We have hypothesized that these PGE2 dependent effects on firing rate are due to changes in the inherent electrical properties of VMPO neurons, which are regulated by the activity of specific ionic currents.Physiological and morphological evidence suggests that activation of the ventromedial preoptic area of the hypothalamus (VMPO) is an essential component of an intravenous LPS-dependent fever. In response to the endogenous pyrogen prostaglandin E2 dependent firing rate responses were not the result of changes in resting membrane potential, action potential amplitude and duration, or local synaptic input. However, PGE2 reduced the input resistance of all VMPO neurons, while increasing the excitability of temperature insensitive neurons and decreasing the excitability of warm sensitive neurons. In addition, the majority of temperature insensitive neurons responded to PGE2 with an increase in the rate of rise of the depolarizing prepotential that precedes each action potential. This response to PGE2 was reversed for warm sensitive neurons, in which the prepotential rate of rise decreased.To characterize the electrical properties of VMPO neurons, whole-cell recordings were made in tissue slices from male Sprague-Dawley rats. Our results indicate that PGE2 is having an effect on the ionic currents that regulate firing rate by controlling how fast membrane potential rises to threshold during the prepotential phase of the action potential.We would therefore suggest that PGE This criterion is based on previous studies that indicate a functional difference for neurons which show this degree of inherent thermosensitivity .HJR, carried out the majority of cellular recordings and data analysis. JDG conceived of the study and participated in its design, coordination and completion. Both authors contributed equally to the drafting of this manuscript.


What can we learn about human autoimmune disorders that have a genetic component -- such as systemic lupus erythematosus -- from mouse models? Autoimmune diseases afflict a large segment of the population in Western countries. Many of them have been described, and rheumatoid arthritis, type I diabetes , multiple sclerosis, and systemic lupus erythematosus (SLE) are among the most common. Although tremendous progress has been made in disease management over the last decade, cures for these diseases have not yet been found. Consequently, a large research effort is sustained in this field. In addition, autoimmunity has intrigued basic immunologists since the early realization that the ability to discriminate self from non-self was at the core of the immune system's ability to protect an organism from pathogens while avoiding self-destruction. A failure of this mechanism results in autoimmune reactions that often lead to clinical disease. In spite of massive research efforts, the mechanisms by which autoimmune diseases develop are not clearly understood. Genetic predisposition as well as environmental triggers plays a role, but the identity of these factors has been largely elusive. The identification of the most common genetic and environmental factors that set off autoimmunity may lead to a better understanding of the ensuing pathogenesis, and offers the best hope for improved therapies, and ultimately, cures.So far, animal models have proved the best way to probe the mechanisms of disease in general, and autoimmune diseases in particular. In the past few decades, the mouse has become the model of choice for experimental medicine, and the rat is following close behind. Starting in the early twentieth century at the Jackson Laboratory , the production of inbred strains of mice and the systematic collection and characterization of naturally occurring mutants have created the building blocks on which much of the research using animal models is now based. Inbred strains are collections of genetically identical animals obtained through selective breeding. These strains have provided homogenous experimental groups, with interindividual variability reduced to environmental (and stochastic) factors. In addition, inbred strains have an assortment of distinct phenotypes that have then been exploited as models of human diseases.Since the 1980s, techniques have been developed to manipulate the mouse genome. Specific genes now can be routinely over-expressed as transgenes, or eliminated by gene targeting, which creates a “knockout” . This apInterestingly, the percentage of mouse genes without any homolog currently detectable in the human genome (and vice versa) has been estimated to be less than 1%, a fact that strengthens the validity of mouse models of human diseases. However, one has to be careful in directly applying data obtained from animal models to human diseases. Most human autoimmune diseases show an extremely heterogeneous clinical presentation, which animal models present as simplified versions. A mouse model, as in any reductionist approach, is both inconvenient, because it provides only a partial representation of the real biological complexity underlying the human disease, and advantageous, because it is a more tractable tool with which to probe mechanistic issues. In addition, a number of differences exist between the human and rodent immune systems . Since iNonetheless, animal models are at the core of autoimmune research, and a large body of literature reflects the many advances brought by these models in terms of deciphering disease mechanisms. The relative lack of progress in certain human autoimmune diseases for which an animal model does not exist, such as neuropsychiatric lupus, corroborates the indispensable role played by animal models. Three basic types of animal models have been used in autoimmune research: spontaneous models, induced models, and genetically engineered models. The latter category has been further subdivided into transgenic and knockout strains .Spontaneous models were produced through fortuitous observations of clinical symptoms reminiscent of a given human autoimmune disease developing in a given mouse strain, or in crosses between mouse strains. This happened, for example, with the nonobese diabetic (NOD) mouse, which developed type 1 diabetes, and the hybrid between the New Zealand Black (NZB) and the New Zealand White (NZW) mouse, (NZB × NZW)F1, which developed a lupus-like disease.Unfortunately, spontaneous models are not available for all human autoimmune diseases. Therefore, scientists have created induced models, often by exposing the animals to high doses of a suspected autoantigen at the same time as stimulating the immune system. Interestingly, marked differences exist between strains in their responses to these autoimmune inductions that reflect genetic variation associated with susceptibility to autoimmune diseases. For this reason, induced models have been limited to a small number of strains. For example, experimental autoimmune encephalomyelitis has been widely used as a model of multiple sclerosis. In this model, spinal cord homogenate or a protein derivative such as myelin basic protein is injected with a mixture of potent immunostimulants, most commonly in mice from the SJL strain. Another example of an induced model is collagen-induced arthritis, which has been used to study rheumatoid arthritis. In this model, type II collagen, a joint component, is injected also with immunostimulants, most commonly into mice from the DBA/1 strain.Finally, the ability to turn specific genes on or off, in specific cell types and/or at specific times, has created a plethora of mouse models limited only by the immunologist's imagination. Genes suspected to play a role in the pathogenesis of various autoimmune diseases have been evaluated this way, and those studies have been very informative in mapping out functional pathways that are targeted in these diseases. However, these models have intrinsic problems that have become more apparent in the past few years, and require careful controls to avoid possible misinterpretation. These problems are a result of the fundamental way in which transgenic and knockout strains are produced. When a piece of DNA carrying the gene of interest is injected as a “transgene” into fertilized eggs, it integrates randomly into the genome, and in doing so, potentially modifies the expression of the gene it integrates into. Since all genetic studies have recognized that a large number of genes are involved in autoimmune disease susceptibility, the potential for a transgene to hit one of those susceptibility genes is not negligible. This potentially confounding factor is usually controlled by producing and comparing several independent transgenic lines.More problematic is the interpretation of results obtained with knockout models. A gene is “knocked out” (KO) by homologous recombination of a disrupting piece of DNA within that gene. This genetic manipulation takes place in embryonic stem (ES) cells, which once mutated, are introduced into the inner cavity of a blastocyst (very early mouse embryo), creating chimeric embryos that are put back into female mice that carry the pregnancy to term. After multiple trials (and errors), ES cell lines from the 129 strain and blastocysts from the C57BL/6 (B6) strain have shown themselves to be superior in terms of efficiency and reliability. Consequently, this strain combination is at the origin of the overwhelming majority of knockout strains. Although the chimeric mice are usually backcrossed to B6 to dilute the contribution of the 129 genome, the knockout strains are always a mixture of the two genomes . Most imThe production of autoantibodies and mild antibody-related renal pathology, highly relevant to autoimmune diseases, especially SLE, has been reported independently in four mouse models using the (129 × B6) genetic background . It is gPLoS Biology, Marina Botto and her colleagues have tested this hypothesis by taking one of their knockout models for SLE, the serum amyloid P component deficient mouse (Apcs−/−) (Apcs−/− mice on a (129 × B6) genetic background develop a lupus-like disease, even after repeated backcrosses to B6 , carrying the same 129 region on Chromosome 1, but expressing Apcs. Amazingly, no difference was found between the two strains regarding the production of autoantibodies, clearly eliminating Apcs deficiency as a mechanism for this autoimmune process. Apcs deficiency was however associated with markedly increased renal damage, suggesting that this gene may be involved in preventing pathological consequences of autoantibody production.In this issue of pcs−/−) . This gres to B6 . Serum an humans . As mentIt has been reported anecdotally that the most common outcome of a genetic knockout is a lupus-like disease. Botto and her colleagues may have identified the reason behind this somewhat surprising observation as a spurious consequence of the gene-targeting process. Gene targeting has been an invaluable tool in understanding the mechanisms of immunological diseases, and has still a very important role to play with increasingly sophisticated techniques of selective targeting. The immediate consequence of this work should be an increased scrutiny for appropriate controls, which may include congenic mice carrying the same 129 flanking region, but expressing the targeted gene .The past few years have shown that genetic susceptibility to autoimmune diseases involves a large number of genes with small individual contributions. In spite of this great complexity, advances have been made, and a small but growing number of susceptibility genes have been identified . A commoUsing mouse models has its pitfalls; many differences, both obvious and subtle, exist between mice and humans. Those differences are, however, outweighed by the power of the experimental system offered by the mouse. What the new study of Botto and colleagues reminds us is that the appropriate control is still crucial to meaningful data interpretation. Keeping that in mind, one can predict that many of the keys to human autoimmune diseases are still in the mouse room.


