Figure 8 suggests that at high amplitudes of stimulation the ROI in SI becomes more homogeneously activated with longer stimulus duration. Some evidence for this is indicated by the large discrepancy (between low- and high-amplitude surface plots) in the standard deviations measured within the ROI. Further studies are required to investigate absorbance distribution and patterning within the ROI.
Examination of spatial histograms (figure 2) and the maps in figures 5 &7 also reveal that the size of the SI region that undergoes an increase in absorbance does not increase with increasing stimulus amplitude, but rather remains relatively constant. Regardless of stimulus amplitude, the activated cortical region appears circular in shape and occupies an area approximately 2 mm in diameter (figures 4 &6). Within the ROI average absorbance increases progressively with increasing stimulus duration. The dimensions of the SI region activated by flutter stimulation observed in this study contrast sharply with results presented in previous studies which demonstrated activation within a 1 mm region in diameter [13,22]. One possible explanation for this discrepancy is the level and type of general anesthesia used in the different studies. Previous studies have reported that anesthetics (e.g. ketamine) which block NMDA receptors or enhance GABAA receptor mediated inhibition (barbiturates), significantly reduce the dimensions of the receptive field of individual SI neurons; actions that would reduce the size of the responding SI neuronal population [23]. Chen et al. previously reported similar (~2 mm) sized regions of activation in response to flutter stimulation of the digit tips in squirrel monkey anesthetized with isofluorane, as well as showing in the same report that use of pentothal anesthetic confined the response to a much smaller region (~1 mm) [24].
It has been suggested that the amplitude of skin flutter stimulation is coded by both the number of activated SI neurons as well as by their level of spike discharge activity [1]. This suggestion is based largely in part on the fact that larger-amplitude stimuli, through transduction of the laterally-transmitted mechanical wave produced by sinusoidal skin displacement, recruit larger numbers of RA afferents and therefore lead to a spatially more widely distributed pattern of afferent input to SI cortex. Combined metabolic tracer and neurophysiological studies have shown that the initial response to a repetitive tactile stimulus occupies an extremely large cortical territory. As the repetitive mechanical stimulation is continued, however, the response is quickly sculpted by cortical inhibitory mechanisms, leading to an activity pattern that becomes confined to a relatively restricted region in SI [25-27]. The results obtained in the present study and results previously reported by other researchers, lead us to suggest that stimulus amplitude contributes importantly to the shaping (via lateral inhibitory mechanisms) of the SI response to protracted skin flutter.
Conclusion
This study investigated the SI response to flutter stimulation of the skin using the OIS. An increase of the amplitude of the flutter stimulus was associated with an increase in absorbance within the region of SI cortex that receives its input from the stimulated skin field. The relationship between the maximal change in absorbance and stimulus amplitude is well characterized by a linear function within the range of amplitudes studied. Measurement of the spatial extent of the activated SI region showed that higher amplitudes of stimulation do not produce a more extensive region of SI activation. Instead, as amplitude is increased, while average peak absorbance within the same ~2 mm diameter SI region increases with amplitude of stimulation, the region of surrounding cortex undergoes a prominent decrease (frequently to levels well below background) in absorbance. Further studies are required to establish the relationship between the effect of different amplitudes of skin flutter stimulation on SI absorbance and SI neuroelectrical activity.
Methods
Subjects & preparation
All methods and procedures are consistent with USPHS policies and guidelines on animal care and welfare in biomedical research. They were reviewed and approved by an institutional committee prior to initiation of the experiments. Experiments were conducted in 10 squirrel monkeys. Following induction of anesthesia with 4% halothane in a 50/50 mix of nitrous oxide (N2O) and oxygen, the trachea was intubated. A veterinary anesthesia machine (Forreger Compac-75) provided an anesthetic gas mix whose composition could be adjusted (typically 1.5–3.0% halothane in 50/50 N2O/oxygen) to maintain a stable level of surgical anesthesia. Methylprednisolone sodium succinate (20 mg/kg) and gentamicin sulfate (2.5 mg/kg) were injected intramuscularly to lessen the probability of halothane-induced cerebral edema and prevent bacterial septicemia, respectively. Placement of a valved catheter into a superficial hindlimb vein enabled administration of 5%glucose, 0.9%NaCl, and drugs.
A 1.5 cm opening was trephined in the skull overlying SI cortex. A recording chamber (25 mm i.d.) was placed over the opening and cemented to the skull with dental acrylic. Wound margins were infiltrated with local anesthetic, closed with sutures and bandaged, and the dura overlying SI was resected. After the completion of all surgical procedures subjects were immobilized with norcuron (loading dose 0.25–0.5 mg/kg,i.v.; maintenance dose 0.025–0.05 mg/kg/hr). From this point on, the animal was ventilated with a 50/50 mix of N2O and oxygen and the concentration of halothane was adjusted (typically between 0.5 and 1.0%) to maintain heart rate, blood pressure, and the EEG at values consistent with general anesthesia. Rate and depth of ventilation were adjusted to maintain end-tidal CO2 between 3.0 and 4.5%. Under these experimental/anesthetic conditions both SI neuron spontaneous and stimulus-evoked spike discharge activity patterns are highly reproducible over even prolonged (>1 hr) time periods.
OIS imaging and stimulus protocol
After obtaining a photograph of the exposed cortical surface, the recording chamber was filled with artificial cerebrospinal fluid, and hydraulically sealed using a clear glass plate. In each of five experiments, the OIS evoked in SI by cutaneous flutter stimuli on the thenar region of the forelimb was recorded. The flutter stimulus was delivered at 50, 100, 200 and 400 μm and was interleaved in order to prevent conditioning of the response. The imaging system consisted of a computer-interfaced CCD camera (Quantix 540 from Roper Scentific), light source, guide and filters required for near-infrared (833 nm) illumination of the cortical surface, a focusing device, and a recording chamber capped by an optical window (for additional methodological details see Tommerdahl et al, 1999a,b). Images of the exposed anterior parietal and surrounding cortical surface were acquired 200 ms before stimulus onset ("reference images") and continuously thereafter for 22 s after stimulus onset ("post-stimulus images") at a rate of one image every 0.9–1.4 s. Exposure time was 200 ms. Difference images were generated by subtracting each pre-stimulus image from its corresponding post-stimulus image. Averaged difference images typically show regions of both increased light absorption (decreased reflectance) and decreased light absorption (increased reflectance) which are believed widely (e.g., Grinvald 1985; Grinvald et al. 1991a,b) to be accompanied by increases and decreases in neuronal activation, respectively.
Although onset of the OIS is delayed at longer wavelengths [19,20], use of near-infrared illumination minimizes the contributions to OIS images of the changes in blood flow and flow/volume that normally accompany cortical neuronal activation, and thus may be more useful in the description of spatial characteristics of the signal [20]. All images were examined prior to their inclusion for analysis. Images containing random high amplitude noise were excluded, and the remaining trials (typically ~20) were averaged to improve the signal to noise ratio. OIS images were analyzed using custom routines written in Matlab.
Correlation methodology
Correlation maps were constructed for comparison of spatial characteristics of the OIS response. This method of analysis has been previously described in detail [28]. Briefly, maps were constructed by choosing a reference region within the imaged field and computing the intensity correlation rij between the reflectance value of each pixel (i, j) and the average reflectance value within the reference region over the time from stimulus onset to stimulus offset. The region selected as the reference was defined by a boxel (π mm2 area) centered on the region of interest (ROI). Each pixel (i, j) on the correlation map is represented by a correlation coefficient rij (-1 <r < 1; - 1 indicates negative correlation; + 1 indicates positive. The statistical significance of each of the correlations was tested with the standard t-test.
Histological procedures/identification of cytoarchitectural boundaries
At the conclusion of the experiment, the imaged cortical region was removed immediately following intracardial perfusion with saline and fixative. The region then was blocked, postfixed, cryoprotected, frozen, sectioned serially at 30 μm, and the sections stained with cresyl fast violet. The boundaries between adjacent cytoarchitectonic areas were identified by scanning individual sagittal sections separated by no more than 300 mm and were plotted at high resolution using a microscope with a drawing tube attachment. The resulting plots then were used to reconstruct a two-dimensional surface map of the cytoarchitectonic boundaries within the region studied with optical and neurophysiological recording methods. The locations of microelectrode tracks and electrolytic lesions evident in the histological sections were projected radially to the pial surface and transferred to the map of cytoarchitectonic boundaries reconstructed from the same sections. As the final step, the cytoarchitectonic boundaries (along with the locations of microelectrode tracks and lesions whenever present) identified in each brain were mapped onto the images of the stimulus-evoked intrinsic signal obtained from the same subject, using fiducial points (made by postmortem applications of india ink or needle stabs) as well as morphological landmarks (e.g., blood vessels and sulci evident both in the optical images and in histological sections). Locations of cytoarchitectonic boundaries were identified using established criteria [29-31].
Authors' contributions
SS participated in acquisition of optical data, performed analysis of the data and drafted the manuscript. VT and JC assisted in the data collection and the analysis of the optical imaging data. OF and BW participated in the design of the study, the conduct of the experiments and the drafting of the manuscript. MT participated in the design, conduct, and analysis of the experiments, and in the manuscript preparation.
Acknowledgements
This work was supported, in part, by US Army Research Office grant P43077-LS (M. Tommerdahl, P.I.), NIH NS050587 (M. Tommerdahl, P.I.) and NIH NS35222 (B. Whitsel, P.I.).
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| 15969752 | PMC1166561 | CC BY | 2021-01-04 16:39:09 | no | BMC Neurosci. 2005 Jun 21; 6:43 | utf-8 | BMC Neurosci | 2,005 | 10.1186/1471-2202-6-43 | oa_comm |
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BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-6-431596975210.1186/1471-2202-6-43Research ArticleAmplitude-dependency of response of SI cortex to flutter stimulation Simons Stephen B [email protected] Vinay [email protected] Joannellyn [email protected] Oleg V [email protected] Barry L [email protected] Mark [email protected] Departments of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA2 Cellular and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA2005 21 6 2005 6 43 43 7 2 2005 21 6 2005 Copyright © 2005 Simons et al; licensee BioMed Central Ltd.2005Simons et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
It is established that increasing the amplitude of a flutter stimulus increases its perceived intensity. Although many studies have examined this phenomenon with regard to the responding afferent population, the way in which the intensity of a stimulus is coded in primary somatosensory cortex (SI) remains unclear.
Results
Optical intrinsic signal (OIS) imaging was used to study the evoked responses in SI of anesthetized squirrel monkeys by 25 Hz sinusoidal vertical skin displacement stimulation. Stimuli were 10 sec duration with a 50 sec inter-stimulus interval. Stimulus amplitude ranged from 50 to 400 microns and different amplitudes were interleaved. Control levels of activity were measured in the absence of stimulation, and used to compare with activation levels evoked by the different stimulus amplitudes. Stimulation of a discrete skin site on the forelimb evoked a prominent increase in absorbance within the forelimb representational region in cytoarchitectonic areas 3b and 1 of the contralateral hemisphere. An increase in stimulus amplitude led to a proportional increase in the magnitude of the absorbance increase in this region of areas 3b and 1 while surrounding cortex underwent a decrease in absorbance. Correlation maps revealed that as stimulus amplitude is increased, the spatial extent of the activated region in SI remains relatively constant, and the activity within this region increases progressively. Additionally, as stimulus amplitude is increased to suprathreshold levels, activity in the surround of the activated SI territory decreases, suggesting an increase in inhibition of neuronal activity within these regions.
Conclusion
Increasing the amplitude of a flutter stimulus leads to a proportional increase in absorbance within the forelimb representational region of SI. This most likely reflects an increase in the firing rate of neurons in this region of SI. The relatively constant spatial extent of this stimulus-evoked increase in absorbance suggests that an increase in the amplitude of a 25 Hz skin stimulus does not evoke a larger area of SI neuronal activation due to an amplitude-dependent lateral inhibitory effect that spatially funnels the responding SI neuronal population.
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Background
The way in which stimulus intensity is represented in primary somatosensory (SI) cortex has remained an intriguing question in the study of the cortical correlates of perception. Although there have been numerous studies of the SI response to changes in stimulus intensity, few have focused on the response at the population level of analysis. Thus, a dearth of information about the global SI response to changes in stimulus intensity exists – current knowledge of the subject depends almost exclusively on reconstruction of predictions of the SI neuronal population response from afferent recordings [1-5] and single unit cortical recordings [6,7].
A number of studies have examined the global SI response using imaging techniques such as fMRI (functional Magnetic Resonance Imaging) [8-10] and MEG (MagnetoEncephaloGraphy)[11,12]. In general, results from these studies indicate that increases in stimulus intensity are accompanied by increases in the intensity of the evoked signal as well as increases in the activated volume of cortex. As a result these studies predicted that amplitude might be coded not only by the average firing rates of individual SI neurons, but also by the total aggregate of responding neurons. However, although each of these imaging techniques provide measures which are indirectly related to neuronal activity, their resolution is limited in two important ways. First, it is difficult to determine the nature of the neuronal activity being imaged (whether or not it is excitatory or inhibitory); and second, both fMRI and MEG studies have limited spatial resolution, which is typically on the order of ~1 mm2 [8,11].
Recently, Chen and colleagues used the optical intrinsic signal (OIS) to demonstrate that a proportionally greater (larger magnitude) response is evoked in SI of squirrel monkeys as the amplitude (as measured by force) of a skin stimulus is increased[13]. However, the primary focus of their report was that the response to simultaneous stimulation of multiple adjacent sites on the skin produced a smaller, more intense region of SI activation than would be normally predicted by summation of the two, and their findings did not detail the effects of amplitude on the dynamics of the response to a flutter stimulus at a single site on the skin. In this report, we extend the aforementioned work by imaging the OIS evoked in SI cortex of squirrel monkeys by a range of amplitudes of skin flutter stimulation. The results suggest that increasing the amplitude of a skin flutter stimulus evokes a proportionally larger absorbance increase in SI that remains confined to the same SI territory. In addition, it was found that increasing the amplitude of flutter evokes a large decrease in absorbance in the territory that borders the activated region of SI. Neurons in the SI region that demonstrate decreased absorbance in response to flutter stimulation are proposed to undergo stimulus-evoked inhibition and to contribute importantly to the SI processing of high-amplitude skin flutter stimuli.
Results
Figure 1(A–C) illustrates typical examples of the OIS response in SI of three different subjects in the absence of stimulation (control), and during low (50 μm) versus high (400 μm) amplitude stimulation. Each image shown in Figure 1 represents the sum of frames taken from the time of stimulus onset to 5 seconds after stimulus offset (frames 1–16). Areas of high absorbance are indicated by dark patches within each image; regions of high absorbance in each case correspond to the SI locus that represents the stimulated site on the skin. SI in each experiment underwent a larger increase in absorbance within the region of interest (ROI) in response to the 400 μm stimulus than evoked in the same region by the 50 μm stimulus. Moreover, in each subject the increase in absorbance appears more evenly distributed and less diffuse throughout the ROI under the 400 μm condition.
Figure 1 OIS response to low vs. high amplitude stimulation. OIS images taken from three subjects (A, B, C). All images are anatomically oriented as shown in the top left image. Images were obtained by averaging across 10 experimental trials and then summing frames taken from the time of stimulus onset to 5 seconds after stimulus offset to better show regions of high absorbance indicated by dark pixels. The left column shows responses acquired in the absence of stimulation (control), while the middle and right columns show the stimulus-evoked response to 50 and 400 μm respectively in the same subjects.
To identify the boundaries of the regions of increased absorbance spatial histograms were constructed. Figure 2 illustrates the average results obtained from all experiments (n = 5) as well as the methodology used to evaluate the spatial extent of the stimulus-evoked activation. In each experiment, the image was segmented along a line 6 mm long and roughly centered on the area of increased absorbance, as shown in the top panel. Pixels along the line were binned (bin size 40 × 200 μm) and absorbance values averaged and plotted as a function of distance along the line. The plots demonstrate that at all amplitudes of stimulation, the spatial extent of the region of above-background absorbance (ie. absorbance values larger than control) is similar and at every stimulus amplitude occupies a circular-shaped territory in SI between 1.8 – 2.24 mm in diameter. The ROI (to be used for further analysis) was therefore defined as the region displaying above background levels of absorbance within the activated region of SI.
Figure 2 Spatial histograms of activity at different amplitudes. Absorbances were measured at each amplitude along the red line shown in the OIS image at top left. Each plotted value represents an average of pixels spanning 100 μm above and below the line and a distance of 40 μm along the line (bin size was 40 × 200 μm). The control condition is plotted on each graph to indicate "background" levels absorbance. Dashed lines on plots indicate where stimulus-evoked activity crosses background absorbance levels (indicating the boundaries of above background absorbance). Histograms indicate no significant change in cortical territory displaying above background absorbance with respect to stimulus amplitude. Higher amplitude stimulation produces regions of below-background absorbance directly outside of the regions of above-background absorbance.
Figure 3 demonstrates (for one exemplary subject) the method used to evaluate the time course of stimulus-evoked SI absorbance. Panel A shows a green filter image of the cortical surface, which highlights the vasculature. Panels B&C are the OIS responses (dark regions) evoked by the low (50 μm) and high (400 μm) stimulus amplitudes respectively. The ROI is the circular territory enclosed by the dashed white lines. Absorbance values within the ROI were averaged for each amplitude of stimulation and plotted as a function of time. The time course of the absorbance values measured between 1 and 22 sec after stimulus onset is plotted for each of the stimulus conditions, and in the absence of stimulation ("control"). Arrows along x-axis of plots at bottom left of Figure 3 indicate stimulus onset (1 sec) and stimulus offset (11 sec), and reveal how absorbance increased with increasing amplitude of stimulation. For each stimulus amplitude absorbance is maximal near to stimulus offset.
Figure 3 Absorbance time course and anatomical registration in SI. A) Green filter image of SI cortex in vivo, used for anatomical registration of OIS images. B&C) Resulting OIS image obtained from averaging across 10 experimental trials and then summing frames taken from the time of stimulus onset to 5 seconds after stimulus offset: B) at stimulus amplitude 50 μm, C) at 400 μm. Dashed circles enclose the ROI within SI. Bottom left) Plot of the averaged absorbance. Arrows indicate time of stimulus onset (1s) and stimulus offset (11s). Absorbance values within the enclosed ROI were averaged and plotted at each amplitude as a function of time.
The analysis approach illustrated in Figure 3 was performed on all experiments (n = 5) and the resulting absorbance plots were averaged (Figure 4). Similar to Figure 3, the plots in Figure 4 demonstrate that absorbance increases with increasing stimulus amplitude. To quantify this relationship a measure of ΔAbsorbanceevoked was used. ΔAbsorbanceevoked was defined as the difference between the absorbance measured at 1 sec (prior to stimulus onset) and 11 sec (point of stimulus offset), and is shown in the plot at the bottom of Figure 4. The plot of ΔAbsorbanceevoked vs. amplitude is well described (coefficient of determination R2 = 0.9921) by the linear function (solid line) ΔAbsorbanceevoked = (4 × 10-6)*d + 0.0005. This type of analysis, however, gives little or no information about the spatial properties of the response.
Figure 4 A) Plots of absorbance and standard deviation averaged across all experiments (n = 5). All data was normalized prior to being averaged. Plotted absorbances were measured within the ROI which was defined as all pixels within a 1 mm radius of the center of activation (as defined from spatial histogram analysis). Controls in which no stimulus was administered are plotted simultaneously for comparison to test conditions. B) Plot of ΔAbsorbanceevoked which was defined as the change in absorbance measured from frame 1 (prior to stimulus onset) to frame 11 (point of stimulus offset). The plot is fit with a linear function (solid line) described by the equation A = (4 × 10-6)d + 0.0005 where A is absorbance and d is stimulus amplitude (displacement). Coefficient of determination for the linear regression (R2) is shown below.
Radial histograms were constructed to better visualize the spatiotemporal relationship of the OIS response at different amplitudes. The radial histograms shown in Figure 5 are representative time-space plots at each amplitude. From the ROI center (as determined by spatial histogram analysis), average absorbance values were determined for the pixels within concentric rings located at progressively larger distances from the center at each frame acquired. Absorbance values are color coded (red indicating areas of high absorbance, blue indicating areas of low absorbance) and plotted as a function of time and radial distance from the center of the ROI. Figure 5 demonstrates that the major differences that exist in the SI global responses to different amplitudes of stimulation are differences in the magnitude of absorbance and not the spatial properties of the absorbance pattern (this also is apparent in the spatial histogram analysis of Figure 2). As would be expected based on the absorbance curves shown in Figures 3 &4, higher stimulus amplitudes evoked a more intense and discrete region of increased absorbance than did the lower amplitudes. Interestingly, one of the more robust differences between low- and high-amplitude stimulation, is the magnitude of decreasing absorbance detected in the territory that surrounds the region in which absorbance increases. This response is most pronounced under the 400 μm condition where it can be seen to occur much sooner after stimulus onset at radial distances as small as 1.5 mm from the ROI center. Spatially, the regions of above- and below-background absorbance are nearly the same at each stimulus amplitude, with the above-background portion extending nearly 1 mm away in all directions from the center of activity, whereas the below-background portion of the response comprises a ring beginning at a radial distance of 1.5 mm from the ROI center and extending out beyond the area that was analyzed.
Figure 5 Radial time space plots. Radial histograms were performed on OIS data at all stimulus amplitudes. Radii were measured from the center of activation as demonstrated in the image at top-left. The dashed blue line is the maximum radial distance used in the maps shown. The schematic at bottom left indicates the anatomical orientation of the cortex in the image above. Absorbance values in maps shown represent an average at each radial distance at each frame after stimulus onset. Green arrows indicate stimulus onset (1s) while red arrows indicate stimulus offset (11s). Absorbance values are color coded as per the color bar shown at the right.
Using similar techniques to those we used to analyze above-background activity in the ROI (as in Figures 2 &3), regions outside the designated ROI were examined to determine whether a similar amplitude-dependency could be established for the time courses of the below-background absorbance observed in the surround. Figure 6 shows plots constructed from averaging the absorbance values in pixels lying 1.5 – 2 mm away from the center of the ROI. Data were normalized and then averaged across experiments (n = 5). It is apparent that the time courses at different amplitudes of stimulation are different with respect to the stimulus timing, (compared with above background levels of activity, which all show maximum absorbance at the point of stimulus offset). Accordingly, a different measure was adopted to quantify this relationship: In this case ΔAbsorbancemax was defined as the difference between the minimum absorbance and the maximum absorbance value obtained at any point during the recording. Interestingly, the relationship between stimulus amplitude and ΔAbsorbancemax in the surround is not linear (Figure 6b). Instead, each of the higher stimulus amplitudes employed (100–400 μm) evoked a very similar level of below-background absorbance. The sole difference between the different curves (Figure 6a) is the time required to reach the peak of the decrease in absorbance. That is, as amplitude is increased from 100 to 400 μm the point of minimum absorbance was attained earlier in time. This is also apparent in Figure 4.
Figure 6 Absorbance trends surrounding the ROI across five experiments. A) Plots of absorbance and standard deviation averaged across all experiments (n = 5). Plotted absorbances were measured at radial distances between 1 and 1.5 mm away from the center of the ROI (defined earlier). B) Plot of the maximal change in absorbance (solid line) as a function of stimulus amplitude. The linear trend, obtained from averaged absorbance measured within the ROI (dashed line from figure 3) has been simultaneously plotted on the axes to demonstrate the significant differences between the two regions.
Correlation maps were constructed to further characterize the spatial properties of the SI response to 25 Hz flutter. A correlation map compares every pixel in the image with the signal referenced from the ROI, and assigns a correlation coefficient (r) to the location of the pixel being compared. This gives a fairly good approximation of the signal at all locations in the image. Since there is no significant difference in the spatial properties between stimuli at intermediate amplitudes (as demonstrated by radial histograms) only the 50 and 400 μm amplitudes will be compared with this technique. Figure 7 shows correlation maps of the OIS responses to stimulus amplitudes of 50 and 400 μm (Top panels). The bottom panels of the figure show the input signal (solid dark red line) used for correlation of each pixel in the map, and the negative (opposite) of the input signal (dotted blue line). A coefficient of +1 (although it never appears in the map) indicates that a pixel's time course perfectly matches the input signal while a coefficient of -1 indicates that pixel's time course perfectly matches the opposite of the input signal (dotted blue line). At 50 μm the correlation map (color-coded image) shows that the correlation is weak and more dispersed within the ROI in area 3b. At the highest amplitude, however, there is a pronounced and well-defined positive correlation within the ROI that is more evenly distributed throughout the ROI. A large region of negatively correlated activity (corresponding to strong below background activity) surrounds the ROI in the high-amplitude map.
Figure 7 Correlation maps for stimulus amplitudes of 50 (left) and 400 (right) microns. The schematic at top-right indicates the anatomical orientation of the OIS image as well as all maps: A-anterior, M-medial, P-posterior, L-lateral. Color bars show coefficient of determination values for each map. The correlated signal, obtained from averaging of absorbance values within the ROI (encircled in the image at top-left) at each frame, is shown under each map (time course in solid red). Only the portion of the signal enclosed within the dashed lines (corresponding to the stimulus duration, green-on, red-off) is correlated. r values describe the degree of similarity between a given pixel's time course and the corrselated signal. Negative r values indicate that absorbance decreases to below-background levels. Thus, the signal in solid red represents the time course that would be observed in a pixel with an r value of +1 and the dotted blue line indicates the time course that would be observed in a pixel with an r value of -1.
To examine the spatial dynamics of the SI response in more detail we examined the patterns of activity generated by low- and high-amplitude stimulation in a 16 mm2 (4 × 4) area centered around the ROI. Figure 8 demonstrates the patterns of activity evoked at three time intervals during the delivery of the stimulus. The 3D surface plots show activity measured within the boxel indicated by the dashed box in the image at the top. In each 3D plot absorbance is plotted in two-dimensional space and is indicated by two measures: height of the peak along the z-axis (as shown in the schematic at the top right), and the color (indicated by the color bar to the right of each row of 3D plots). These data make it apparent that after a short period of stimulation (1 sec) the activity pattern is very similar for the different amplitudes. That is, at this early time interval both patterns are diffuse and occupy much of the ROI. However as stimulus duration increases, the pattern of increased absorbance evoked by high-amplitude stimulation tends to become restricted to the center of the ROI and within this region becomes homogeneous. Standard deviation was used to measure the variability within the ROI at low and high amplitudes. At 10s after stimulus onset, standard deviations for low- and high-amplitude surface plots were 0.1415 and 0.1166 respectively. Average standard deviation across all sets of maps (n = 5) differs very little from these values (0.1448 at low amplitude vs. 0.1201 at high amplitude). These differences suggest that within the ROI evoked absorbance levels are more homogeneous in response to high- vs low-amplitude stimulation. In addition, at 5 and 10s after onset of high-amplitude (but not low-amplitude) stimulation the territory surrounding the ROI becomes dominated by below-background changes in absorbance.
Figure 8 Spatial plots of activity evoked by low- (50 μm) and high-amplitude (400 μm) stimulation. The schematic at the top-left indicates the anatomical orientation of the cortex: A-anterior, M-medial, P-posterior, L-lateral. Absorbances were measured within the boxel shown in the top-middle panel. The schematic (top-right) shows how each frame is spatially represented with respect to the boxel of interest. Both stimulus amplitudes are mapped 1, 5 and 10 seconds after stimulus onset. Absorbance values at each pixel (x, y location) are represented two fold: both by their height along the z-axis, and by their color.
Discussion
This study evaluated the global SI response to different amplitudes of flutter stimulation by imaging the optical intrinsic signal (OIS). The OIS indirectly reflects both cortical neuronal spike discharge activity and the local, subthreshold changes in neuronal membrane potential evoked by sensory stimulation [14-16]. As a result, the observed tendency for absorbance in the same localized region of area 3b to increase with increasing stimulus amplitude (Figures 3 &4) most likely is due to the amplitude-dependence of the average firing rate of neurons in the same region [17]. An increase in SI absorbance in response to an increase in stimulus intensity has been reported previously by Chen et al [13]. The observed increase we observed in the stimulus-evoked SI OIS that accompanies increases in stimulus amplitude is well described by a linear function.
One important distinction between previous work done using the OIS and the present study is our use of near-infrared wavelengths for illumination during acquisition of the OIS. The OIS obtained using infrared light has been shown to be highly correlated with light scattering effects that accompany astrocyte swelling subsequent to the clearance of extracellular K+ and neurotransmitter [14,15,18] and local increases in blood volume [19,20]. Although the OIS at this wavelength may be influenced by changes in hemoglobin concentration and oxygenation, it is likely that contributions from these factors are small when compared with light scattering effects[20]. Additionally, OIS imaging using near-infrared illumination not only minimizes the contributions of artifacts introduced by changes in the vasculature (which can dominate the OIS at lower wavelengths) [19] but the time course of the OIS detected at shorter wavelengths (600 nm) is markedly different (shorter) than the protracted OIS observed in this study [13,15,19].
Previous studies conducted by this laboratory have reported that the SI optical response evoked by an extended period (>1 sec) of flutter stimulation not only consists of an increase in absorbance in the region that receives its input from the skin site that was stimulated, but also decreases in absorbance (frequently to levels well below-background) that occur in the surrounding cortex [21]. The present study demonstrates that the below-background component of the SI optical response to flutter stimulation is particularly evident at large stimulus amplitudes (figures 5, 7 &8). However, unlike the increase in absorbance evoked by flutter, the relationship between the magnitude of the stimulus-evoked decrease in absorbance and stimulus amplitude is not satisfactorily described by a linear function. Indeed, the results shown in figure 6 suggest that this component of the optical response to skin flutter is either absent or extremely small at small stimulus amplitudes, and remains maximal or near-maximal across a wide range of suprathreshold stimulus amplitudes. Interestingly, this stimulus-evoked decrease in absorbance (in the surround) appears later in time than the increases in absorbance (within the ROI) and as amplitude is increased it tends to develop earlier after stimulus onset.