Tuberculosis (TB) is increasing in the world and drug-resistant (DR) disease beckons new treatments.6 IU of recombinant human IFN gamma intramuscularly, daily for one month and then three times per week up to 6 months as adjuvant to the indicated chemotherapy, according to their antibiograms and WHO guidelines. Sputum samples collection for direct smear observation and culture as well as routine clinical and thorax radiography assessments were done monthly.To evaluate the action of interferon (IFN) gamma as immunoadjuvant to chemotherapy on pulmonary DR-TB patients, a pilot, open label clinical trial was carried out in the Cuban reference ward for the management of this disease. The eight subjects existing in the country at the moment received, as in-patients, 1 × 10Sputum smears and cultures became negative for acid-fast-bacilli before three months of treatment in all patients. Lesion size was reduced at the end of 6 months treatment; the lesions disappeared in one case. Clinical improvement was also evident; body mass index increased in general. Interferon gamma was well tolerated. Few adverse events were registered, mostly mild; fever and arthralgias prevailed.These data suggest that IFN gamma is useful and well tolerated as adjunctive therapy in patients with DR-TB. Further controlled clinical trials are encouraged. Mycobacterium tuberculosis. Every year, around 8 – 10 million new cases occur . LacTherefore a pilot clinical study was done with the aim to evaluate IFN gamma effect on drug resistant pulmonary TB patients regarding their clinical, bacteriological and radiological evolutions. The results show that the use of this protein in eight DR-TB patients (four of them MDR) as adjuvant to antibiotics had short and middle-term beneficial effects.An open-label, non-randomized, non-controlled, pilot trial was carried out at the "Benéfico -Jurídico" Hospital, Havana, which is the national reference unit for TB and other respiratory diseases. According to the national TB program, all patients with unfavorable response to treatment are remitted to this center.The study population was constituted by eight Cuban patients, both sexes, more than eighteen years old, with diagnosis of TB without a favorable response to the usual therapy, who gave their written, informed consent to participate. The diagnosis comprised clinical findings such as cough and expectoration, pulmonary lesions at thorax radiography, and positive DR-TB sputum-smear and culture. To confirm drug resistance the Canetti's multiple proportions method  was usedThe previous drug therapy received by the patients included 4 drugs  daily during 2 months, then isoniazid and rifampin twice per week for 2 additional months. Since their sputum tests had not become negative at this point they were returned to the 4 drugs regime plus ethambutol daily for 3 months, and finally isoniazid, rifampin, and ethambutol three times per week for 5 months.The trial was done in compliance with the Helsinki Declaration. The protocol was approved by the hospital's Ethics Committee and by the Cuban Regulatory Authority. Data from nineteen historical control cases were obtained from the hospital's archives.®, Heber Biotec, Havana), intramuscularly, daily during 4 weeks and then 3 times per week for the next 20 weeks. Participants stayed as in-patients during the study period. They received anti-TB drugs (WHO schemes) [Patients received 1 000 000 IU of human recombinant IFN gamma (Heberon Gamma Rschemes) , accordiEvaluations were carried out at entry and monthly during IFN gamma treatment. A complete physical examination was done. Sputum samples were taken for acid-fast-bacilli smear and culture, as well as blood samples for hematological counts, globular sedimentation rate, alanine aminotransferase, and creatinin determinations. Thorax radiographies were also recorded. Afterwards, patients were followed up with half-yearly evaluations during one year.Treatment efficacy evaluation included clinical, bacteriological and radiological outcomes. Complete response was defined as total disappearance of all signs and symptoms, negative sputum acid-fast-bacilli smear and culture, and pulmonary lesions improvement at X-ray. Partial response included signs and symptoms decrease, negative sputum smear and culture and stable X-ray lesions. No response consisted in signs and symptoms persistence, positive bacteriological examinations, and lesions stabilization or progression. Safety and tolerability of the IFN gamma treatment were monitored by means of a rigorous control of the adverse events that could be presented.2. Their main symptoms were cough, expectorations, dyspnea, stertors, distal cyanosis, and finger clubbing. Bacteriological tests codification was mostly high and all patients showed active lesions at thorax radiography. Most of them had accelerated globular sedimentation rates (GSR). Anemia or other hematological alterations were not recorded.Eight patients were enrolled in the study. Those were all the pulmonary DR-TB cases in the country during the inclusion period, from December 1999 to February 2002. They had not responded to the usual Directly Observed Treatment Short-course (DOTS) chemotherapy regime. Strain resistance was acquired in all cases. This was well determined since in Cuba all detected cases are screened for resistance on their first isolate. The patients did not have any extra-pulmonary manifestation of the disease. Their demographic and baseline characteristics are shown in Table A rapid favorable evolution was obtained after treatment with IFN gamma Table . ClinicaThe treatment with IFN gamma was safe and well tolerated. Four patients presented at least one adverse event. These events were arthralgias, fever, headache and asthenia. All adverse events were mild, except for one moderate fever, which was efficiently controlled with acetaminophen. Significant differences were not detected in other clinical laboratory tests.After completion or the IFN gamma 6-months treatment the patients continued with the corresponding chemotherapy schedule. Seven of the eight patients remained bacteriologically, clinically and radiologically negative at least twelve months after the treatment with IFN gamma concluded. However, patient number five relapsed six months after the end of IFN therapy. He developed additional resistance to rifampin and ethionamide and a chronic obstructive respiratory disease that contributed negatively to his evolution.Table In spite of the reduced size of the population studied, the results suggest the efficacy of IFN gamma on DR-TB, when used as adjuvant to chemotherapy. All eight patients were considered as complete responders at the end of IFN treatment with disappearance of the disease signs and symptoms, sputum tests conversion, and pulmonary lesions improvement. Bacteriological and radiological improvement correlated with the clinical evolution; BMI increased and manifestations as cough and expectoration did not recur after IFN gamma treatment. Clinical practice demonstrates that these results are very difficult to obtain in such a short period of time with the chemotherapy alone.Any conclusion from this study is also limited by the fact that it was not a controlled trial. This was not possible due to the low incidence of TB   and DR-TThe fact that four patients received rifampin, according to the strain sensitivity, which is one of the election drugs for the treatment of TB with excellent results in patients treated with DOTS regime, does not diminish the consideration regarding the possible benefit exerted by IFN gamma since these same patients had already not responded to this antibiotic as part of the DOTS. Moreover, one patient recurred after he had finished IFN gamma treatment for 6 months, despite still being under chemotherapy regime. This suggests that in his case a new IFN gamma cycle combined to the antibiotics would have been necessary, but this was not previewed in the protocol. However, only a controlled trial can definitely clarify the role of IFN gamma and second line antibiotics in this kind of patient's improvement.The radiological results demonstrated IFN gamma antifibrotic properties as well. All patients had a reduction in the pulmonary lesions size, while one showed a complete resolution. This effect cannot be attributable to the antibiotics, since it is well known that DR-TB patients only develop radiological improvement long time after sputum smears and culture become negative. In many cases extensive fibrotic lesions never improve, and stay stable for life. This antifibrotic action agrees with that obtained with IFN gamma in idiopathic lung fibrosis  and suggTherapy was well tolerated. It was not necessary to suspend the combined treatment due to adverse events; mostly mild. Adverse events such as arthralgias, fever and headache coincide with those reported for interferons .M. tuberculosis [Mycobacteria. It stimulates macrophages to produce tumor necrosis factor alpha (TNF α), oxygen free radicals and nitric oxide, increase surface display of MHC antigens and Fc receptors, decrease lysosomal pH, and increase the intracellular concentration of some antibiotics [Immunity to TB depends on the development of CD4+ cells- and macrophages-mediated Th1 response. The role of IFN gamma as the main macrophage-activator Th1 cytokine has been clearly established in animal models infected with rculosis ,26,27. Iibiotics -14,28.Regarding its antifibrotic effect, IFN gamma inhibits lung fibroblast proliferation and chemotaxis in a dose dependent manner, and reduces collagen synthesis ,19,29. FOn the other hand, mutations in the IFN gamma gene , or in tMycobacteria [IFN gamma therapy has been shown effective for cerebral tuberculosis caused by a multidrug-resistant