The correlation maps shown in figure 7 provide a comprehensive overview of the time course of absorbance at each location in the image. The optical signal at each pixel is cross-correlated with a known input signal. In this case, it is the average absorbance measured within the ROI. The assigned coefficient of determination indicates the degree of similarity between a pixel's time course of absorbance and the input signal. Therefore pixels with a large positive correlation undergo increases in absorbance very similar to the input signal, while pixels with a large negative correlation undergo a decrease in absorbance which more closely resembles the opposite (negative) of the input signal. Figure 8 suggests that at high amplitudes of stimulation the ROI in SI becomes more homogeneously activated with longer stimulus duration. Some evidence for this is indicated by the large discrepancy (between low- and high-amplitude surface plots) in the standard deviations measured within the ROI. Further studies are required to investigate absorbance distribution and patterning within the ROI.
Examination of spatial histograms (figure 2) and the maps in figures 5 &7 also reveal that the size of the SI region that undergoes an increase in absorbance does not increase with increasing stimulus amplitude, but rather remains relatively constant. Regardless of stimulus amplitude, the activated cortical region appears circular in shape and occupies an area approximately 2 mm in diameter (figures 4 &6). Within the ROI average absorbance increases progressively with increasing stimulus duration. The dimensions of the SI region activated by flutter stimulation observed in this study contrast sharply with results presented in previous studies which demonstrated activation within a 1 mm region in diameter [13,22]. One possible explanation for this discrepancy is the level and type of general anesthesia used in the different studies. Previous studies have reported that anesthetics (e.g. ketamine) which block NMDA receptors or enhance GABAA receptor mediated inhibition (barbiturates), significantly reduce the dimensions of the receptive field of individual SI neurons; actions that would reduce the size of the responding SI neuronal population [23]. Chen et al. previously reported similar (~2 mm) sized regions of activation in response to flutter stimulation of the digit tips in squirrel monkey anesthetized with isofluorane, as well as showing in the same report that use of pentothal anesthetic confined the response to a much smaller region (~1 mm) [24].
It has been suggested that the amplitude of skin flutter stimulation is coded by both the number of activated SI neurons as well as by their level of spike discharge activity [1]. This suggestion is based largely in part on the fact that larger-amplitude stimuli, through transduction of the laterally-transmitted mechanical wave produced by sinusoidal skin displacement, recruit larger numbers of RA afferents and therefore lead to a spatially more widely distributed pattern of afferent input to SI cortex. Combined metabolic tracer and neurophysiological studies have shown that the initial response to a repetitive tactile stimulus occupies an extremely large cortical territory. As the repetitive mechanical stimulation is continued, however, the response is quickly sculpted by cortical inhibitory mechanisms, leading to an activity pattern that becomes confined to a relatively restricted region in SI [25-27]. The results obtained in the present study and results previously reported by other researchers, lead us to suggest that stimulus amplitude contributes importantly to the shaping (via lateral inhibitory mechanisms) of the SI response to protracted skin flutter.
Conclusion
This study investigated the SI response to flutter stimulation of the skin using the OIS. An increase of the amplitude of the flutter stimulus was associated with an increase in absorbance within the region of SI cortex that receives its input from the stimulated skin field. The relationship between the maximal change in absorbance and stimulus amplitude is well characterized by a linear function within the range of amplitudes studied. Measurement of the spatial extent of the activated SI region showed that higher amplitudes of stimulation do not produce a more extensive region of SI activation. Instead, as amplitude is increased, while average peak absorbance within the same ~2 mm diameter SI region increases with amplitude of stimulation, the region of surrounding cortex undergoes a prominent decrease (frequently to levels well below background) in absorbance. Further studies are required to establish the relationship between the effect of different amplitudes of skin flutter stimulation on SI absorbance and SI neuroelectrical activity.
Methods
Subjects & preparation
All methods and procedures are consistent with USPHS policies and guidelines on animal care and welfare in biomedical research. They were reviewed and approved by an institutional committee prior to initiation of the experiments. Experiments were conducted in 10 squirrel monkeys. Following induction of anesthesia with 4% halothane in a 50/50 mix of nitrous oxide (N2O) and oxygen, the trachea was intubated. A veterinary anesthesia machine (Forreger Compac-75) provided an anesthetic gas mix whose composition could be adjusted (typically 1.5–3.0% halothane in 50/50 N2O/oxygen) to maintain a stable level of surgical anesthesia. Methylprednisolone sodium succinate (20 mg/kg) and gentamicin sulfate (2.5 mg/kg) were injected intramuscularly to lessen the probability of halothane-induced cerebral edema and prevent bacterial septicemia, respectively. Placement of a valved catheter into a superficial hindlimb vein enabled administration of 5%glucose, 0.9%NaCl, and drugs.
A 1.5 cm opening was trephined in the skull overlying SI cortex. A recording chamber (25 mm i.d.) was placed over the opening and cemented to the skull with dental acrylic. Wound margins were infiltrated with local anesthetic, closed with sutures and bandaged, and the dura overlying SI was resected. After the completion of all surgical procedures subjects were immobilized with norcuron (loading dose 0.25–0.5 mg/kg,i.v.; maintenance dose 0.025–0.05 mg/kg/hr). From this point on, the animal was ventilated with a 50/50 mix of N2O and oxygen and the concentration of halothane was adjusted (typically between 0.5 and 1.0%) to maintain heart rate, blood pressure, and the EEG at values consistent with general anesthesia. Rate and depth of ventilation were adjusted to maintain end-tidal CO2 between 3.0 and 4.5%. Under these experimental/anesthetic conditions both SI neuron spontaneous and stimulus-evoked spike discharge activity patterns are highly reproducible over even prolonged (>1 hr) time periods.
OIS imaging and stimulus protocol
After obtaining a photograph of the exposed cortical surface, the recording chamber was filled with artificial cerebrospinal fluid, and hydraulically sealed using a clear glass plate. In each of five experiments, the OIS evoked in SI by cutaneous flutter stimuli on the thenar region of the forelimb was recorded. The flutter stimulus was delivered at 50, 100, 200 and 400 μm and was interleaved in order to prevent conditioning of the response. The imaging system consisted of a computer-interfaced CCD camera (Quantix 540 from Roper Scentific), light source, guide and filters required for near-infrared (833 nm) illumination of the cortical surface, a focusing device, and a recording chamber capped by an optical window (for additional methodological details see Tommerdahl et al, 1999a,b). Images of the exposed anterior parietal and surrounding cortical surface were acquired 200 ms before stimulus onset ("reference images") and continuously thereafter for 22 s after stimulus onset ("post-stimulus images") at a rate of one image every 0.9–1.4 s. Exposure time was 200 ms. Difference images were generated by subtracting each pre-stimulus image from its corresponding post-stimulus image. Averaged difference images typically show regions of both increased light absorption (decreased reflectance) and decreased light absorption (increased reflectance) which are believed widely (e.g., Grinvald 1985; Grinvald et al. 1991a,b) to be accompanied by increases and decreases in neuronal activation, respectively.
Although onset of the OIS is delayed at longer wavelengths [19,20], use of near-infrared illumination minimizes the contributions to OIS images of the changes in blood flow and flow/volume that normally accompany cortical neuronal activation, and thus may be more useful in the description of spatial characteristics of the signal [20]. All images were examined prior to their inclusion for analysis. Images containing random high amplitude noise were excluded, and the remaining trials (typically ~20) were averaged to improve the signal to noise ratio. OIS images were analyzed using custom routines written in Matlab.
Correlation methodology
Correlation maps were constructed for comparison of spatial characteristics of the OIS response. This method of analysis has been previously described in detail [28]. Briefly, maps were constructed by choosing a reference region within the imaged field and computing the intensity correlation rij between the reflectance value of each pixel (i, j) and the average reflectance value within the reference region over the time from stimulus onset to stimulus offset. The region selected as the reference was defined by a boxel (π mm2 area) centered on the region of interest (ROI). Each pixel (i, j) on the correlation map is represented by a correlation coefficient rij (-1 <r < 1; - 1 indicates negative correlation; + 1 indicates positive. The statistical significance of each of the correlations was tested with the standard t-test.
Histological procedures/identification of cytoarchitectural boundaries
At the conclusion of the experiment, the imaged cortical region was removed immediately following intracardial perfusion with saline and fixative. The region then was blocked, postfixed, cryoprotected, frozen, sectioned serially at 30 μm, and the sections stained with cresyl fast violet. The boundaries between adjacent cytoarchitectonic areas were identified by scanning individual sagittal sections separated by no more than 300 mm and were plotted at high resolution using a microscope with a drawing tube attachment. The resulting plots then were used to reconstruct a two-dimensional surface map of the cytoarchitectonic boundaries within the region studied with optical and neurophysiological recording methods. The locations of microelectrode tracks and electrolytic lesions evident in the histological sections were projected radially to the pial surface and transferred to the map of cytoarchitectonic boundaries reconstructed from the same sections. As the final step, the cytoarchitectonic boundaries (along with the locations of microelectrode tracks and lesions whenever present) identified in each brain were mapped onto the images of the stimulus-evoked intrinsic signal obtained from the same subject, using fiducial points (made by postmortem applications of india ink or needle stabs) as well as morphological landmarks (e.g., blood vessels and sulci evident both in the optical images and in histological sections). Locations of cytoarchitectonic boundaries were identified using established criteria [29-31].
Authors' contributions
SS participated in acquisition of optical data, performed analysis of the data and drafted the manuscript. VT and JC assisted in the data collection and the analysis of the optical imaging data. OF and BW participated in the design of the study, the conduct of the experiments and the drafting of the manuscript. MT participated in the design, conduct, and analysis of the experiments, and in the manuscript preparation.
Acknowledgements
This work was supported, in part, by US Army Research Office grant P43077-LS (M. Tommerdahl, P.I.), NIH NS050587 (M. Tommerdahl, P.I.) and NIH NS35222 (B. Whitsel, P.I.).
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| 15967041 | PMC1166562 | CC BY | 2021-01-04 16:03:49 | no | BMC Ophthalmol. 2005 Jun 20; 5:13 | latin-1 | BMC Ophthalmol | 2,005 | 10.1186/1471-2415-5-13 | oa_comm |
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BMC PediatrBMC Pediatrics1471-2431BioMed Central London 1471-2431-5-171595816010.1186/1471-2431-5-17Research ArticleThe effect of functional splinting on mild dysplastic hips after walking onset Windhagen Henning [email protected] Fritz [email protected] Heinrich [email protected] Thomas [email protected] Dieter [email protected] Christina [email protected] Department of Orthopaedic Surgery, Hannover Medical School, Annastift, Anna-von-Borries-Str.1, 30625 Hannover, Germany2 Trauma Surgery, Städtisches Krankenhaus Hildesheim, Weinberg 1, 31134 Hildesheim, Germany3 Department of Pediatric Orthopaedic Surgery, Clinic 1, Annastift, Anna-von-Borries-Str.1, 30625 Hannover, Germany2005 15 6 2005 5 17 17 20 12 2004 15 6 2005 Copyright © 2005 Windhagen et al; licensee BioMed Central Ltd.2005Windhagen et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
For treatment of Graf class IIb dysplastic hips at walking onset a treatment concept with abduction splints allowing patterns as walking and crawling under constant abduction control was investigated. However, as the splint still incapacitates child movements the research question remains whether the physiologically progressing maturation of hips can be significantly altered using such abduction splints for walking children.
Methods
Of 106 children showing late hip dysplasia, 68 children treated with the Hoffman-Daimler (HD-splint) abduction splint were compared with 38 children with neglect of the abduction treatment in this retrospective study. Radiographic analyses were performed measuring the development of the age dependent acetabular angle.
Results
The regression analysis for splint treatment showed a significant linear regression for both splint treatment and no splint treatment group (r2 = 0,31 respectively r2 = 0,33). No statistical difference between both treatment groups was apparent.
Conclusion
Considering the characteristics of this study, there seems to be no strong rationale supporting the use of an abduction device in growing children. As no significant difference between treatment groups is apparent, a future controlled prospective study on splinting effects can be considered ethically allowed.
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Background
Developmental dysplasia of the hip is a gradually progressing disorder reflecting anatomically different situations reaching from mild subluxation of the femoral head to full luxation of the hip. The disorder is caused by malformations of anatomic structures that have developed during the embryologic period. The pathology of developmental dislocation of the hip is associated with a loose hyperelastic capsule, elongated ligamentum teres and slight eversion of the hypertrophied acetabular rim. While the femoral head is normal in shape, excessive femoral and acetabular antetorsion may be present causing anatomic instability of the hip joint [1-5].
Subluxation and luxation are conditions that always lead to symptomatic degenerative hip disease [5-7]. Depending on the severity of dysplasia pain onset is observed already in the second decade for severely subluxed hips while minor subluxation leads to pain starting in the fifth or sixth decades. Considering this time course of disease early treatment regimens for developmental dysplasia of the hip were recommended.
While dislocated hips after birth present clinical features as the Ortolani's and Barlow's sign [8], subluxated hips present significant changes in the sonographic morphology of the hip. Therefore, especially in Europe, ultrasound is considered to play a pivotal role in the early diagnosis of developmental hip diseases [9,10]. Especially in babies with risk factors associated with developmental dysplasia a careful examination is needed [11]. General screening concepts remain controversial due to added costs [12,13]. The treatment of dysplastic hips depends on the degree of subluxation. Based on the sonographic appearance the Graf classification has gained wide acceptance [14-18]. While class I hips need no follow up and treatment, class II hips form a group in which the degree of abnormality and the need for treatment are less clear and remain controversial. While some authors treat class II hips showing instability [19], others report about spontaneous recovery [20]. For treatment purposes authors introduced abduction devices such as harnesses providing abduction and flexion [20-22]
Graf Class II b hips are defined as hips of babies older than three month, exhibiting an alpha angle of 50–59 degrees. Radiographs show an acteabular angle of more than 30 degrees. The morphology shows a stable, but deficient bony shape of the acetabulum and femur and a broadened cartilage roof. Class IIb hips show a deficit of bony maturation and therefore need treatment options. Usually, the use of abduction devices is expanded until walking onset at approximately age 8 month. With the increased mobility of the baby, an abduction and flexion harness becomes an increasing handicap.
For treatment of Graf class IIb hips at age of walking onset an abduction splint with ball and socket joints was introduced, allowing patterns as walking and crawling under constant abduction control. However, the splint still incapacitates child movements and is generally not liked by parents and custodians. Thus, an estimated number of untreated cases can be considered leading to the research question, whether the physiologically progressing maturation of hips can be significantly improved using such abduction splints for walking children.
Methods
Between1998 and 2004 106 children were treated with the Hoffman-Daimler (HD-splint) abduction apparatus (Fig. 1) and included in this retrospective study. The age at diagnostics was 6–18 month. Indications of treatment were based on sonography for children under 8 month and plain a.-p. radiographs over age 8 month. To allow a combination of ultrasound and radiographic measurements, the dysplasia classification of Tönnis and Brunken [1] was used.
Figure 1 Hoffman-Daimler abduction splint. The Hoffman-Daimler splint consists of a individually shaped leg sleeve and a variable bar connected by ball and socket joints.
A treatment algorithm based on the Graf Classification was introduced in the Department of Orthopaedic Surgery in 1989. The use of HD-splints in Class IIb hips was fully established prior to this retrospective analysis. From 1998 68 children were treated using the HD-splint, while no splint was used in 38 children. All children were monitored using radiographic follow ups of the hips. Reasons for no splint treatment were solely based on parents or custodians declared intention. The decision, not to use a splint was argued with pain, incapacitation, poor splint fitting and continuous neglect.
Radiographs were digitized using a radiographic scanner and analyzed using ImageJ -software (NIH, Bethesda, USA). The acetabular angle (AC) following Hilgenreiner was measured for all hips and assigned to the respective radiographic age. (Fig. 2).
Figure 2 Acetabular angle (AC – Hilgenreiner). The AC- angle arises from a horizontal line trough the triangular cartilage at the right and left pelvis and a second line connecting the corner of the triangular cartilage and the lateral acetabular rim.
Regression analyses were performed between age as dependent variable and AC-angle as independent variable. Differences in regressions between the no splint group and the abduction splint group were compared using an analysis of covariance.
Results
Classification
88% of all hips were graded mild dysplastic (1 standard deviation, [1]) following screening data of mid-European newborn hips, while 12% were graded severely dysplastic (2 standard deviations, [1]). Those hips with treatment start under 8 month were sonographically classified Graf IIb, those over 8 month were graded by x-ray anaylsis (AC-angle).
Regression analysis
The regression analysis for splint treatment showed a significant linear regression for splint treatment. (y = 29,9x-0,17, p = 0,03; r2 = 0,31). Similarly, the regression analysis for the no splint treatment group showed a significant regression for splint treatment (y = 28,7x-0,176, p = 0,03; r2 = 0,33). There was no difference between both treatment groups regarding regression quotients as analyzed with ANOVA. (Fig 3.)
Figure 3 Regression analysis of treatment groups. Regressions of the splint and no splint groups are displayed with age as the dependent and AC-angle as the independent variable. For illustrative purposes the pathological limits for age dependent AC-angles from literature data [2] are displayed. Side- and gender-related differences were averaged. Regression equation and coefficient of determination (r2) are displayed.
Initial AC-angles in the splint treatment group were higher than in the no splint group (mean 29 ± 8 vs. 26 ± 6)
Over time both groups showed a decline of the acetabular angle (AC) from 26+-6 degrees at 12 month to 17 ± 5 degrees at 70 month.
Discussion
The goal of this study was to assess the effect of an abduction splint during onset of walking age in children on the maturation of dysplastic hips. In a retrospective study on 106 children no difference was obtainable between a group of children treated with this abduction device and a group of children neglecting such devices. From the data of this study no rationales were found indicating a sufficient effect of this abduction device in the presented treatment context.
The concept of continuous abduction in growing children with mild dysplastic hip symptoms was introduced before [23,24]. As much as the effect of early abduction devices (harness etc.) is known, several authors further recommend the use of rocking horses and toys with abduction for late cases of hip dysplasia [25]. However, comparing abduction devices of young babies with the presented abduction splint one has to consider a major difference: Abduction splints in young babies always combine abduction with hip flexion. With walking onset the children's legs are abducted by approximately 60 degrees using the HD-splint, however no flexion is apparent. Thus, one reason for no visible effects of the HD-splints may be the missing flexion component.
Another reason for the analogy of results in both groups may be based on the group assortment and group size. The design of this study was purely retrospective and differences between both groups were developed by the behavior of children and parents. Furthermore the groups were not balanced regarding the initial magnitude of the AC-angles. With slightly higher initial AC-angles in the HD-splint group an influence of the splint in decreasing AC-angles could be underestimated in this analysis.
The reason, why babies reach walking onset with symptomatic hips despite detection and treatment may have several reasons: First, the diagnosis may be late and abduction therapy introduced several month after birth; second, initial errors in ultrasound screening may have underestimated the hip condition and led to neglect of sufficient therapy. As a third reason the insufficient development of hips initially graded class I has been described. In these patients the maturation of the acetabular roof does not progress sufficiently. Causes of this development remain unclear.
When interpreting the results of this study several shortcomings have to be addressed: first, the treatment group were different with 36 to 68 group size. Second, the radiographic follow ups for each patient were different, thus there is a tendency to overweight patients with multiple follow ups in this study. Third, the initial therapeutic decision for splint treatment happened during an age range form 6 to 18 months. Thus, the comparison of patients with different ages of treatment initiation may influence the validity of the presented data.
Conclusion
The effect of an abduction splint during onset of walking in children on the maturation of dysplastic hips remains unclear. From data of this study there is no strong rationale supporting the use of an abduction device in growing children, however, the results have to be weighted according to the characteristics of the study design. On the other hand, this study gives strong support for the ethical validity of a controlled prospective study, as no siginifcant difference between treatment groups can be expected.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
HW and HK designed the study profile, participated in the patient selection, data assessment and drafted the manuscript. TP provided radiographic digitization and angle measurements. FT participated in mathematical analysis. DH participated in the design of the study. CS participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
==== Refs
Tonnis D Brunken D [Differentiation of normal and pathological acetabular roof angle in the diagnosis of hip dysplasia. Evaluation of 2294 acetabular roof angles of hip joints in children] Arch Orthop Unfallchir 1968 64 197 228 5730180
Tonnis D [Conservative and surgical early treatment of congenital femoral dysplasia and defects to obtain early normal growth] Z Orthop Ihre Grenzgeb 1995 133 543 550 8571658
Ponseti IV Morphology of the acetabulum in congenital dislocation of the hip. Gross, histological and roentgenographic studies J Bone Joint Surg Am 1978 60 586 599 681377
Ponseti IV Early diagnosis and pathology of congenital dislocation of the hip Pediatr Ann 1982 11 512 5, 517 7099738
Weinstein SL Natural history of congenital hip dislocation (CDH) and hip dysplasia Clin Orthop Relat Res 1987 62 76 3315382
Wedge JH Wasylenko MJ The natural history of congenital disease of the hip J Bone Joint Surg Br 1979 61-B 334 338 158025
Wedge JH Wasylenko MJ The natural history of congenital dislocation of the hip: a critical review Clin Orthop Relat Res 1978 154 162 743823
Barlow TG Neonatal hip dysplasia--treatment, results and complications Proc R Soc Med 1975 68 475 128003
Graf R Fundamentals of sonographic diagnosis of infant hip dysplasia J Pediatr Orthop 1984 4 735 740 6392336
Graf R Classification of hip joint dysplasia by means of sonography Arch Orthop Trauma Surg 1984 102 248 255 6712426 10.1007/BF00436138
Westberry DE Davids JR Pugh LI Clubfoot and developmental dysplasia of the hip: value of screening hip radiographs in children with clubfoot J Pediatr Orthop 2003 23 503 507 12826951 10.1097/00004694-200307000-00017
Elbourne D Dezateux C Arthur R Clarke NM Gray A King A Quinn A Gardner F Russell G Ultrasonography in the diagnosis and management of developmental hip dysplasia (UK Hip Trial): clinical and economic results of a multicentre randomised controlled trial Lancet 2002 360 2009 2017 12504396 10.1016/S0140-6736(02)12024-1
Lehmann HP Ultrasonography in the diagnosis and management of development hip dysplasia (UK Hip Trial): clinical and economic results of a multicentre randomised controlled trial J Pediatr 2003 143 138 12921082
Kohler G Hell AK Experiences in diagnosis and treatment of hip dislocation and dysplasia in populations screened by the ultrasound method of Graf Swiss Med Wkly 2003 133 484 487 14652804
Holen KJ Tegnander A Eik-Nes SH Terjesen T The use of ultrasound in determining the initiation of treatment in instability of the hip in neonates J Bone Joint Surg Br 1999 81 846 851 10530848 10.1302/0301-620X.81B5.9502
Tegnander A Holen KJ Anda S Terjesen T Good results after treatment with the Frejka pillow for hip dysplasia in newborns: a 3-year to 6-year follow-up study J Pediatr Orthop B 2001 10 173 179 11497357 10.1097/00009957-200107000-00003
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Bernau A [The Tubingen hip flexion splint in the treatment of hip dysplasia] Z Orthop Ihre Grenzgeb 1990 128 432 435 2147328
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| 15958160 | PMC1166563 | CC BY | 2021-01-04 16:31:06 | no | BMC Pediatr. 2005 Jun 15; 5:17 | utf-8 | BMC Pediatr | 2,005 | 10.1186/1471-2431-5-17 | oa_comm |
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-691596322910.1186/1471-2458-5-69Research ArticleDown syndrome, paternal age and education: comparison of California and the Czech Republic Dzurova Dagmara [email protected] Hynek [email protected] Faculty of Science, Charles University, Czech Republic2 School of Public Health, University of California, Berkeley, USA3 Department of Epidemiology and Public Health, University College London, UK2005 17 6 2005 5 69 69 16 12 2004 17 6 2005 Copyright © 2005 Dzurova and Pikhart; licensee BioMed Central Ltd.2005Dzurova and Pikhart; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The association between maternal age and risk of Down syndrome has been repeatedly shown in various populations. However, the effect of paternal age and education of parents has not been frequently studied. Comparative studies on Down syndrome are also rare. This study evaluates the epidemiological characteristics of Down syndrome in two culturally and socially contrasting population settings, in California and the Czech Republic.
Methods
The observed live birth prevalence of Down syndrome was studied among all newborns in the California counties monitored by California Birth Defects Monitoring Program from 1996 to 1997, and in the whole Czech Republic from 1994 to 1998. Logistic regression was used to analyze the data.
Results
A total of 516,745 (California) and 475,834 (the Czech Republic) infants were included in the analysis. Among them, 593 and 251, respectively, had Down syndrome. The mean maternal age of children with Down syndrome was 32.1 years in California and 26.9 years in the Czech Republic. Children born to older mothers were at greater risk of Down syndrome in both populations. The association with paternal age was mostly explained by adjusting for maternal age, but remained significant in the Czech Republic. The association between maternal education and Down syndrome was much stronger in California than in the Czech Republic but parental age influences higher occurrence of Down syndrome both in California and in the Czech Republic.
Conclusion
The educational gradient in California might reflect selective impact of prenatal diagnosis, elective termination, and acceptance of prenatal diagnostic measures in Californian population.
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Background
Down syndrome (also called trisomy 21 or trisomy of chromosome 21), is the most common chromosome abnormality in newborns. The disease is associated with mental retardation, immune system disorders, autoimmune problems, premature aging and Alzheimer disease at the age of 30–40 years [1,2]. The association between maternal age and risk of having a Down syndrome pregnancy was first published in 1933 [3]. In 1959 the presence of an extra chromosome 21 was identified [4]. In 1966, the first chromosome analysis of amniotic fluid cells was published [5]. The first report of the antenatal diagnosis of Down syndrome was published two years later [6].
Many studies have been conducted to increase understanding of the Down syndrome epidemiology and its geographic variations [7-11]. The relation of advanced maternal age to an increased risk of Down syndrome has been established, but the effects of other risk factors have not been confirmed [12,13]. Given the current level of knowledge, neither the conception of children with Down syndrome nor their birth can be prevented but several screening programs do exist.
Although Down syndrome affects a relatively small number of families directly, internationally it is discussed with millions of parents every year when they are offered prenatal screening. This is often the first time the individuals are confronted with questions about the usefulness and value of genetic testing [14,15]. The diagnosis of Down syndrome is made by chromosomal analysis, which can be initiated prenatally (in the first or second trimester of pregnancy) due to given risk factors for pregnancy, or postnatally due to the characteristic appearance of the newborn child.
Because routine prenatal screening is based on the assumption that it is reasonable for prospective parents to choose to prevent a life with Down syndrome, the proportion of people with Down syndrome in the future will be based both on the development of prenatal screening and on the personal choices of prospective parents [14-17].
In this study, we analyze the epidemiological characteristics of Down syndrome in two culturally and socially contrasting settings, in California and the Czech Republic. The Californian population represents an advanced democratic society with a long tradition of positive attitudes towards people with disabilities [18]. The second is a post-communist society in transition, the Czech population. In the Czech Republic, children with Down syndrome were often referred at birth to residential institutions before year 1989 (the "Velvet revolution"). The communist party propaganda promoted better health through removing people with disabilities from mainstream society. During the past 15 years of societal transformation, negative attitudes have changed. Most of the positive improvements, social acceptance, and the quality of life of people with disabilities have been the results of parental support. As a result of parental support and activity, children with Down syndrome are gradually integrated to the general population. The same trend was recognized in the United States, but it was 30–40 years earlier [19].
The objective of this study is to quantify the effects of different demographic factors on the prevalence of live births with Down syndrome, to examine possible interactions between them, and to compare effects of these factors in two different populations. To do this, we used data from a population-based registry in California, and from birth and congenital anomalies registers in the Czech Republic. Our main focus was on parental age and education. Although the effect of maternal age as a risk factor for Down syndrome is well known, the role of paternal age and maternal education has not been clearly established. Additionally, there have not been many epidemiological studies assessing the association between Down syndrome and parental demographic factors in Central and Eastern Europe.
Methods
Samples
Individual anonymous records of births and congenital anomalies were collected from two populations.
The Californian sample comes from the California Birth Defects Monitoring Program data (CBDMP), representing all births from January 1, 1996, to December 31, 1997 in hospitals in the California counties monitored by the registry (n = 516,745). In addition to birth data, the CBDMP, a regional population-based registry of congenital anomalies, recorded information about birth defects within the first year of life for all newborns registered by this program.
The Czech sample includes all live births reported to the Czech Statistical Office between January 1, 1994 and December 31, 1998 (n = 475,834). For the purpose of this study, birth registry data were linked to the Czech Congenital Anomalies Register by national personal identity numbers. The mandatory statistical records are kept for all children with congenital anomalies up to 15 years of age. The birth/congenital anomalies data set was linked at the Institute of Health Informatics and Statistics of the Czech Ministry of Health (for research project No. 403/00/1521). The linkage was successful for 95% of Down syndrome cases.
For our analysis, we used all live births with gestational age 25 weeks or longer (25 weeks was the lowest recorded gestational age in the Czech sample). Therefore, 2,366 Californian babies with recorded gestational age lower than 25 weeks (including 4 Down syndrome cases) were excluded from the analysis.
Variables
We used only variables available in both datasets. For this reason we did not use, for example, ethnicity because this variable is not available in the Czech register. Some variables were categorized for the purpose of the analysis.
Maternal and paternal age at child birth was used as continuous or categorical variable. When used as categorical, the age was classified into ten 3-year age groups (19 years or below; 20–22, 23–25, 26–28, 29–31, 32–34, 35–37, 38–40, 41–43, and over 44 years). We used 10 categories because we wanted to assess possible non-linear trends between parental age and prevalence of Down syndrome, and our large samples allowed the use of such number of categories. Maternal education at the birth of child was classified into four categories: primary, vocational, secondary and university.
Analysis
All live-born cases of Down syndrome (diagnosed either pre- or postnatally) were used as the outcome in our analysis. The effect of parental characteristics on occurrence of Down syndrome was quantified by logistic regression. First, crude odds ratios were calculated for each of the independent variables available in both datasets (maternal and paternal age, education of mother, and sex of infants). Then, all characteristics, except paternal age, were entered into one model to assess their independent effects, and adjusted odds ratios were calculated. Finally, we included paternal age in the model, and fully adjusted odds ratios were estimated.