strain . The aerbacteria ,39-42.These results can suggest a beneficial effect of IFN gamma when it is used as adjuvant in the treatment of tuberculosis patients that have resistance to standard chemotherapy, and encourage carrying out more extensive, controlled studies. Combination with second-line drugs can reduce the time of treatment, diminishing toxicities and possible relapses; in many cases could reduce the application of recessional surgery. Further controlled clinical trials are needed to confirm these results.Authors IGG, CMVS, and PALS are employees of the Center for Biological Research, which is part of the Center for Genetic Engineering and Biotechnology, Havana network, where IFN gamma is produced. The rest of the authors have no competing interests at all.RSM conceived the study and carried out the bacteriological determinations. IGG participated in the study design and coordinating, and wrote the manuscript draft. NFO, MVQ, MTM, DC, and DMM took care of patient recruitment, management, and follow-up. CMVS participated in the study design and result analysis. PALS took part in the design, results analysis and manuscript writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Back to Sleep campaign in the United States, which encouraged parents to use a supine sleeping position for infants.Parental smoking and prone sleep positioning are recognized causal features of Sudden Infant Death. This study quantifies the relationship between prenatal smoking and infant death over the time period of the This retrospective cohort study utilized the Colorado Birth Registry. All singleton, normal birth weight infants born from 1989 to 1998 were identified and linked to the Colorado Infant Death registry. Multivariable logistic regression was used to analyze the relationship between outcomes of interest and prenatal maternal cigarette use. Potential confounders analyzed included infant gender, gestational age, and birth year as well as maternal marital status, ethnicity, pregnancy interval, age, education, and alcohol use.We analyzed 488,918 birth records after excluding 5835 records with missing smoking status. Smokers were more likely to be single, non-Hispanic, less educated, and to report alcohol use while pregnant (p < 0.001). The study included 598 SIDS cases of which 172 occurred in smoke-exposed infants. Smoke exposed infants were 1.9 times (95% CI 1.6 to 2.3) more likely to die of SIDS. The attributed risk associating smoking and SIDS increased during the study period from approximately 50% to 80%. During the entire study period 59% (101/172) of SIDS deaths in smoke-exposed infants were attributed to maternal smoking.Back to Sleep campaign. Mothers should be informed of the 2-fold increased rate of SIDS associated with maternal cigarette consumption.Due to a decreased overall rate of SIDS likely due to changing infant sleep position, the attributed risk associating maternal smoking and SIDS has increased following the  Back to Sleep campaign, has had a major role in reducing SIDS rates. This public health campaign encourages parents to place infants in a supine rather than prone sleeping position. Early studies investigating the effects of supine sleeping revealed significantly reduced SIDS rates [Previous literature has shown a relationship between maternal smoking and the Sudden Infant Death Syndrome (SIDS). Published studies vary in size and methodology but consistently demonstrate a two-fold increased odds of SIDS with both prenatal and postnatal maternal smoking -10, inclDS rates , but smoBack to Sleep campaign in the United States for the ten-year period from 1989 to 1998. We hypothesized that maternal prenatal smoking confers a clinically significant risk of SIDS and that an increased attributed risk of SIDS associated with smoking could be identified in the wake of the supine sleeping campaign.The aim of this study was to confirm the relationship between reported prenatal maternal smoking and SIDS and to examine the effect of sleeping position changes on the attributed risk of SIDS and smoking. The analysis spans the rollout of the Since 1969, the State of Colorado has collected data on multiple items through a birth registry. This registry includes extensive demographic data such as maternal report of the number of cigarettes smoked per day during pregnancy. We used the Colorado Infant Death Registry over a 10-year period (1989–1998) to identify causes of infant death.We conducted a retrospective cohort study utilizing Cochran Mantel-Haenszel univariate analysis of risk factors and multiple logistic regression on the outcomes of infant death, SIDS, and respiratory deaths. We excluded non-singleton births (14978 records), infants born at less than 2500 grams (36779 records), and birth records where a mother's smoking status was unknown (5835 records). Additional records with missing data were coded so as to place the record in the presumed lower risk group: 42 records with unclear marital status were coded as "married," 67 records missing a maternal age were coded as age "18–34 years," 865 records with unclear gestational age were coded as "term" infants, and 4 records missing a gender assignment were coded as "female" in the analysis. Missing data for education (9659 records) and ethnicity (582 records) were coded as such and included in the analysis directly. We linked the birth and death registries using a unique birth number present in both registries. Utilizing multiple logistic regression, we modeled the exposure of interest, maternal smoking, as both a dichotomous and a continuous variable. Other variables analyzed included infant gender and gestational age , as well as maternal marital status, ethnicity, time between pregnancies (less than 12 months or 12 months and greater), maternal age , education, and self-reported use of alcohol in pregnancy. In the analyses for SIDS and respiratory causes of death, deaths from other causes were excluded from the analysis.Power to detect a 20% difference in a baseline disease occurrence of 3 per 1000 was calculated at 99% for a population of 500,000. SAS version 8.0 (SAS Institute) on a PC was utilized for statistical analyses. We included the interaction between ethnicity and cigarette use in the modeling and requested the Hosmer-Lemeshow statistic from the model.We compared cases of SIDS (ICD 9 codes 798.0 to 798.9) between cohorts of infants born to mothers who reported prenatal smoking versus mothers who reported no prenatal smoking. Secondary outcomes included total infant mortality and respiratory deaths (ICD 9 codes 033.0 to 033.9 and 460 to 490). We used unadjusted annual rates to calculate the attributable risk of SIDS associated with maternal cigarette consumption.Over the 10-year study period, 1573 infants died, 598 SIDS cases occurred (1.2/1000 live born infants), and 34 infants died due to a respiratory etiology. Table We calculated adjusted odds ratios by analyzing smoking as a dichotomous exposure (mother smoked or did not smoke) and as a continuous variable (number of reported cigarettes per day during the pregnancy). Dichotomous outcomes of reported smoking during pregnancy yielded adjusted odds ratios of 1.9 (95% CI: 1.6 to 2.3) for death due to SIDS, 1.5 (95% CI: 1.3 to 1.7) for infant deaths from all causes, and 3.0 (95% CI: 1.4 to 6.3) for deaths due to respiratory etiologies . Table per cigarette are 1.042 . In this model, consumption of 10 cigarettes per day is associated with a 51% increased odds of SIDS (95% risk limit 35% to 70%). The Hosmer-Lemeshow test statistic was non-significant (p > 0.1948) suggesting adequate model fit.Analyzing cigarette consumption as a continuous variable shows the associated odds 12. In this analysis, 101 infant deaths due to SIDS bear an association with maternal smoking among the exposed cohort of 172 infants. Figure Back to Sleep campaign. In the final year of the analysis, 80% of the SIDS deaths in the smoke-exposed infant cohort are attributed to maternal smoking.Table Back to Sleep public health campaign in the United States, we noted an increasing attributed risk of SIDS with smoking. As a major etiology of SIDS, prone sleeping position, decreased over the study period due to the national educational campaign, the remaining SIDS deaths may now be due to the more isolated effects of tobacco exposure. Due to a decreased overall rate of SIDS, almost 80% of SIDS among smoke-exposed infants now bear a relationship to cigarette smoking.Smoking is deleterious and has negative effects on children born to mothers who smoke -16. We cBack to Sleep campaign in the United States, suggest that up to 30% of SIDS deaths may be attributed to maternal smoking [Taylor and Sanderson, using data prior to the  smoking . We noteOur study cannot establish a causal role for maternal smoking in SIDS. Others have argued for a causal relationship based on prospective data, demonstrable dose-response relationships, and analysis using multiple potential confounders . The cauThe published literature reports a 20% to 30% smoking rate among pregnant women . ApproxiBack to Sleep campaign in the United States and our dataset did not assess whether parents positioned their infants prone or supine. We therefore could not control for this ecological association with SIDS. The very nature of retrospective data creates limitations in that we cannot carefully control the setting in which the data are collected, or the individual collecting the data. Missing data records are a third limitation, although this appears to have not been a major issue with this dataset.This study has several important limitations related to design and the nature of the data set used. Given the rich nature of the Colorado Birth Registry, we controlled for many potential confounding