All analyses were carried out using the SPSS (SPSS Inc, Chicago, USA) and STATA (Stata Corp, College Station, Texas, USA) statistical packages. The use of the data was in accordance with the statutory obligations to protect confidentiality. Individuals could not be identified from the data provided for analysis.
Results
The crude observed live birth prevalence of Down syndrome in California was 11.5 per 10,000 births in 1996–97 (Table 1). This figure represents 1 case of Down syndrome in every 870 live births. In the Czech Republic, the live birth prevalence of Down syndrome was 5.3 per 10,000 live births in 1994–98 (Table 1). This figure represents 1 case of Down syndrome in every 1,900 live births. There was a slightly higher proportion of boys with Down syndrome in California (12.4 boys vs. 10.5 girls per 10,000 live births) and in the Czech Republic (5.9 boys vs. 4.6 girls per 10,000 live births).
Table 1 Numbers of newborns, children with Down syndrome, and prevalence rates of Down syndrome, California (1996–97) and the Czech Republic (1994–98)
California Czech Republic
All children Children with DS All children Children with DS
No. % of births No. % of births Prevalence per 10 000 No. % of births No. % of births Prevalence per 10 000
Sex of infants
Boy 264 321 51.2 328 55.3 12.4 244 503 51.4 144 57.4 5.9
Girl 252 408 48.8 265 44.7 10.5 231 331 48.6 107 42.6 4.6
Unknown 16 0 0 0
Education of mother
Primary 85 615 16.6 157 26.5 18.3 64 774 13.6 34 13.5 5.2
Vocational 107 633 20.8 96 16.2 8.9 199 260 41.9 110 43.8 5.5
Secondary 234 312 45.3 251 42.3 10.7 168 659 35.4 80 31.9 4.7
University 83 681 16.2 77 13.0 9.2 43 135 9.1 27 10.8 6.3
Unknown 5 504 12 6 0
Maternal age
-19 65 058 12.6 31 5.2 4.8 46 236 9.7 17 6.8 3.7
20–22 73 102 14.1 46 7.8 6.3 118 195 24.8 57 22.7 4.8
23–25 79 444 15.4 55 9.3 6.9 121 827 25.6 50 19.9 4.1
26–28 83 791 16.2 56 9.4 6.7 85 173 17.9 40 15.9 4.7
29–31 78 435 15.2 64 10.8 8.2 52 998 11.1 28 11.2 5.3
32–34 64 442 12.5 101 17.0 15.7 28 689 6.0 27 10.8 9.4
35–37 42 301 8.2 75 12.6 17.7 14 171 3.0 14 5.6 9.9
38–40 21 289 4.1 83 14.0 39.0 6 431 1.4 7 2.8 10.9
41–43 7 173 1.4 68 11.5 94.8 1 818 0.4 6 2.4 33.0
44+ 1 527 0.3 14 2.4 91.7 296 0.1 5 2.0 168.9
Unknown 183 0 0 0
Paternal age
-19 24 635 4.8 14 2.4 5.7 6 670 1.4 3 1.2 4.5
20–22 47 498 9.2 30 5.1 6.3 49 493 10.4 14 5.6 2.8
23–25 64 126 12.4 41 6.9 6.4 87 675 18.4 43 17.1 4.9
26–28 74 838 14.5 54 9.1 7.2 88 677 18.6 41 16.3 4.6
29–31 75 282 14.6 82 13.8 10.9 68 831 14.5 29 11.6 4.2
32–34 69 024 13.4 76 12.8 11.0 42 989 9.0 24 9.6 5.6
35–37 51 791 10.0 75 12.6 14.5 23 953 5.0 21 8.4 8.8
38–40 32 849 6.4 46 7.8 14.0 14 105 3.0 14 5.6 9.9
41–43 18 406 3.6 66 11.1 35.9 7 402 1.6 7 2.8 9.5
44+ 18 286 3.5 66 11.1 36.1 6 610 1.4 9 3.6 13.6
Unknown 40 010 43 79 429 46
Total 516 745 100 593 100 11.5 475 834 100 251 100 5.3
In California, mean maternal age (SD) was 27.2 (6.5) years for mothers of all children and 32.1 (7.3) years for mothers of children with Down syndrome, whereas in the Czech Republic, it was 25.1 (4.9) and 26.9 (6.3) years. The maternal age difference between non-Down syndrome and Down syndrome children was significant in both Californian and Czech samples (t = 18.84, p < 0.001 for California, and t = 6.11, p < 0.001 for the Czech Republic). In California, the highest proportion of all children was born to mothers aged 23–31, and of children with Down syndrome to mothers aged 32–40. The live birth prevalence rate of Down syndrome was higher among older mothers (Table 1). In the Czech Republic the largest proportion of newborns and newborns with Down syndrome was among mothers aged 20–25 years. Prevalence rates of Down syndrome substantially increased with increasing maternal age (Table 2). There is a large difference in proportion of Down syndrome babies born to younger women (< 35 years); in California it was 59.5% and in the Czech Republic 87.3% of Down syndrome babies.
Table 2 Odds ratios (95% confidence interval) of occurrence of Down syndrome California, 1996–97
Crude 95% CI Fully adjusted* 95% CI
Lower Upper Lower Upper
Sex of infants
Boy 1 1
Girl 0.85 0.72 1.00 0.84 0.71 0.99
Education of mother
Primary 1.99 1.52 2.62 2.80 2.12 3.72
Vocational 0.97 0.72 1.31 2.24 1.62 3.10
Secondary 1.16 0.90 1.50 1.87 1.44 2.44
University 1 1
P for trend <0.001 <0.001
Maternal age
-19 1 1
20–22 1.32 0.84 2.08 1.66 0.95 2.92
23–25 1.45 0.94 2.26 2.05 1.14 3.68
26–28 1.40 0.90 2.17 2.18 1.20 3.99
29–31 1.71 1.12 2.63 2.74 1.49 5.04
32–34 3.29 2.20 4.92 5.47 3.00 9.98
35–37 3.72 2.45 5.66 6.27 3.37 11.64
38–40 8.21 5.43 12.40 12.34 6.61 23.05
41–43 20.07 13.12 30.71 28.42 14.98 53.91
44+ 19.40 10.30 36.55 27.57 12.22 62.21
Paternal age
-19 1 1
20–22 1.11 0.59 2.10 0.85 0.44 1.67
23–25 1.13 0.61 2.06 0.69 0.35 1.37
26–28 1.27 0.71 2.29 0.65 0.32 1.31
29–31 1.92 1.09 3.38 0.83 0.41 1.66
32–34 1.94 1.10 3.43 0.67 0.33 1.37
35–37 2.55 1.44 4.51 0.68 0.33 1.40
38–40 2.47 1.36 4.49 0.54 0.26 1.13
41–43 6.33 3.55 11.27 1.02 0.49 2.11
44+ 6.37 3.58 11.34 0.86 0.41 1.79
Note: * -Adjusted for all variables in the Table.
In California, mean age (SD) of fathers of all children and children with Down syndrome was 35.4 (19.7) and 38.7 (18.6) years, whereas in the Czech Republic it was 28.2 (5.7) and 30.2 (6.9). The difference in paternal age between non-Down syndrome and Down syndrome babies was highly significant in both samples: t = 12.94 (P < 0.001) in California and t = 5.04 (P < 0.001) in the Czech Republic. In California, the highest percentage of all newborns was among fathers 26–34 years old, while the highest percentage of children with Down syndrome among fathers 29–37 years old. In the Czech Republic it was among 23–31 year olds for both all newborn babies and babies with Down syndrome (Table 1). The proportion of children with Down syndrome born to young fathers (< 35 years) was 54% in California and 75% in the Czech Republic.
Paternal and maternal age of children with Down syndrome was, however, highly correlated: more in the Czech Republic (r = 0.75) than in California (r = 0.71) but both were highly significant (p < 0.001). Similar correlations were observed among non-Down syndrome children: correlation between paternal and maternal age was 0.74 in California and 0.72 in the Czech Republic. This strong correlation creates great potential for residual confounding of maternal age and the association between paternal age and Down syndrome.
In California, the highest proportion of infants was among mothers with secondary education (45.3% for all newborn babies and 42.3% for babies with Down syndrome; Table 1), whereas in the Czech Republic, it was among mothers with vocational education (41.9% for all newborn babies and 43.8% for babies with Down syndrome). The live birth prevalence of Down syndrome was the highest among mothers with the lowest education in California, and with the highest education level in the Czech Republic (Table 1). In California, the odds ratio of having child with Down syndrome was 1.99 (95% CI 1.52–2.62) for mothers with completed primary education compared to mothers with university education. In the Czech Republic, it was 0.84 (0.51–1.39).
Tables 2 and 3 show crude and adjusted odds ratios for live birth prevalence of Down syndrome by the sex of infants, education of mothers, maternal and paternal age for both study populations. After simultaneously controlling for 4 covariates, the effects of maternal education and maternal age increased in California, while in the Czech Republic there was no change in the effect of maternal education and a decrease in the effect of maternal age on the occurrence of Down syndrome. When additionally adjusted for paternal age (right panel in tables 2 and 3), the effect of maternal age is slightly reduced but remains highly significant. The effect of paternal age on live birth prevalence of Down syndrome is less clear. When adjusted for maternal age and education, the effect completely disappeared in California and was substantially reduced in the Czech Republic.
Table 3 Odds ratios (95% confidence interval) of occurrence of Down syndrome Czech Republic, 1994–98
Crude 95% CI Fully adjusted* 95% CI
Lower Upper Lower Upper
Sex of infants
Boy 1 1
Girl 0.79 0.61 1.01 0.67 0.51 0.89
Education of mother
Primary 0.84 0.51 1.39 1.03 0.57 1.86
Vocational 0.88 0.58 1.34 1.06 0.66 1.70
Secondary 0.76 0.49 1.17 0.85 0.53 1.36
University 1 1
P for trend 0.98 0.44
Maternal age
-19 1 1
20–22 1.31 0.76 2.25 0.89 0.46 1.69
23–25 1.12 0.64 1.93 0.67 0.33 1.33
26–28 1.28 0.72 2.25 0.76 0.37 1.60
29–31 1.44 0.79 2.62 0.61 0.27 1.41
32–34 2.56 1.39 4.69 1.31 0.57 3.03
35–37 2.68 1.32 5.45 1.39 0.55 3.56
38–40 2.96 1.23 7.13 1.00 0.28 3.52
41–43 8.99 3.54 22.82 4.61 1.38 15.40
44+ 46.65 17.10 127.25 11.26 2.14 59.36
Paternal age
-19 1 1
20–22 0.63 0.18 2.19 0.68 0.19 2.41
23–25 1.09 0.34 3.51 1.33 0.40 4.50
26–28 1.03 0.32 3.32 1.34 0.39 4.63
29–31 0.94 0.29 3.08 1.23 0.35 4.40
32–34 1.24 0.37 4.12 1.49 0.41 5.46
35–37 1.95 0.58 6.53 1.98 0.52 7.44
38–40 2.21 0.63 7.69 2.02 0.51 7.96
41–43 2.10 0.54 8.14 1.68 0.38 7.42
44+ 3.03 0.82 11.20 2.03 0.47 8.77
Note: * -Adjusted for all variables in the Table.
Discussion
The main focus of the present study is to compare the epidemiological characteristics of Down syndrome in two contrasting populations, in California and the Czech Republic. Overall, prevalence of Down syndrome was significantly higher in Californian population. Most children with Down syndrome had parents younger than 35 years, however we found that the highest risk of having child with Down syndrome is among older parents (and particularly older mothers). We have also found that the risk of Down syndrome associated with increasing age increases more dramatically in California than in the Czech Republic. The association with paternal age was mostly explained when adjusted for maternal age. The association between maternal education and Down syndrome was much stronger in California than in the Czech Republic.
The effect of maternal age shown in this study is in agreement with several previous studies [7,20,21,23]. The removal of the effect of paternal age after adjustment for age of mother is very similar to recent analysis of Norwegian data [21] which showed relatively strong effect of paternal age almost completely explained by adjustment for maternal age. The association between paternal age and Down syndrome was also influenced by high proportion of missing information about age of fathers (about 8% in California and 17% in the Czech population). We looked at the association between education of mother and maternal age and proportion of missing data on paternal age. There was relationship between maternal education and missing paternal data: paternal data were available for 94% of children with university educated mother compared to only 52% available data for children with primary educated mothers. Paternal data were available for 87% of children with mothers aged 25–34 compared to 71% of children with mothers aged 40 and more, and 60% of children with mothers aged 19 or less.
Maternal age is, however, associated very strongly with Down syndrome. The association seems to be non-linear (much higher increase in prevalence of Down syndrome in older age than in younger ages). We tested for several polynomial functions but at the end we decided to use 3-years age groups as the model using age as categorical variable described the association between two variables the best.
The data on demographic and social characteristics are collected by the medical staff from medical records, identification cards, or self-reported by the new mothers. Although some misclassification of the independent variables could have occurred, it was probably small and random. The international comparison is based on data from two different sources. Thus, this study has the following potential limitation: the diagnosis of Down syndrome is slightly different in both populations. In California, Californian Birth Defect Monitoring program collects the information about congenital anomalies only for children up to the age of one year; in the Czech Republic this information is entered to the Register for all children up to 15 years of age. However, a high proportion of Down syndrome identification occurs in a very short period after birth; in the Czech Republic 95% of cases are diagnosed during the infant time period. It is not clear how this would bias our results.
The advanced health care systems – prenatal care in California and the Czech Republic provide a chance to compare the epidemiology of Down syndrome between these populations – the clinical practice is based on an approach that combines routine offer of maternal serum screening or amniocentesis to women with age 35 as a cut off for this procedure, or both. In the Czech Republic, prenatal diagnostics of Down syndrome (DS) is based on second trimester screening biochemical and ultrasound methods. An efficiency of DS prenatal diagnostics is around 66 – 67 % in the last years, that is, probably, a maximum for these methods. In order to increase the prenatal screning efficiency and to move the diagnostics towards earlier stages of pregnancy, methods of first trimester screening are gradually introduced. At present, first trimester screening is available at some prenatal dianostics departments and is expected to be implemented at other departments in early future. An increase of pre- and postnatally diagnosed DS cases at present is caused by several demographic and medical factors, such as by an increase of mean maternal age in the country along with increasing proportion of mothers 35 years of age old and over and increased number of multiple pregnancies. The current US prenatal testing guidelines recommend offering amniocentesis to women aged 35 years or older, or women who have been found by serum and ultrasound screening to be at a similarly high risk of giving birth to an infant with DS or another chromosal abnormality [24]. Although the prenatal testing for chromosomal disorders is of very high standards in both societies, other factors, such as the use of prenatal diagnostic services and accessibility of prenatal care may highly influence the prevalence levels. It is clear that abortion of affected fetuses play the important role in the occurrence of Down syndrome in the newborns. The selective use of prenatal diagnostic testing can have many implications in both comparative settings.