variables, but others may exist. This analysis likely represents an under-estimate of the actual associated risk with prenatal smoking, given that it relies on maternal report of a behavior widely known to be harmful. We suspect that self-report of maternal smoking underestimates the true rate of maternal smoking. Fetal and infant exposure to tobacco smoke occurs in manner ways, including prenatal maternal smoking, prenatal maternal second-hand smoke exposure, and postnatal smoke exposure by one or more care providers in the home. In this study we cannot distinguish among these exposure pathways, which may result in an erroneous under or over estimate of risk. The time period spanned by this study includes the rollout of the Back to Sleep campaign in the United States we examined the increasing attributed risk associating smoking with SIDS. As SIDS rates declined, the attributed risk of SIDS with smoking has increased. In the final year of analysis, we demonstrated a link between 80% of the SIDS deaths and maternal smoking. A major, preventable exposure remains for infants in the United States and providers should redouble counseling efforts toward reducing this exposure.As prone infant sleeping has decreased, other causes of SIDS such as maternal smoking assume increased importance in the effort to protect infants from SIDS. Spanning the rollout of the The author(s) declare that they have no competing interests.MA participated in the study conception, background research, study design, statistical analysis, and drafting of the manuscript. DJ participated in the study conception, study design, and proofreading of the manuscript. HB participated in the study conception, study design, statistical analysis, and proofreading of the manuscript.The pre-publication history for this paper can be accessed here:


Drug industry insiders share their tips on using celebrities to market drugs and diseases Drug industry insiders share their tips on using celebrities to expand markets As the Hispanic world well knows, the word in Spanish for advertising is ‘propaganda’, its meaning derived literally from the propagation of the faith, the antithesis of science's Enlightenment ideals. The old word somehow seems perfect for describing the new world of drug promotion and its growing use of the famous face. Like the catholic cardinals of the 17th century, many of the feted celebrities of the 21st are now engaged in spreading the word. Now, as then, the religion promises miraculous breakthroughs, wonder cures, and sometimes even eternal life. The difference is that this time around, the stars are earning fat fees from the marketing departments of giant pharmaceutical companies. And if the latest revelations from industry insiders are anything to go by, their hefty investments in celebrity selling are well worth it.Oprah and The Today Show. According to celebrity brokers, the star's remuneration package, though always confidential, can range from $20,000 to $2 million.The epicentre of this phenomenon is of course the United States, where companies routinely hire celebrities to attract attention to the latest drugs and the diseases that go with them. Pfizer famously paid presidential hopeful Bob Dole to promote awareness of erectile dysfunction as sildenafil (Viagra) was hitting the market. Wyeth hired supermodel Lauren Hutton to hawk hormone replacement therapy and menopause. GSK contracted football star Ricky Williams to sell social anxiety disorder, helping make paroxetine (Paxil)—briefly—the world's top-selling antidepressant. Even the dead are raising awareness, with the estate of Errol Flynn now enlisted to help promote cardiovascular disease as a household name . The cel‘A partnership between a celebrity and a brand has an intangible sort of magic’, writes a senior marketing executive at Amgen, in an extremely candid piece published recently in an industry trade magazine . Amgen iThe Rosie Show can be better forums for celebrities than straight advertisements, which are governed by regulations. ‘The great advantage over advertising is that the airtime is practically free, and there is no fair balance to worry about’ she writes [Benshoshan then reveals why on-air talk-show appearances on ‘top-tier media venues’ like e writes . The dowe writes .Another recent report from within the industry draws on public opinion survey data to guide drug company marketers on the selection and ‘effective use’ of celebrity spokespersons . The surThe issue of credibility is important, the NexCura Inc. researchers point out, because ‘the credibility rating is used as a surrogate for “buying” behavior’—an intermediate measure of whether the star can persuade people to request the target drug from their doctor. The survey found that Bob Dole was still the most recognisable celebrity marketer with the United States public, but that the skater Dorothy Hamill—currently promoting Merck's arthritis medication rofecoxib (Vioxx)—took the lead in the credibility stakes. Significantly though, almost three-quarters of those surveyed were correctly able to identify Bob Dole with Pfizer's Viagra, despite the fact that the advertisements in which he appeared were ‘unbranded’ ads for erectile dysfunction. The researchers concluded by recommending that drug companies choose a celebrity with personal experience of the target condition; choose someone trustworthy—perhaps a newsreader or sports figure; and choose someone who will promote a single cause or brand rather than multiple ones.Ironically, the NexCura survey also found two-thirds of medical consumers agreed with the proposition that celebrities were ‘just doing it for the money and can't be trusted’.Frasier star Kelsey Grammer and his wife were promoting irritable bowel syndrome on top-rating TV shows, viewers thought the pair were speaking on behalf of an independent foundation. In fact the couple's fee had flowed from GSK, which was at that time preparing the market for alosetron (Lotronex), a controversial new drug that carried modest benefits and severe side effects, including possible death [The first problem here is that the public is often not even informed whether a celebrity is receiving money from a drug company. In the case of TV star Rob Lowe, there was no mandated requirement for him to disclose his link with Amgen when appearing on media shows watched by millions. According to one industry insider familiar with the case, who did not want to be named, ‘it depended if he remembered to say it, and whether he was asked’. The media's failure to disclose relevant conflicts of interest when covering healthcare is well established . When Frle death .Equally as serious is the lack of any formal requirement for stars or media outlets to spell out drug side effects along with benefits when celebrities are pushing products or conditions. Lauren Hutton can be quoted, in magazine articles read by millions of readers, as saying, ‘My No. 1 secret is estrogen’ without any need for her, or the magazine, to list the dangers of the hormone replacement therapy made by her sponsor . But perWhile Prince Charles's companion Camilla Parker Bowles takes no money from drug companies, she did choose to make an important public statement about the bone condition osteoporosis at an international conference funded by Lilly, a company promoting a medication for the condition . CamillaWith pharmaceutical marketing, it is clear that nothing short of a Vatican II-style reform is required, though there are already encouraging signs of change. Scientific journals are slowly disentangling themselves from unhealthy industry influence over what they publish, and public access to clinical trial data is daily a closer reality . However


The shift handover forms an important part of the communication process that takes place twice within the nurses' working day in the gynaecological ward. This paper addresses the topic of implementing a new system of bedside handover, which puts patients central to the whole process of managing care and also addresses some of the shortcomings of the traditional handover system.A force field analysis in terms of the driving forces had shown that there was dissatisfaction with the traditional method of handover which had led to an increase in the number of critical incidents and complaints from patients, relatives and doctors. The restraining forces identified were a fear of accountability, lack of confidence and that this change would lead to more work. A 3 – step planned change model consisting of unfreezing, moving and refreezing was used to guide us through the change process. Resistance to change was managed by creating a climate of open communication where stakeholders were allowed to voice opinions, share concerns, insights, and ideas thereby actively participating in decision making.An evaluation had shown that this process was successfully implemented to the satisfaction of patients, and staff in general.This successful change should encourage other nurses to become more proactive in identifying areas for change management in order to improve our health care system. The shift handover in this ward was conducted as "a ritual inheritance," [This study was undertaken in a 28 – bedded gynaecological ward catering for female patients aged 16 and above. There were 21 nurses based in this ward of whom 14 were qualified and the remaining were health care assistants, with experience ranging from 1 The verbal handover was derived from written information on the office white board which included the patient's name, bed number, medical diagnosis and the treating doctor. This was in line with the findings of Sherlock  who arguThe root cause of the problem identified was the model of handover used to communicate clinical information. As a