In the Czech Republic, very low live birth prevalence rate of Down syndrome (5.3 cases per 10,000 newborn infants) and a low proportion of children with Down syndrome born to women after 35-years of age (about 13%) supports consistent detection of this type of birth defect during pregnancy (almost 1 from 6 newborns had invasive diagnostic testing procedures during pregnancy, for example in 2003 14,984 from 93,185 newborns had diagnostic test) and a high ratio of terminated pregnancies. Congenital anomalies database (aggregated data by age of mother) for years 1994–1998 shows that only 42% of positively diagnosed Down syndromes were born. Among DS pregnancies of mothers 19 years old or younger, 55% children were born (and for age groups 20–22, 23–25 and 26–28 years, the proportions were 66%, 53% and 54%). However, only 13% were born to those aged 35 years or more [22] (+ Vladimir Gregor, personal communication). Rates of pregnancies with Down syndrome in 1994–98 are shown in table 4. No significant differences in prevalence of Down syndrome by maternal education in the Czech population are consistent with the fact that prenatal care is offered free and on the same qualitative level to all women.
Table 4 Prevalence rates of pregnancies with Down syndrome (per 10000), Czech Republic (1994–98)
Age of mother 1994 1995 1996 1997 1998 Total
-19 6.26 8.45 4.90 5.74 8.25 6.70
20–22 5.36 6.16 8.14 7.66 11.01 7.36
23–25 5.62 8.13 9.15 8.90 7.35 7.80
26–28 7.60 9.27 6.23 8.14 11.82 8.69
29–31 12.96 6.68 6.80 9.60 16.35 10.57
32–34 26.79 13.14 26.73 14.56 31.52 22.66
35–37 21.02 36.90 48.87 45.26 52.05 40.93
38–40 125.28 96.85 86.68 102 92.88 101.07
41–43 306.12 187.2 305.6 292.4 400 297.03
44+ 333.33 615.4 169.5 204.1 317.5 337.84
Total 10.94 11 12.34 12.32 16.29 12.50
The educational gradient found in Californian sample might reflect selective impacts of maternity care, prenatal diagnosis, elective termination, and acceptance of prenatal diagnostic measures. We tried to test whether the association between education and Down syndrome can be confounded by ethnicity. When additional analysis including ethnicity of mother was conducted (results not presented), the association between maternal education and prevalence of Down syndrome was reduced but remained significant. Our assumption, that parents with lower socio-economic status often have few options for maternity care and little knowledge of prenatal testing is supported by the data from the birth certificates analysis (Table 5). The proportion of the amnio-utilization during pregnancy increased not only with maternal age but also with maternal education level. These results suggest that special support before and during the pregnancy would help to reduce social inequalities in prevalence of Down syndrome in California.
Table 5 Births with Amniocentesis by Mother's education and age, California, 1998 (in %)
Mother's education: Mother's age:
(in years) - 19 years 20–34 years 35 + years Total
0 – 8 years 0.3 0.4 2.0 0.6
9 – 11 years 0.3 0.5 4.0 0.6
12 – 15 years 0.5 0.9 11.1 2.2
16 + years - 1.7 16.5 6.1
Total 0.4 0.9 11.5 2.5
Source: CDC, 1998, Birth Cohort Data Set
Previous results suggest that the rate of detection of Down syndrome may be higher in the Czech Republic than in the United States. The evaluation of the prenatal screening programs in Iowa and California showed that only approximately 40 to 50% of the cases were detected [25-27]. In the Czech Republic, 58% of pregnancies with DS were aborted in 1994–1998.
In California, the higher live birth prevalence rate of Down syndrome (11.5 cases per 10,000 newborn infants) and higher proportion of children with Down syndrome born to women older than 35 years (41%) might also reflect different attitudes towards prenatal diagnosis and abortion, different social and familial background and, maybe, a much more favorable opinion towards people with disabilities. Therefore it seems that the study results are strongly related to regional social context and abortion behavior.
As stated in the result section, the distribution of maternal age in two populations is different (more advanced maternal age in California). When birth prevalence rates in both countries are standardized by age of mother, difference in live birth prevalence of DS reduces (6.0 cases per 10,000 newborn infants in the Czech Republic and 10.0 in California) however it is still relatively large. It can be seen from table 1, that (with exception of 44+ age group), live birth prevalence of DS is higher in California in every age category.
At the beginning of the 1990s, the Czech Republic had one of the highest rates of therapeutic abortions in the world. The societal changes in the Czech Republic after 1989 have had a positive influence on the abortion situation. The abortion rate has significantly decreased and in 1994–1998 the induced abortion rate was 18.1 per 1,000 women of reproductive age (15–49). In the study period the number of newborns with Down syndrome was lower than the number of abortions with affected fetuses. A total of 251 newborns with Down syndrome and 396 electively aborted fetuses with Down syndrome were ascertained (data from Czech Congenital Anomalies Register); 61% of pregnancies with Down syndrome are electively aborted [22]. Although abortion incidence is the subject of research in the United States, nationally valid data are available from only two sources: the federal Centers for Disease Control and Prevention (CDC) and The Alan Guttmacher Institute. However the CDC does not collect abortion information from California specifically. In California, very limited statistics exist on abortion. The Alan Guttmacher Institute estimates the induced abortion rate was 31.2 per 1,000 women of reproductive age in California in 2000 [28]. Since December 2002 there have been restrictions on abortion in California – a woman must receive mandatory state-directed counseling before an abortion is provided. In the 1996–1997 California Genetic Disease Branch data set, a total of 456 positive pregnancies with Down syndrome were diagnosed via amniocenteses and 267 from them were electively aborted; the termination rate for these pregnancies was 58.5% (information obtained from the Department of Health Services, Genetic Disease Branch, Richmond, USA).
It is clearly recognized that Down syndrome prenatal screening is driven by several primary forces: (i) effort to reduce "the costs of life-long care" of people with Down syndrome through prenatal screening; (ii) clinical support for individual choices of mothers or couples; (iii) public health strategies designed to reduce birth defects and improving reproductive outcomes [14].
The social benefits of prenatal screening of Down syndrome are very important: (i) the prospective parents demand to be well-informed about their pregnancy outcomes, and (ii) the prospective parents need time to make informed decisions about selective prenatal termination of affected pregnancies or follow-up with it. When the pregnancy for Down syndrome affected child continues, the main goal is to support socially disadvantaged families, and to help to start lives of children born with Down syndrome.
Conclusion
This study supports previous research showing that most children with Down syndrome are born to parents below 35 years of age and that significant risk levels for Down syndrome are not only in advanced maternal age categories. However risk of births with Down syndrome significantly increases with increasing paternal age, and, in particular, with increasing maternal age. Additionally, educational effects on maternal age-specific risk rates of Down syndrome were found for California mothers. The educational gradient might reflect selective impacts of maternity care, prenatal diagnosis, elective termination, and acceptance of prenatal diagnostic measures in the Californian population. On average, parents with lower socio-economic status often have few options for maternity care and little knowledge of prenatal testing, and they need special support for their start with parenthood and well-being of future generations. To prevent births of unwanted children with Down syndrome, comprehensive maternity care services must be available to all pregnant women regardless of socio-economic status. Prenatal diagnostic testing is also important for pregnant women at any age who would not consider abortion because babies with Down syndrome can need specialized care at delivery. Individuals with Down syndrome can live full, productive, and quality lives with help from modern medicine and lifetime educational/support programs. Access to the prenatal testing of chromosomal disorders to all pregnant women may be one possible task in strategy to reduce social inequalities in health.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DD designed the study, collected the data, participated in data analyses and preparation of manuscript. HP analyzed the data and participated in preparation of manuscript. Both authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
DD work was supported by Fogarty Program for Czech Post-Doctoral Scholars, School of Public Health, UC Berkeley, D43 TW05810-01 and MSM 0021620831, the Czech Republic. The authors thank Vladimir Gregor, head of Medical genetics department of Thomayer Faculty Hospital in Prague, the Czech Republic, for information on prenatal diagnostics in the Czech Republic. The authors thank Karen Lutfey and Amanda Nicholson for help with proof reading. Finally, the authors want to thank Babak Khosnood for stimulating comments during review process.
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| 15963229 | PMC1166564 | CC BY | 2021-01-04 16:28:56 | no | BMC Public Health. 2005 Jun 17; 5:69 | utf-8 | BMC Public Health | 2,005 | 10.1186/1471-2458-5-69 | oa_comm |
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BMC Womens HealthBMC Women's Health1472-6874BioMed Central London 1472-6874-5-71590451610.1186/1472-6874-5-7Research ArticleChanges in elderly women's health-related quality of life following discontinuation of hormone replacement therapy Heller Debra A [email protected] Carol H [email protected] Frank M [email protected] Kristine E [email protected] Theresa V [email protected] Margaret R [email protected] First Health Services/The PACE Program, 4000 Crums Mill Road, Suite 301, Harrisburg, PA 17112 USA2 Department of Biobehavioral Health, The Pennsylvania State University, 315 East Health and Human Development, University Park, PA 16802 USA3 Pennsylvania Department of Aging/The PACE Program, 555 Walnut Street, 5th Floor, Harrisburg, PA 17101 USA2005 16 5 2005 5 7 7 27 12 2004 16 5 2005 Copyright © 2005 Heller et al; licensee BioMed Central Ltd.2005Heller et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Many women have discontinued hormone replacement therapy (HRT) in view of recent findings. The goal of this study was to determine if HRT discontinuation is associated with changes in health-related quality of life (HRQOL) in elderly women.
Methods
We studied women enrolled in Pennsylvania's Pharmaceutical Assistance Contract for the Elderly (PACE) program, linking prescription claims with data from a longitudinal mail survey. HRQOL measures included the number of days out of the last 30 that physical health was not good and analogous measures for mental health, pain, and interference with activities, as well as a composite "healthy days" measure developed by CDC. Longitudinal analyses focused on 2,357 women who completed surveys in both 2002 and 2003, and who used HRT at baseline (mean age = 75.5, range = 65–102). Propensity scores were used to match HRT continuers and discontinuers according to HRT type, demographics, and baseline HRQOL. Analysis of covariance was used to compare HRQOL change in continuers and discontinuers.
Results
Between 2002 and 2003, 43% of HRT users discontinued therapy. Analysis of covariance to examine HRQOL change revealed complex interactions with age. Discontinuers aged 65–74 reported greater increases in days in which mental health was not good (p < .05), fewer "healthy days" (p < .05), more days in which health interfered with activities (p < .01), and more days with pain (p < .01). Among women aged 75–84, HRT discontinuers reported more days in which physical health was not good (p < .01); no other significant effects were observed in this group. Relative to HRT continuers, discontinuers aged 85 and older experienced apparent HRQOL improvements following cessation, with fewer days in which physical health was not good (p < .01), fewer days of poor mental health (p < .05), and more "healthy days" (p < .01).
Conclusions
These results suggest that there are substantial age differences in response to HRT discontinuation. While women aged 65–74 experienced apparent declines in HRQOL following HRT cessation, women aged 85 and older experienced relative improvements. The HRQOL declines observed among younger women underscore the importance of communication between clinicians and patients throughout the discontinuation process. These results also demonstrate the value of HRQOL surveillance as a component of health program administration.
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Background
Few health topics are currently associated with as much confusion as the issue of hormone replacement therapy (HRT) for postmenopausal women. Historically, the results of observational studies spanning several decades have suggested that HRT confers important cardiovascular benefits for postmenopausal women [1,2]. In July of 2002, however, the combination HRT component of the Women's Health Initiative (WHI) study-a randomized placebo-controlled trial-was halted due to an excess incidence of coronary events, stroke, pulmonary embolism, and breast cancer among women receiving estrogen and progestin in combination [3,4]. In February of 2004, the estrogen-only arm of the WHI study was similarly halted due to observed increases in the risk of stroke among women with prior hysterectomy who received unopposed estrogen [5].
The well-publicized halting of the WHI combination therapy trial, along with emerging evidence from other studies, sparked intense debate and ultimately led to an important paradigm shift with respect to HRT. Guidelines such as those released in 2002 by the U.S. Preventive Services Task Force stress that the harmful effects of HRT related to breast cancer and cardiovascular risk outweigh potential benefits such as increased bone density, and that HRT should therefore not be used for the prevention of chronic conditions in postmenopausal women [6]. The newer guidelines and warnings are in sharp contrast to HRT's cultural history and early marketing, which portrayed hormone replacement as fundamental for the preservation of health, youth, and femininity. Many older women who are now long past menopause initiated HRT decades ago with the belief that they would continue with the therapy for life. The recent shifts in guidelines and practice have therefore presented a dilemma for clinicians and patients, and many women have opted to discontinue HRT [7].
Enhanced quality of life is a frequently-cited benefit of HRT [8], but recent studies addressing quality of life have produced mixed results. A number of observational studies comparing HRT users and non-users have observed better psychological health and health-related quality of life among HRT users [9-11]. Recent randomized clinical trials, however, have reported fewer apparent benefits for postmenopausal women. For example, comparing HRT users and non-users in the Heart and Estrogen/Progestin Replacement Study (HERS), Hlatky and his colleagues found no evidence of a general benefit in quality of life associated with HRT [12]. Among women with menopausal symptoms such as flushing, however, HRT use produced improvements in mental health and depressive symptoms. Similar findings were reported by Hays et al. using WHI data [13]. Both the WHI and HERS studies concluded that, aside from women experiencing acute menopausal symptoms, HRT users did not differ from non-users with respect to quality of life. No published studies to date, however, have specifically examined the impact of HRT discontinuation on quality of life. In addition, relatively few HRT studies have focused explicitly on elderly women, but have instead focused on younger postmenopausal women, who are most likely to experience acute menopausal symptoms. Elderly women comprise an important segment of the population of HRT users, and more research on elderly HRT users is needed. It is important for clinicians to understand the potential changes in quality of life that may be associated HRT discontinuation, and how women of different ages may respond to HRT discontinuation.
The goal of this study was to determine if HRT discontinuation is associated with changes in health-related quality of life in a population of elderly women. We studied women enrolled in a state pharmaceutical assistance program for the elderly, linking prescription claims with data from a longitudinal mail survey that addressed health-related quality of life. The availability of longitudinal survey data for HRT continuers and discontinuers provides an important opportunity to examine the impact of HRT discontinuation on the quality of life of elderly women.
Methods
Study population
Subjects included women who were enrolled during 2002 and 2003 in Pennsylvania's Pharmaceutical Assistance Contract for the Elderly (PACE), a state program providing prescription drug assistance to elderly with low to moderate incomes. All PACE applicants complete a detailed application form prior to enrollment. Depending on their income, enrollees are then required to re-enroll either annually or biannually. For purposes of research and evaluation, PACE includes an optional two-page survey – the Survey on Health and Well-Being – with all new and renewal enrollment applications. The survey was approved by the Institutional Review Board of the Pennsylvania State University. Historical data suggest that most PACE cardholders view the survey positively, with annual response rates exceeding 70% [14].
In order to examine longitudinal associations, the present study focuses on women who completed both the 2002 and 2003 Surveys on Health and Well-Being, and who were HRT users at baseline (i.e., the time that they completed the 2002 survey) based on prescription claims review.
Measurement of health-related quality of life
Health related quality of life (HRQOL), although variously defined, refers generally to those aspects of quality of life that are clearly related to either physical or mental health [15,16]. The U.S. Centers for Disease Control and Prevention (CDC) has described HRQOL as "an individual's or group's perceived physical and mental health over time" [15]. Over the last three decades, HRQOL has been increasingly recognized as an important component of health status that should be considered when evaluating the effectiveness of health programs and medical treatments [17].