benchmark, the findings from evidence on the bedside handover were used to give meaning and strength to the proposed change. Bedside handovers offer an immediate solution to the many problems that are associated with the traditional handover ,15,16. IUnfreezing, Moving and Refreezing provided us with useful frameworks for our change management [An adaptation of Spradley's 8-step model and Lewin's 3-step model of nagement ,14.Unfreezing is about encouraging people think about the current situation and helping them recognise the need for change . Change The first move therefore was to create awareness by communicating the proposed change to all those who were going to be affected by the new practice: the nurses, patients and the ward manager so that they all had a shared vision of an improved handover system. A goal-seeking behavior with a clear logical sequence of action, were demonstrated throughout the process as advocated by Lancaster and Lancaster ,17. ReseThe proposed change was announced in advance by using different communication channels, e.g. personal contact with individual nurses, staff information/notice board by the authors. This initiated informal discussion among nurses of the ward by creating a cognitive dissonance which led to a quest for more information about the new handover. This consultation phase allowed the nurses to discuss various clinical scenarios and analyse the constraints and benefits of the new proposal in the local context. They were also involved in group work to identify and make proposals on how to deal with some of the problems that we may encounter in our local context e.g. handover coinciding with ward rounds or emergency situations and patients too distressed to talk. Case studies and research articles on this topic were used for discussion and to further reinforce the beliefs of staff of the ward that the current practice had shortcomings and could be improved. The status quo was therefore unsettled and this enabled us to rule out the first resistance through a normative re-educative strategy. A group of senior nurses who had experience in this particular area agreed take turn to act as mentors in order to facilitate this process and offer support to their junior colleagues in the first week until they become confident to carry out the process without supervision.The extensive literature search also provided us with options for alternatives to bedside handover. These were thoroughly debated before reaching a decision. The options considered were the following:1) Tape recorded handover2) Computer generated handover using information technology3) Bedside handover, based on individualized care planThe 'SMART' criteria were used to evaluate the feasibility of the alternatives to bedside of handover. The tape recorded handover would require a tape recorder being taken around to each of the patients and the interaction recorded. An informal discussion with the patients revealed that this method was distractive and the majority of them did not feel comfortable about their conversation being recorded. With regards to the computer generated handover using information technology, the patients felt this system will not enable them to engage fully in the process. It was also felt that since the first two options required extra financial, technical resources for implementation, these would not be feasible in the first instance whereas the bedside handover gained unanimous support from both patients and staff. This was also more realistic in term of its applicability in our practice area. It was specific, measurable in terms of its performance and achievable within existing resources and a defined time frame. Its foundation rested on evidence base practice, which showed theoretical soundness.There was a shared vision about the worth of the proposed change by the team and consequently bedside handover was logically considered as the best option for change. The vision formulated was that in three months' time, bedside handover would become the normal shift handover process of the ward. The mission statement agreed was "all handovers would be carried out at the patients' bedside between the incoming and outgoing nursing staff with the patients' involvement."A force field analysis, as shown in table Careful planning is essential if trauma is to be minimized . It was th of February 2003 up to 8th May 2003. One-month time was judged sufficient to unfreeze the situation and the remainder to implement and evaluate the change.The time frame earmarked to implement the change was three months starting from the 8Choosing a strategy for the change process depended upon various factors and good interpersonal relationship was a critical factor. It has been proposed that strong leadership and excellent communication skills were essential if an atmosphere of trust was to be engendered ,8. With Confidential issues related to matters that the patients brought up during the admission procedure and during their stay, certain issues that were brought up during ward rounds and from the patients own requests.In cases of occasional lateness in resuming work, the handover would proceed with the other patients in first the instance and if the staff was still late, then some other colleague would step in her place. Reassurance was given with respect to 'no substantial overlapping' of shift in that it would not have major bearing on the handover process by explaining that shorter handover can reduce the likelihood of information overload and result in concise and pertinent information being exchanged as per care plans. There was a general agreement that fifteen minutes as officially allocated for handover would be sufficient for this purpose. Assurance was also given that confidentiality of patients' clinical information would be taken into consideration in drafting a protocol for bedside handover, as shown in table Several meetings were organized with different groups of nurses to explain and clarify any shortcomings and to reach a consensus. This approach was recommended by Driscol , as it eth of March 2003. For the first week, six senior staff who had experience in this area volunteered and took turn to continue to be present in as many handovers as observers and participants, to monitor and reinforce the established protocol step by step.Following a pilot handover session involving senior staff in a participant and an observer capacity over 2 morning and 2 evening handover sessions, which did not require any major changes, implementation of the bedside handover was started on 8They also provided clarification and support to staff in cases of difficulty, and helped evaluate the extent of change that had taken place in an effective manner. The nurses present during the handover had no difficulty in adapting to this new situation, using a care plan incorporating a more psychosocial and patient-centered approach to bedside handover with the patients' participation.The evaluation of the implementation of bedside handover was carried out in two distinct phases. A protocol, as shown in table th criteria at 90%. In one of the sessions, the nurse had left the patient whilst the bedside handover was in progress to attend to another patient without explaining the reason for this short absence to him which accounts for the 90%.Analysis of results of the observational data on 10 handovers, Table Analysis of the results of semi-structured interviews with 40 patients, Table The result was evaluated at a full staff meeting and the ward manager and colleagues recognized the change. Despite unlearning of the old practice had taken place two nurses still displayed some difficulties with the new handover as they were always eager to report everything themselves rather than allowing the patients to have a say. After a reassessment of the situation, accurate feedback was given to them. With the group support, they became used to the new system by observing their colleagues in action during the handover and doing it in turn. After a couple of sessions they became fully conversant with the new system. By this time, this project was ready for the refreezing stage.One of the major difficulties encountered was to rally everybody behind this project. The normative re-education, in line Bernhard and Walsh , was useManaging change in a hospital set up is a daunting task as it involves a change in the attitude and behavior of staff in a complex environment in order to gain their collaboration. The concept of no pain no gain was very evident throughout the process. Lewin's 3 – stage model was useful in implementing the change in a planned and structured way. Resistance was overcome by creating a climate which encouraged open communication. The support of the ward manager and key stakeholders were significant. Evaluation has shown that the new system of handover is working well but monitoring will be ongoing with evaluation of a larger sample of patients. This change has been an enriching experience for the staff, and has generated enthusiasm and given them confidence to question some of the practices on the ward. This new approach to handover can therefore be implemented in other areas of practice and evaluated to ensure that they are meeting patients' satisfaction. Further studies can be undertaken to explore how the multidisciplinary team could further consolidate this process.The author(s) declare that they have no competing interests.HKK taught this module and supervised this change management project. ZBJ implemented this change in the gynaecological ward as part of her assessment in the "Management Development" module. Both authors wrote this manuscript, read and approved the final version.The pre-publication history for this paper can be accessed here:


Perineodynia , anal and urinary incontinence are the main symptoms of the pudendal canal syndrome (PCS) or entrapment of the pudendal nerve. The first aim of this study was to evaluate the effect of bilateral pudendal nerve decompression (PND) on the symptoms of the PCS, on three clinical signs  and on two neurophysiological tests: electromyography (EMG) and pudendal nerve terminal motor latencies (PNTML). The second aim was to study the clinical value of the aforementioned clinical signs in the diagnosis of PCS.In this retrospective analysis, the studied sample comprised 74 female patients who underwent a bilateral PND between 1995 and 2002. To accomplish the first aim, the patients sample was compared before and at least one year after surgery by means of descriptive statistics and hypothesis testing. The second aim was achieved by means of a statistical comparison between the patient's group before the operation and a control group of 82 women without any of the following signs: prolapse, anal incontinence, perineodynia, dyschesia and history of pelvi-perineal surgery.When bilateral PND was the only procedure done to treat the symptoms, the cure rates of perineodynia, anal incontinence and urinary incontinence were 8/14, 4/5 and 3/5, respectively. The frequency of the three clinical signs was significantly reduced. There was a significant reduction of anal and perineal PNTML and a significant increase of anal richness on EMG. The Odd Ratio of the three clinical signs in the diagnosis of PCS was 16,97 .This study suggests that bilateral PND can treat perineodynia, anal and urinary incontinence. The three clinical signs of PCS seem to be efficient to suspect this diagnosis. There is a need for further studies to confirm these preliminary results. The objective of perineology is to treat each defect of the perineum with the right procedure -3. PudenBefore going into details of this procedure, it is necessary to remember the anatomy of the pudendal nerve. This anatomy is still controversial.While summarizing the data of the literature and the results of our dissections, the likeliest anatomy of the pudendal nerve presents itself as follows. The pudendal nerve is a mixed nerve carrying motor and sensory fibers. Its fibers are derived from the sacral roots S2, S3 and S4 . Once thThe pudendal canal syndrome (PCS) and its surgical treatment have been described by Shafik in 1991 . This syThe cause of the PCS is not always clear but it is often possible to find a compression  or a stretching  of the pudendal nerve in the Alcock's canal -19 in thThe clinical signs and investigation results proposed by Shafik  to confiThe trans-gluteal approach proposed by Robert to treat pudendal neuralgia aimed to open also the "clamp" between the sacro-spinal and the sacro-tuberous ligament by cutting one or two of them .Since Shafik's study in 1991, some questions about the effect of PND on the PCS remained open. No peer reviewed publications confirming the efficacy of this surgery on anal incontinence or on urinary incontinence could be found. Even the existence of a genuine PCS has never been validated.The aim of this study was to answer the following questions:Is there any effect of the bilateral PND according to Shafik on:- three main symptoms of PCS ?           - perineodynia             - anal incontinence           - urinary incontinence- three clinical signs of PCS ?           - abnormal sensibility           - painful Alcock's canal           - painful "skin rolling test" - two neurophysiological tests ?           - electromyography (EMG) of the anal sphincter and of the bulbocavernosus muscles.           - pudendal nerve terminal motor latencies (PNTML) of the anal and perineal branches.What is the clinical value of the aforementioned three clinical signs in the diagnosis of PCS ?A retrospective analysis to study the effects of a bilateral PND. The studied sample comprised 74 female patients who underwent a bilateral PND between 1995 and 2002 done by the same surgeon. The average age was 56.1 years (range: 28 – 77). All these patients underwent a complete history and clinical examination following the three perineal axis  according to the concept of perineology .The frequency of the 3 main symptoms of the PCS  in the 74 patients before surgery is presented in Figure Associated surgical procedures were performed at the same time as the PND to treat all the defects revealed by the clinical examination, and are presented in Table The following variables were used:For perineodynia, four situations were encountered: no pain, proctalgia, unilateral pain, bilateral pain. The effect of surgery was estimated by the patient using one of the following proposals: cured, improved, unchanged or worsened.For anal incontinence, a four levels ordinal scale was used: no incontinence, gas incontinence, liquid incontinence, solid incontinence. "Cured" was defined as "no incontinence". The patient was considered "improved" if there was a change of at least one level in the scale going from "solid" to "gas" incontinence. The patient was defined as "worsened" if there was a change of at least one level in the opposite direction.For urinary incontinence, a four levels ordinal scale was used: no incontinence, mild incontinence, moderate incontinence and severe incontinence. The two types of urinary incontinence, stress and urge incontinence, were evaluated separately even if both were present in the same patient (mixed incontinence). "Cured" was defined as "no incontinence". The patient was considered "improved" if there was a change of at least one level in the scale going from "severe" to "mild" incontinence. If the change observed was in the opposite direction the patient was considered "worsened".Sensibility was tested with a needle comparing the left and the right sides of the vulva and of the skin 2 cm lateral to the anus. The interpretations of the results were done using a four levels ordinal scale: 0 = total anaesthesia, 1 = reduced sensibility, 2 = normal sensibility, 3 = hypersensibility. 0, 1 and 3 were considered as "abnormal sensibility".The pain induced by the palpation of the pudendal canal by rectal examination was evaluated using a seven levels ordinal scale : 0 = no pain, 1 = mild pain, 2 = mild pain with Tinel sign (irradiation of the pain), 3 = moderate pain, 4 = moderate pain with Tinel sign, 5 = severe pain, 6 = severe pain with Tinel sign. The Alcock's canal was considered "painful" if the pain was 4 or more.Beginning from 5 cm behind the level of anus the skin was pinched and then rolled to the front until the skin fold was at the level of the clitoris. The skin rolling test was considered "painful" if it induced a severe pain at least at one level Figure .Concentric needle EMG was done at rest and during voluntary contraction on both sides of the external anal sphincter and on each bulbocavernosus muscle. The richness of the EMG was grossly evaluated using a six levels scale: 1 = simple, 2 = poor, 3 = intermediate poor, 4 = intermediate, 5 = intermediate rich and 6 = rich.Anal and perineal PNTML were measured before and after the operation using the St Marks electrode to stimulate the pudendal nerve by the rectal route just under the ischial spine. For anal PNTML the electrical potentials induced in the striated anal sphincter were collected using the ring of this electrode. For the perineal PNTML the method described by Kiff  and SnooAt least one of the 3 following symptoms resistant to conservative treatments :a. Anal incontinenceb. Perineodyniac. Urinary incontinenceAssociated with at least two of the five following criteria:a. increased anal or perineal PNTMLb. pathological EMG of the anal sphincter or bulbocavernosus muscles .c. painful Alcock's canal on rectal examination (at least on one side)d. abnormal perineal sensibility (at least at one level)e. painful "skin rolling test" (at least on one side).Surgical procedure as described by Shafik in 1991 . The opeThe different steps of the procedure were:- Vertical incision of the skin between the anus and the ischial tuberosity.- Opening of the ischio-rectal fossa with scissors.- The inferior rectal nerve is hooked under the finger and followed to the entrance of the Alcock's canal Figures  and 4.- Opening of this canal .- Control of the haemostasis.- Self draining closing of the skin with nylon.The efficacy on the symptoms, on the clinical signs and on the neurophysiological tests was evaluated during a follow up consultation one year or more after the surgical procedure because the nerve healing can be very slow .Firstly, the efficiency of the PND on the symptoms and clinical signs was studied by means of descriptive statistics. Tests of hypothesis were done to compare the mean values of the neurophysiological tests before versus after PND.Secondly, the diagnostic value of the clinical signs was evaluated in a "case control" setting. A subject belongs to the "controls group" when PCS is considered to be absent, namely if the patient does not present any of the following symptoms, signs or risk factors for PCS: perineodynia, anal incontinence, prolapse, previous surgery in the area, dyschesia. The clinical signs are not used to decide if a subject belongs to the controls or cases group. The statistical comparison was done between the patient's group before ("cases group") and after the operation, and the "controls group" of 82 women .Statistical analysis of differences was performed using chi-square testing for categorical variables and t-tests for continuous variables.The effect of PND on the symptoms of PCS is presented in Table In order to treat completely the patient, PND was frequently associated with other procedures which might have an effect on the symptom studied. Therefore, the results for each symptom were presented in two columns: the first corresponds to the entire sample  and the second to the small group of patients in which the symptom was treated by PND only ("without").On the 26 patients with pain before the operation, 18 were reviewed 12 months or more after the operation. The pain had disappeared in 11 and was reduced or had another origin  in 3. The cure rate with a mean follow-up of 22,2 months was 61,1 % .As none of the surgical associated procedures used in this study were known to improve or cure perineodynia, the only procedure removed was levatorplasty. Theoretically this operation can reduce the stretching on the pudendal nerves by reducing the sagging of the levator plate. The results were similar in the group without levatorplasty.On the 46 patients with anal incontinence before the operation, 36 were reviewed 12 months or more after the operation. 