The PACE Survey on Health and Well-Being includes a series of HRQOL questions that were adapted from the CDC's Behavioral Risk Factor Surveillance System (BRFSS) telephone survey. The BRFSS HRQOL measures include a core set of four questions as well as an optional set of questions related to pain perception and activity limitation [15]. Moderate to excellent retest reliabilities for the HRQOL module questions have been reported [18]. Although brief, the BRFSS HRQOL module has produced results comparable to the SF-36 in prior studies, and has successfully distinguished groups varying in clinical diagnoses [19,20]. Recent research has also demonstrated that the instrument is responsive to changes in health status and is well-accepted by older adults [21,22].
Table 1 displays the BRFSS HRQOL questions included in the PACE survey. The present analysis focuses on the number of days out of the last 30 that physical health was not good and analogous measures for mental health, interference of health with activities, and pain. The present study also utilized composite measures of "unhealthy days" and "healthy days" developed by CDC. The total number of unhealthy days in the last month was estimated by summing the number of "not good" physical and mental days, with a logical maximum of 30 days. CDC's measure of healthy days, a positive complementary form of unhealthy days, was computed by subtracting the number of unhealthy days from 30 [15,23]. Prior studies have demonstrated that the healthy and unhealthy days composite variables are valid and responsive measures of perceived health [15,19].
Table 1 Health-related quality of life questions included in survey
Core HRQOL module questions
1. Would you say that in general your health is: excellent, very good, good, fair or poor?
2. Now thinking about your physical health, which includes physical illness and injury, for how many days during the past 30 days was your physical health not good?
3. Now thinking about your mental health, which includes stress, depression, and problems with emotions, for how many days during the past 30 days was your mental health not good?
4. During the past 30 days, for about how many days did poor physical or mental health keep you from doing your usual activities, such as self-care, work, or recreation?
Optional HRQOL module questions (pain and activity limitation)
1. During the past 30 days, for about how many days did PAIN make it hard for you to do your usual activities, such as self-care, work, or recreation?
2. Are you limited in any activities because of any impairment or health problem?
3. What is the MAJOR impairment or health problem that limits your activities?
4. For HOW LONG have your activities been limited because of your impairment or health problem? Please give the length of time.
5. Because of any impairment or health problem, do you need the help of other persons with your PERSONAL CARE needs, such as eating, bathing, dressing, or getting around the house?
6. Because of any impairment or health problem, do you need the help of other persons in handling your ROUTINE needs, such as everyday household chores, doing necessary business, shopping, or getting around for other purposes?
Measurement of HRT and other prescription drug use
PACE utilizes a point-of-sale claim payment system; at the time of dispensing, pharmacies submit electronic claims to PACE for adjudication and reimbursement. Complete data on all prescription medications obtained by study participants were therefore available for study, provided that participants filled the prescriptions by using their PACE cards. All HRT prescriptions, and details regarding the specific type of HRT, were identified by linking claim records for study participants to a database of drug attributes (Red Book®) provided by Medical Economics/Thomson Healthcare (Montvale, New Jersey). Systemic forms of HRT, including oral and transdermal products, were distinguished from topical forms on the basis of each dispensed product's formulation code.
HRT status was evaluated by identifying all systemic HRT claims dispensed between October 1, 2001 and March 31, 2004. PACE allows only a 30-day supply of medication to be dispensed per prescription refill. In order to allow for early refills and potential prescription overlap, baseline HRT users were defined as individuals who filled one or more HRT prescriptions during the 45 days preceding their 2002 baseline survey response date. For follow-up status evaluation, claims were examined for the entire time interval between the baseline survey date and up to 90 days after the 2003 follow-up survey date. Discontinuers were defined as individuals whose last HRT prescription was filled more than 45 days prior to their follow-up survey response date. Women who filled one or more HRT prescriptions during the 45 days immediately preceding the follow-up survey date were identified as continuers. A small number of baseline HRT users (n = 143) who did not have any HRT claims during the 45 days prior to the follow-up survey, but who filled HRT prescriptions within 90 days after their 2003 survey date, were also categorized as continuers. Baseline and follow-up HRT use for each study participant was further categorized as either unopposed estrogen (estrogen without progestin) or combination therapy (estrogen with progestin).
Statistical analyses
Descriptive statistics
All statistical analyses were conducted using the Statistical Analysis System (SAS) for Windows, Version 8.2 (SAS Institute, Cary, NC). Descriptive statistics were used to describe HRT continuers and discontinuers in terms of demographic characteristics, self-reported health behaviors, HRT and other drug utilization, and HRQOL survey measures.
Propensity scores
The goal of the present study was to compare HRQOL change in HRT continuers and discontinuers. A limitation, however, is that women who choose to continue HRT may differ fundamentally from women who discontinue HRT. If so, then these fundamental differences may also be reflected in differential change in HRQOL, leading to biased estimates of the effect of HRT discontinuation on HRQOL. Such bias is inherent in many observational studies, in contrast to randomized trials in which subjects are assigned randomly to treatment or non-treatment groups.
Propensity scores have been proposed as a methodological strategy for bias control in observational studies [24-26]. The propensity score is defined as the conditional probability that an individual is a member of a treatment group, given all available covariate values [26]. Compared to traditional stratification, matching, or covariate adjustment methods, propensity scores offer the advantage of reducing a large number of background covariates to a single scalar value. Once propensity scores have been created, traditional procedures such as stratification or matching can then be applied to the propensity scores [26].
In the context of the present study, the propensity score represents the conditional probability (ranging from 0 to 1) that a woman using HRT at baseline will discontinue HRT, given her baseline characteristics. Propensity scores were created through a multivariate logistic regression analysis to predict the binary outcome of HRT discontinuation at the time of the follow-up (2003) survey, based on multiple explanatory variables measured at baseline (2002). The baseline explanatory variables included demographic measures (age, race, income, marital status, education, long-term care residence, and urban/rural residence), self-reported health behaviors (alcohol use and smoking), type of HRT, and non-drug utilization.
Matching of continuers and discontinuers
Continuers and discontinuers were matched using a SAS macro provided by researchers at the Mayo Clinic Division of Biostatistics [27]. The propensity score generated in the multivariate logistic regression described above was used as the primary matching factor. To control further for potential baseline differences in HRQOL, the baseline HRQOL measures were also subjected to principal components analysis, a data reduction technique that summarizes the variance shared by a set of variables [28,29]. The first principal component, which accounted for 71.9% of the total variance in the baseline HRQOL measures, was then used as a secondary matching variable. The rationale for including the principal component score in the matching algorithm was to ensure that the matched samples of continuers and discontinuers were well-balanced in terms of all baseline HRQOL measures. In addition to the propensity and baseline HRQOL principal component scores, continuers and discontinuers were further matched by baseline HRT type (estrogen alone or combination) and age group (65–74 years, 75–84 years, and 85 or older). Using the above-described selection factors, a single best-matching HRT continuer was identified for each HRT discontinuer. All subsequent analyses were conducted using the reduced sample of matched HRT continuers and discontinuers.
Analysis of covariance (ANCOVA)
Once the final matched sample of continuers and discontinuers was created, analysis of covariance (ANCOVA) was used to examine the impact of HRT continuation or discontinuation on HRQOL change. As discussed by Vickers and Altman [30], ANCOVA may be used to compare change in one or more groups by predicting follow-up scores from baseline scores and a treatment group indicator. In the present study, the ANCOVA model equation may be visualized as:
HRQOL2= Constant + β1·HRQOL1+ β2·HRT Group
where HRQOL1 and HRQOL2 are baseline and follow-up HRQOL scores, β1 and β2 are coefficients to be estimated, and HRT Group is a binary variable coded 1 for HRT discontinuation and 0 for HRT continuation. Using the SAS GLM procedure, separate ANCOVA analyses were performed for each of the HRQOL questions. To explore age differences in response to HRT discontinuation, separate analyses were conducted within three age groups: 65–74 years, 75–84 years, and 85 years or older.
Results
A total of 4,236 women completed a survey in 2002 and used HRT at baseline, based on one or more claims for systemic HRT products during the 45 days preceding their 2002 survey return date. Of these women, 1,076 (25.4%) had incomes low enough to qualify them for two years of PACE coverage rather than only one year, and were therefore not required to reapply for coverage in 2003. Among the remaining 3,160 women, 2,899 reapplied for PACE coverage in 2003, and 2,357 of those reapplying also completed the follow-up survey in 2003. The mean interval between the baseline and follow-up surveys was 367 days. Of the 2,357 baseline HRT users who completed surveys in both 2002 and 2003, 1,015 women (43.1%) discontinued HRT between the time of their baseline and follow-up surveys.
Determinants of HRT discontinuation
Table 2 summarizes the results of a multivariate logistic regression analysis to predict HRT discontinuation from multiple baseline measures, including demographic factors, self-reported health behaviors, type of HRT, non-HRT prescription drug use, and baseline HRQOL. Percentage frequencies for each categorical measure and means for each continuous measure are shown for HRT continuers and discontinuers. Table 2 also presents the parameter estimates, standard errors, adjusted odds ratios with associated confidence limits, and probability values for all independent variables. The strongest predictor of HRT discontinuation was type of HRT used at baseline, with combination users being three times as likely as unopposed estrogen users to discontinue HRT (O.R. = 3.03, p < .0001). This result is not surprising given the fact that initial media attention focused on the WHI combination therapy arm that was halted in 2002; the unopposed estrogen arm of the WHI study was not halted until 2004. On average, HRT discontinuers had higher annual incomes than HRT continuers ($13,999 vs. $13,805, O.R. per $1,000 = 1.06, p = .0283). Relative to women living in urban areas, women residing in rural or semi-rural areas appeared more likely to discontinue HRT, although this result did not achieve conventional significance (O.R. = 1.17, p = .0948). Women who had any baseline use of cardiovascular drugs were significantly more likely than non-users to discontinue HRT (O.R. = 1.25, p = .0471), as were women who had any baseline use of medications used to treat osteoporosis (O.R. = 1.36, p = .0146). The total number of non-HRT medication classes used during the 45 days preceding the baseline survey was also significantly associated with discontinuation – women who used higher numbers of medications were less likely to discontinue HRT (O.R. for each additional therapeutic class = 0.95, p = .0021).
Table 2 Determinants of HRT discontinuation: results of propensity analysis
Multivariate Logistic Regression Results*
Variable HRT Continuers (N = 1342) HRT Discontinuers (N = 1015) Parameter Estimate (β) Std. Error of β Adjusted Odds Ratio 95% CI for Odds Ratio P-value
Age
Age in years (mean) 75.5 75.5 0.0081 0.0075 1.041 0.993–1.023 .2847
Race
White (%) † 96.7 95.6 -- -- 1.000 -- --
Black (%) 2.7 3.5 0.3536 0.2527 1.424 0.868–2.337 .1616
Other race (%) 0.6 1.0 0.2903 0.4992 1.337 0.503–3.556 .5609
Income
Annual income in thousands (mean) $13.805 $13.999 0.0537 0.0245 1.055 1.006–1.117 .0283
Marital status
Widowed, divorced, or never married (%) † 80.6 80.5 -- -- 1.000 -- --
Currently married (%) 19.4 19.5 -0.1327 0.1384 0.876 0.668–1.149 .3378
Education
12 or more years of education (%) 60.5 63.5 0.0937 0.0899 1.098 0.921–1.310 .2971
Residence type
Nursing or personal care facility (%) 1.6 1.0 -0.4390 0.4067 0.645 0.291–1.431 .2804
Urban/rural residence
Semi-rural or rural residence (%) 33.6 35.5 0.1526 0.0913 1.165 0.974–1.393 .0948
Alcohol use
Current alcohol user (%) 26.5 24.4 -0.1335 0.1008 0.875 0.995–1.022 .1854
Smoking history
Past or present smoker (%) 34.7 33.0 -0.0573 0.0924 0.944 0.788–1.132 .5352
Type of HRT used at baseline
Unopposed estrogen (%) † 89.5 74.2 -- -- 1.000 -- --
Combination estrogen/progestin (%) 10.5 25.8 1.1100 .1171 3.034 2.412–3.817 .0001
Baseline non-HRT prescription drug use
Any cardiovascular drug use (%) 78.8 79.9 0.2265 0.1141 1.254 1.003–1.568 .0471
Any osteoporosis treatment (%) 12.2 15.4 0.3100 0.1270 1.363 1.063–1.749 .0146
Total number of non-HRT drug classes (mean) 6.3 5.9 -0.0507 0.0165 0.951 0.920–0.982 .0021
Baseline HRQOL
Days that physical health was not good (mean) 6.3 6.3 0.0086 0.0069 1.009 0.995–1.022 .2174
Days that mental health was not good (mean) 3.3 3.0 -0.0042 0.0067 0.996 0.983–1.009 .5323
Healthy days (mean) ‡ 21.9 22.3 -- -- -- -- --
Days that health interfered (mean) 4.2 3.8 -0.0113 0.0077 0.989 0.974–1.004 .1432
Days that pain made it hard (mean) 6.3 6.4 0.0060 0.0064 1.006 0.993–1.019 .3514
* All parameter estimates are adjusted for two-way interaction terms involving age, income, marital status, alcohol use, smoking history, prescription drug use, and baseline HRQOL.
† Reference group
‡ Because the healthy days variable is a composite of the physical and mental days measures, it was omitted from the logistic regression.
Impact of discontinuation on HRQOL
As discussed above, HRT continuers and discontinuers were matched on the basis of the following factors: 1) the propensity scores obtained from the multivariate logistic regression, 2) the first principal component scores for the combined baseline HRQOL measures, 3) age group, and 4) type of HRT used at baseline. Of the 2,357 women present in the original sample, 1,770 respondents (75.1%) were successfully matched using these criteria. The final matched sample included 65.4% of the original sample of HRT continuers, and 86.5% of the original sample of discontinuers. Relaxing the matching algorithm requirements would have resulted in a higher number of matched subjects, but would have provided less control for selection bias.
Table 3 summarizes the results of analysis of covariance to examine associations between HRT discontinuation and changes in HRQOL. For each of the follow-up HRQOL measures, mean values for HRT continuers and discontinuers are shown for three groups: 65–74 years, 75–84 years, and 85 years or older. Each ANCOVA model predicted follow-up HRQOL score from the corresponding baseline HRQOL measure and HRT group. Ancillary information to accompany the ANCOVA results in Table 3 is shown in Figures 1 through 5, which present the adjusted mean change in HRQOL for each measure by age group, while controlling for baseline HRQOL scores.