23 of them were cured, 7 improved, 2 were worse and 4 reported no change. The cure rate with a mean follow-up of 26.4 months was 63.9 % .The results according to the severity of incontinence are presented in Table To study the real impact of PND on anal incontinence, we removed the following cases in which PND was associated with anal sphincteroplasty, levatorplasty or cure of rectocele by fascia and perineal body restoration:- 2 patients had anal sphincteroplasty together with the PND: one was cured and the other improved.- Levatorplasty according to Shafik  was used- 30 cases had a cure of rectocele by fascia and perineal body restoration  and 1 improved (liquid incontinence).Anal ultrasound was done before the operation in 13 of the 36 patients reviewed. Only 4 were normal, 3 showed a rupture of the internal and external sphincters, and 6 presented a disruption of the external sphincter alone.In the 7 patients who had a rupture of the anal sphincter  without anal sphincteroplasty and a follow up of more than 12 months (mean 18.5 months), 4 were cured and 3 were a failure.5 patients who were still incontinent after 1 year follow up (2 incontinences for flatus and 3 for liquid) became continent 2 years after the operation.Five patients presenting urinary incontinence (4 urge and 1 stress incontinence) had PND without any other procedure around the urethra. The mean follow up was 18,5 months for the 4 patients with urge incontinence. In this small group, 3 patients were cured and one was a failure. The patient with stress urinary incontinence was improved (the number of pads used per day was reduced from 9 to 4) one year after surgery.The effect of PND on the three clinical signs is described in Table Because levatorplasty can theoretically reduce the stretching of the pudendal nerve, the evaluation of the effect on the clinical signs has been done for the entire sample  and for the same group without the patients who had a levatorplasty ("without: levat").The cure rate of the 3 clinical signs was between 60 and 70 % depending of the sign and of the type of sample studied.38 patients underwent a complete EMG evaluation before and after surgery. A relevant comparison was possible in 35 patients. 3 patients were excluded because one of the two EMG explorations was insufficient for technical reasons. The average follow up was 16,9 months .Left and right values for one parameter correspond to two different nerves. Therefore, these values were considered independent .The effect of PND on EMG and PNTML is presented in Table The "Anal Richness" on EMG after surgery was significantly higher than before. The mean "Bulbocavernosus Richness" after surgery was slightly higher than before but this difference was not significant. Both anal and perineal PNTML after PND were significantly reduced compared to values before.The box-plots of the 4 studied parameters are presented in Figures We present here the results concerning the evaluation of the three clinical tests: abnormal sensibility, painful Alcock's canal and painful "skin rollling test" as diagnostic tests for PCS.The statistical analysis is based on the following contingency tables presented in Tables The proportions of observations in the "Cases Before Surgery" and "Controls" columns of the contingency table vary significantly from row to row , whereas no significant difference is observed between the proportions of observations between "Cases After Surgery" and "Controls" .Estimated sensibility, specificity, predictive values (positive and negative) and odd ratio  corresponding to each of the three clinical tests and combinations of all of them are presented in Tables The most sensible test is the "Painful Alcock's canal" and the most specific is the "Skin rolling test".Using the three signs altogether, the most sensible combination is "At least 1 positive versus All negative" and the most specific combination is the "All positive versus All negative".During one operation a heavy bleeding coming from the pudendal artery just near the pudendal nerve was very difficult to treat (selective ligature). This patient had a blood transfusion but no long term side effect. Since the operation, one patient has presented sometimes a short lasting clitoridal pain. This patient had also an increase in her anal incontinence (gas incontinence became a liquid incontinence). Three patients had wound healing problems which resolved with simple disinfection.In the literature there is no data available about the prevalence of the PCS. Therefore, it seems to be a rare event. In this study, we evaluated the prevalence of the PCS in an outpatient perineology clinic. By using three different methods during the last 24 months  the estimated prevalence should be around 20%.Before discussing the results of surgery, the first important issue is about the interest of a bilateral decompression. The benefit – risk ratio must be studied. The results of Shafik were obtained after bilateral decompression ,29,30. FThe treatment of pain starts with a holistic approach of the woman  with exclusion of other causes of pain: piriformis syndrome, coccygodynia, interstitial cystitis, endometriosis... The other neurological causes must be excluded by a complete electrophysiological study of the perineum  and imaging of the spinal cord . If the Even with the transgluteal approach where the "clamp" between the sacro-spinal and the sacro-tuberous ligament is opened by sectioning the sacro-spinal ligament, the cure rate remains around 50%. In the 4 cases of proctalgia fugax the results were better (3 cured and 1 improved). Shafik [For Robert early diagnosis appears to be the determining factor in improving results . He usedFor anal incontinence, our results were in the same range as Shafik . In a prMore interesting was the cure rate in the group of patients with a clear rupture of one or the two anal sphincters. The traumatic rupture of the anal sphincter  usually induces an immediate anal incontinence. In the patients who remained continent the power of the broken muscles remain sufficient to avoid flatus or faeces leakage. In the long term the continence is probably maintained with the help of the fibrous tissue located between the two edges of the ruptured muscle which acts like a bridge and therefore enables the sphincter to be efficient during many years. The aging process of the muscle and the pudendal neuropathy reduce the power of the muscle  with time and explain the appearance of an anal incontinence. Therefore it is logical to restore continence by improving the conduction in the pudendal nerve. This fact can also explain why the results of sphincteroplasty decrease with time especially in the non diagnosed or treated pudendal neuropathies .The fact that 5 patients were cured from their anal incontinence only 2 years after surgery emphasized the importance of a long follow up period to obtain relevant cure rates. Surprisingly, the cure rate seems to be not dependant of the degree of anal incontinence but the number of solid incontinences  was too small to validate this impression.The results of the pudendal nerve decompression seem to be equivalent to these of neuromodulation  and the For urinary incontinence the number of cases is too small to give a relevant cure rate but there were enough cases to suggest that this surgery can treat some patients with stress or urge incontinence.In a previous study, 3 of the 7 patients presenting a stress urinary incontinence were cured by bilateral pudendal nerve decompression alone ,37.In Shafik's study 6 patients were cured from their stress urinary incontinence, 5 improved and one was a failure . For thiThis study is the first one dealing with a possible effect of the pudendal nerve decompression on urge incontinence. It is probably due to a better control of the urethral sphincter which can reduce urethral instability  and imprOne weakness of this study is the rough evaluation of the symptoms. We did not use any scoring system, pad test, quality of life questionnaires or "visual analog pain scale". Furthermore the number of anal and urethro-vesical manometries done before and after PND was too small to give relevant results.The objective evaluation of PND was done using two neurophysiological tests and the clinical examination. Like Shafik ,30 we foShafik described many clinical signs of the PCS ,29,30. ICompared to patients with negative clinical signs, those having positive clinical signs have a 4,42 ; 5,52 and 6,56 higher likelihood of PCS for "Abnormal sensibility", "Painful Alcock's canal" and "Painful skin rolling test", respectively Table . When paThe most specific sign was the "Painful skin rolling test" and the most sensitive was the "Painful Alcock's canal". The association of the three positive tests had a very high specificity in the diagnosis of a PCS (89 %). This high specificity was confirmed by the low frequency of