Table 3 Results of ANCOVA to compare HRQOL change in HRT continuers and discontinuers, by age group
Raw Mean Scores
Continuers Discontinuers
Follow-up Measure Age Group N Time 1 Time 2 Time 1 Time 2 Source of Variation Sum of Squares DF F Value Significance of F
Number of days that physical health was not good 65–74 810 5.19 5.27 5.17 6.31 Baseline physical days 19431.83 1 286.97 <.0001
HRT group 218.26 1 3.22 .0730
75–84 820 6.27 6.24 6.18 7.77 Baseline physical days 25852.59 1 348.90 <.0001
HRT group 510.31 1 6.89 .0088
85+ 140 7.49 9.81 7.86 5.39 Baseline physical days 4130.90 1 50.34 <.0001
HRT group 759.71 1 9.26 .0028
Number of days that mental health was not good 65–74 810 2.53 2.67 2.75 3.78 Baseline mental days 12963.01 1 309.92 <.0001
HRT group 190.93 1 4.56 .0329
75–84 820 2.49 3.67 2.52 3.12 Baseline mental days 11864.39 1 256.49 <.0001
HRT group 67.51 1 1.46 .2274
85+ 140 2.93 5.15 2.17 1.99 Baseline mental days 1402.63 1 23.85 <.0001
HRT group 287.32 1 4.88 .0287
"Healthy days" composite 65–74 810 23.43 23.30 23.43 21.97 Baseline healthy days 28043.55 1 340.82 <.0001
HRT group 348.18 1 4.23 .0400
75–84 820 22.41 21.81 22.59 20.89 Baseline healthy days 34431.84 1 388.18 <.0001
HRT group 222.31 1 2.51 .1138
85+ 140 20.81 17.96 20.44 22.82 Baseline healthy days 5494.08 1 53.96 <.0001
HRT group 914.52 1 8.98 .0032
Number of days that physical or mental health interfered with activities 65–74 810 3.22 3.40 3.17 4.89 Baseline interference days 14886.08 1 256.99 <.0001
HRT group 455.88 1 7.87 .0051
75–84 820 3.63 4.70 3.77 5.20 Baseline interference days 19210.32 1 303.58 <.0001
HRT group 35.08 1 0.55 .4568
85+ 140 5.15 7.46 4.36 5.14 Baseline interference days 4440.59 1 54.06 <.0001
HRT group 121.65 1 1.48 .2257
Number of days that pain interfered with activities 65–74 810 5.71 5.29 5.67 6.91 Baseline pain days 29680.32 1 446.86 <.0001
HRT group 539.67 1 8.13 .0045
75–84 820 5.80 7.02 5.88 7.65 Baseline pain days 33113.98 1 425.40 <.0001
HRT group 67.38 1 0.87 .3525
85+ 140 7.54 8.46 8.44 6.74 Baseline pain days 4637.67 1 53.90 <.0001
HRT group 169.95 1 1.98 .1621
Figure 1 Mean number of days that physical health was not good, by study occasion, age group, and HRT status. Raw means by study occasion are presented for each age group (65–74, 75–84, and 85+) and HRT continuation group (continuers vs. non-continuers). For each age group, the statistical significance of the HRT continuation group difference in HRQOL change (from ANCOVA analyses; see Table 3) is also shown.
Figure 2 Mean number of days that mental health was not good, by study occasion, age group, and HRT status. Raw means by study occasion are presented for each age group (65–74, 75–84, and 85+) and HRT continuation group (continuers vs. non-continuers). For each age group, the statistical significance of the HRT continuation group difference in HRQOL change (from ANCOVA analyses; see Table 3) is also shown.
Figure 3 Mean number of "healthy days", by study occasion, age group, and HRT status. Raw means by study occasion are presented for each age group (65–74, 75–84, and 85+) and HRT continuation group (continuers vs. non-continuers). For each age group, the statistical significance of the HRT continuation group difference in HRQOL change (from ANCOVA analyses; see Table 3) is also shown.
Figure 4 Mean number of days that health interfered with activities, by study occasion, age group, and HRT status. Raw means by study occasion are presented for each age group (65–74, 75–84, and 85+) and HRT continuation group (continuers vs. non-continuers). For each age group, the statistical significance of the HRT continuation group difference in HRQOL change (from ANCOVA analyses; see Table 3) is also shown.
Figure 5 Mean number of days that pain made activities difficult, by study occasion, age group, and HRT status. Raw means by study occasion are presented for each age group (65–74, 75–84, and 85+) and HRT continuation group (continuers vs. non-continuers). For each age group, the statistical significance of the HRT continuation group difference in HRQOL change (from ANCOVA analyses; see Table 3) is also shown.
Striking age differences are apparent in the associations between HRT group and follow-up HRQOL, controlling for baseline HRQOL. Within the youngest group of women – aged 65–74 – HRT discontinuation was associated with a significant increase in the days in which mental health was not good (adjusted mean change = 1.1 days, p = .0329), fewer healthy days (adjusted mean change = -1.5 days, p = .0400), more days in which physical or mental health interfered with activities (adjusted mean change = 1.7 days, p = .0051), and more days in which pain made it hard to do routine activities (adjusted mean change = 1.2 days, p = .0045).
Among women aged 75–84, HRT discontinuers reported more days at follow-up in which their physical health was not good (adjusted mean change = 1.6 days, p = .0088). No other significant differences in HRQOL between HRT continuers and discontinuers were found in this age group.
In contrast to the apparent declines in HRQOL experienced by younger women who discontinued HRT, women aged 85 or older who discontinued HRT appeared to experience improvements in several HRQOL measures, while older continuers experienced declines. As shown in Table 2 and Figures 1 through 5, HRT discontinuers in this age group showed fewer days in which physical health was not good at follow-up, compared to baseline, while HRT continuers reported more "not good" physical days relative to baseline (adjusted mean changes for HRT continuers and discontinuers were 2.2 and -2.4, respectively; p = .0028). HRT continuers aged 85 or older also reported an increase in days that mental health was not good, while discontinuers experienced a slight decline (2.4 vs. -0.4, p = .0287). For the composite healthy days measure, HRT continuers experienced a mean decline of 2.8 days healthy days, while discontinuers exhibited a mean increase of 2.3 healthy days (p = .0032). In this oldest age group, HRT continuers and discontinuers did not differ significantly in the number of days at follow-up that health interfered with activities or that pain made it hard to do activities.
Discussion
The goal of this study was to assess changes in health-related quality of life associated with discontinuation of HRT in a sample of elderly women. To our knowledge, no studies to date have specifically examined the impact of HRT discontinuation on quality of life. Given the large number of women who have either discontinued or will discontinue HRT, it is important to understand the associations between HRT cessation and HRQOL. The results of this study suggest that HRT discontinuation is associated with changes in HRQOL, but the direction and magnitude of these changes vary according to age. While HRT discontinuation was associated with apparent HRQOL declines among younger women in this study, women aged 85 or older appeared to experience improvements in HRQOL following HRT cessation.
The HRT discontinuation rate of 43% observed in this study is consistent with other recent reports. A recent study of U.S. national HRT prescribing rates found that overall HRT use declined by 38% between 2002 and 2003, with higher declines observed for combination therapy than for unopposed estrogen therapy [7]. The majority of HRT users in the present study used unopposed estrogen, warranted only for women without uteri. The high observed rate of unopposed estrogen use in this sample is supported by a 2000 PACE survey which found that, while 40% of all female respondents reported a prior hysterectomy, 75% of current HRT users reported having had a hysterectomy. These results suggest that hysterectomy history may play an important role in prescribers' decisions to initiate or continue HRT in elderly women.
Depending on the specific measure examined, HRT discontinuers aged 65–74 averaged an increase of one to two days per month in which HRQOL was suboptimal. The results described here do not explain what factors may have mediated this decline. Possibilities include acute vasomotor symptoms, such as flushing, which have been shown in prior studies to be important mediators of depression in menopausal women [31]. Alternatively, there may be more complex physiological effects of HRT discontinuation that affect mood, pain perception, or other aspects of perceived health.
The BRFSS HRQOL measures employed in this study have been previously shown to predict morbidity and mortality in the PACE population [32]. Based on the significant associations of these measures with HRT discontinuation it is plausible that HRT cessation may affect risk for some adverse outcomes, although data needed to address this question are not yet available. Recent work suggests that HRT discontinuation is associated with reduced bone density and increased fracture risk [33,34]. On the other hand, given the risks for breast cancer and cardiovascular disease that appear to be attributable to HRT, discontinuation may result in reduced risk for these outcomes. Clearly, more research is needed to explore the complex relationships among age, HRT cessation, HRQOL, and specific health outcomes.
A strength of this study is that it takes advantage of a natural experiment afforded by the availability of repeated survey data, which were collected during the general time period in which large numbers of women discontinued HRT. However, an important caveat regarding our findings is that our study design compared women who had either continued or discontinued HRT as of their follow-up survey date, but did not model the time course of HRQOL change following HRT discontinuation. Therefore, although the time interval between the baseline and follow-up surveys was approximately one year for all study subjects, the interval between HRT cessation and the follow-up survey response date could range from approximately one month to approximately one year. We conducted additional analyses to examine the impact of time since HRT cessation on HRQOL among discontinuers. Those analyses found no gradient in HRQOL change associated with the time elapsed since discontinuation. However, sample size considerations limited our statistical power to detect such differences. Further research is needed to model the time course of HRT discontinuation and its impact on HRQOL, and to examine whether short-term changes in HRQOL following cessation persist over time.
Due to the greater age and burden of illness present in the PACE population, the results of this study are limited in their generalizability to other populations, including younger women. These unique features of PACE, however, provide a valuable opportunity to examine the impact of HRT use and HRT discontinuation on elderly women. Most studies of HRT use have focused on women in the immediate postmenopausal years rather than elderly women. The present study, therefore, provides new information about the impact of HRT use and cessation on elderly women. The mean age of HRT users in this study was 75.5 years, and HRT users' ages at baseline ranged from 65 to 102. Although the large number of very old HRT users may be somewhat surprising, it is consistent with unpublished research indicating that 28% of PACE HRT users in 2000 were aged 80 or older. The same study found that, among current HRT users, the mean self-reported duration of use was 17.8 years, and 25% of current users reported that they had used HRT for 28 years or longer. These results highlight the need for awareness regarding the prevalence of very long-term HRT use by elderly women for whom menopause may have occurred several decades in the past.
A number of statistical limitations that could have a bearing on the results reported here should be noted. In this observational study, we sought to reduce selection bias through the use of propensity scores and by matching continuers with discontinuers on the basis of multiple factors. Comparison of the baseline HRQOL means for continuers and discontinuers suggested that the groups were well balanced in terms of baseline HRQOL. One limitation of this study, however, is that despite the statistical procedures employed to reduce bias, it is still possible that women who discontinued HRT differed in unmeasured ways from women who continued HRT. Conversely, because we matched on multiple variables as well as the propensity score, another statistical limitation relates to possible overmatching, as discussed by Rothman and Greenland [35]. To the extent that matching may have been performed on variables related to HRT use but not to HRQOL, statistical efficiency may have been reduced. Of greater concern is the possibility that some matching variables may have been directly related to HRQOL change, which could lead to additional bias [35].
Another limitation of this study is one that is inherent in any study relying on pharmacy claims data – HRT usage was inferred from prescription claim records. It is not known to what extent women who filled HRT prescriptions actually took the medication, or if the data recorded by the pharmacy at the point of sale accurately described patients' dosing instructions from their physicians. It is also possible that some women who did not fill prescriptions for HRT had access to the medication through other sources, such as samples received from physicians. Another important consideration relates to potential age-related measurement errors, such as recall bias, which may have affected women's perceptions of their HRQOL over the last 30 days. Age differences in women's expectations regarding the anticipated effects of HRT discontinuation could also be a factor. These limitations underscore the need for further studies to explicate the determinants and outcomes of HRT discontinuation.
Despite these limitations, the pattern of results observed in this study is an important reminder that even populations defined on the basis of age – such as PACE, for which the minimum eligible age is 65 – may include a broad range of ages with associated heterogeneity. Our results suggest that the response to HRT discontinuation among women aged 85 or older may be quite different from that of women in their 60's or 70's. The etiology of the age differences seen in this study is not known, and may reflect cohort effects, including the age-related measurement issues discussed above, or alternatively, the results may reflect physiological differences related to aging. Other recent work suggests that there are important age differences in the effects of estrogen on various physiological systems. For example, Brownley and her colleagues have recently reported differential associations between HRT and blood pressure according to the time elapsed since menopause [36]. There is also growing evidence from animal studies that the effects of estrogen replacement on neurological function may be attenuated with increasing age [37]. As discussed by Savonenko and Markowska [37], such findings suggest that aging processes may modulate the mechanisms by which estrogen exerts physiological effects.
Regardless of the mechanisms that may explain the pattern of HRQOL changes reported in this study, the declines observed among younger HRT discontinuers emphasize the need for communication between clinicians and patients throughout the discontinuation process. Based on current evidence obtained from clinical trials, HRT increases risk for breast cancer, stroke, and other adverse health outcomes. On that basis, HRT discontinuation is rational and may provide important health benefits. Nevertheless, short-term changes in HRQOL may occur following HRT cessation, and strategies to optimize the discontinuation process are needed. For example, current recommendations for HRT discontinuation advocate a gradual cessation in which dosing is tapered over a three to six month period [38,39]. Ideally, future research efforts will evaluate differences in HRQOL change according to the intensity and duration of the cessation process.
Conclusions
This is the first study to present information about the impact of HRT discontinuation on HRQOL in elderly women. The results of this study suggest that there are significant age differences in response to HRT discontinuation. We believe that the present study furthers knowledge regarding older women's health in several important ways. First, it provides new information about potential declines in quality of life that women in their 60's and 70's may experience shortly after discontinuation of HRT. Awareness of these potential changes may help clinicians and patients to prepare for the discontinuation experience. These results have implications for clinical practice, suggesting that more clinical and social support may be needed for older women discontinuing HRT. These findings also highlight the importance of communication between health care providers and patients throughout the discontinuation process. Secondly, the results of this study suggest that many women aged 85 or older, including long-term users of HRT, may be able to discontinue HRT with little or no negative impact on quality of life. In fact, some women in this age group may experience improvements in quality of life upon HRT cessation, although the mechanisms involved in the observed improvements are not yet understood.
Finally, this study also has implications for health policy in that it illustrates the value of health monitoring and surveillance as a component of health program administration. Health-related quality of life is an important factor that should be considered when making clinical decisions and developing health policies. By adding an optional survey to the application process, the PACE Program has demonstrated a commitment to understanding the health status and needs of its older patient population. The use of survey measures such as those described in this study provide a useful framework for the evaluation of clinical program initiatives, and also provide a valuable resource for addressing relevant research questions. We hope that the work described here may encourage administrators of other programs to consider adopting similar health surveillance strategies.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DAH conceived of the study, carried out the analyses, and drafted the manuscript. CHG assisted in study conception and design, and helped to rewrite the manuscript. FMA contributed to the study design and conception, provided statistical advice, and helped to draft the manuscript. KEP assisted with survey data processing and reviewed the manuscript. TVB assisted in the design of the survey and reviewed the manuscript. MRG assisted with the study conception and provided pharmacological expertise throughout the study. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors would like to thank Pennsylvania's Pharmaceutical Assistance Contract for the Elderly (PACE) and First Health Services Corporation for their support in providing data for this study.
==== Refs
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| 15904516 | PMC1166565 | CC BY | 2021-01-04 16:30:35 | no | BMC Womens Health. 2005 May 16; 5:7 | utf-8 | BMC Womens Health | 2,005 | 10.1186/1472-6874-5-7 | oa_comm |
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