this association after the operation (return to the same level as the control group).Therefore, in some cases the clinical examination should be sufficient to prove the existence of a PCS. For example, a patient presenting anal incontinence, an intact sphincter proven by ultrasound and the three clinical signs positive has almost certainly a PCS. Of course, from a scientific point of view it is still interesting to perform a complete electrophysiological study and a precise neurological examination to exclude a central problem  or a polyneuropathy.However, making the diagnosis of PCS is not usually an easy task. Many times, there is a high degree of suspicion but, as in many illness, all the symptoms or signs are not present. In this study, we decided to operate when at least two of the five clinical and neurophysiological signs described in the methods section were associated with one or more of the 3 classical symptoms . At the beginning of this study, it was usually "increased PNTML" and "painful Alcock's canal". With the introduction of the "skin rolling test" and of the "sensibility test", clinical examination became more important in the decision. The more symptoms  and signs were present, the more confident we were in the diagnosis of PCS. Further studies are necessary to validate this and to define more precisely the minimal criteria needed for the PCS diagnosis.The pudendal nerve decompression by the perineal route is a blind procedure. The search for the inferior rectal nerve and the opening of the Alcock's canal are done under finger control. In our experience it is not easier with retractors. Therefore it is necessary to have a clear anatomical vision of this area before performing the operation.Maybe the use of a laparoscope would help  but the Despite the blind character of the procedure we only had one heavy bleeding probably coming from the pudendal artery. One patient still presents with a mild intermittent clitoridal pain and a worsening of anal incontinence. Because the nerve of the clitoris leaves the pudendal nerve just before the entrance into the Alcock's canal this problem is probably the result of a too large dissection in the upper part of this canal. The two cases of anal incontinence worsening (gas incontinence becoming liquid incontinence), including the aforementioned patient, are difficult to explain. Maybe the neuropathy increased with the stretching involved in the procedure, the scarring process or a too large dissection. It could also be the result of the changes in the posterior level anatomy induced by concomitant procedures (easier expulsion of gas or faeces). For the 2 patients the EMG data and the clinical examination after the operation did not improve therefore showing that the neuropathy was not healing.Because the roughly estimate prevalence of PCS is around 20%, this "defect" seems to be a very frequent problem in Perineology. Therefore it should be logical to search for it in each clinical examination of a patient presenting with prolapse, perineodynia, urinary or anal incontinence.Pudendal neuropathy is probably a frequent "defect" in perineology. Pudendal nerve decompression seems to be the defect specific procedure indicated in such a problem. In fact it can treat perineodynia, anal and probably urinary incontinence. Anal incontinence can be cured by pudendal nerve decompression alone even in the presence of a clear disruption of the anal sphincter on anal ultrasound. Anal richness on EMG increases and PNTML decrease significantly after surgery proving an objective effect on the nerve. The frequency of abnormal puncture sensibility, painful Alcock's canal and painful "skin rolling test" are significantly reduced by the operation. This study suggests that the three clinical tests could be used in practice to confirm or suspect the diagnosis of pudendal neuropathy in case of pain, urinary and/or anal incontinence. However, further studies are necessary to confirm these preliminary results.PCS = pudendal canal syndromePND = pudendal nerve decompressionEMG = electromyographyPNTML = pudendal nerve terminal motor latencyThe authors declare that they have no competing interests.JB conceived the study, carried out surgery and clinical follow up and drafted the manuscript.DC participated in the design of the study and performed the statistical analysis.MB carried out the neurophysiological examinations.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Genome-wide association studies for complex diseases will produce genotypes on hundreds of thousands of single nucleotide polymorphisms (SNPs). A logical first approach to dealing with massive numbers of SNPs is to use some test to screen the SNPs, retaining only those that meet some criterion for futher study. For example, SNPs can be ranked by p-value, and those with the lowest p-values retained. When SNPs have large interaction effects but small marginal effects in a population, they are unlikely to be retained when univariate tests are used for screening. However, model-based screens that pre-specify interactions are impractical for data sets with thousands of SNPs. Random forest analysis is an alternative method that produces a single measure of importance for each predictor variable that takes into account interactions among variables without requiring model specification. Interactions increase the importance for the individual interacting variables, making them more likely to be given high importance relative to other variables. We test the performance of random forests as a screening procedure to identify small numbers of risk-associated SNPs from among large numbers of unassociated SNPs using complex disease models with up to 32 loci, incorporating both genetic heterogeneity and multi-locus interaction.Keeping other factors constant, if risk SNPs interact, the random forest importance measure significantly outperforms the Fisher Exact test as a screening tool. As the number of interacting SNPs increases, the improvement in performance of random forest analysis relative to Fisher Exact test for screening also increases. Random forests perform similarly to the univariate Fisher Exact test as a screening tool when SNPs in the analysis do not interact.In the context of large-scale genetic association studies where unknown interactions exist among true risk-associated SNPs or SNPs and environmental covariates, screening SNPs using random forest analyses can significantly reduce the number of SNPs that need to be retained for further study compared to standard univariate screening methods. Genome-wide association studies for complex diseases such as asthma, schizophrenia, diabetes, and hypertension will soon produce genotypes on hundreds of thousands of single nucleotide polymorphisms (SNPs). Due to the large number of SNPs tested and the potential for both genetic and environmental interactions, determining which SNPs modify the risk of disease is a methodological challenge. While the number of genotypes produced by candidate gene approaches will be somewhat less daunting, on the order of hundreds to thousands of SNPs, it will still be a considerable challenge to weed out the noise and identify the SNPs contributing to complex traits.A logical first approach to dealing with massive numbers of SNPs is to first conduct univariate association tests on each individual SNP, in order to screen-out those with no evidence for disease association. The primary goal of such a procedure is not to prove that a particular variant or set of variants influences disease risk, but to prioritize SNPs for further study. Using a univariate test at this stage will result in low power for SNPs with very small marginal effects in the population, even if the SNPs have large interaction effects. Of course, in addition to taking all individual SNPs, all SNP pairs could also be tested for association. However, when dealing with multiple thousands of SNPs at the outset, such an approach is cumbersome, and raises the question of where to stop: why not all sets of three, four, or even five SNPs as well?Many model-building methods exist for dealing with large numbers of predictors. For example, stochastic search variable selection (SSVS) , a form Multivariate adaptive regression splines (MARS) models have also been explored in the context of genetic linkage and association studies ,6 with sCombinatorial partitioning and multifactor dimensionality reduction -10 are ce.g., [a priori, traditional regression models, which lump all individuals into a single group and estimate average effects over the entire sample, are unlikely to successfully identify the genetic causes of diseases.An additional concern to be considered is genetic heterogeneity. We define genetic heterogeneity to mean that there are multiple possible ways to acquire a disease or trait that can involve different subsets of genes. Traditional regression models are limited in their ability to deal with underlying genetic heterogeneity . If gene.g., . When thTree-based methods consist of non-parametric statistical approaches for conducting regression and classification analyses by recursive partitioning . These The ease of interpretation of classification trees, along with their flexibility in accommodating large numbers of predictors and ability to handle heterogeneity, has resulted in increasing interest in their application to genetic association and linkage studies. Classification trees have been adapted for use with sibling pairs to subdivide pairs into more homogenous subgroups defined by non-genetic covariates , thus inClassification trees are grown by recursively partitioning the observations into subgroups with a more homogeneous categorical response . At eachUsing ensembles of trees built in this manner increases the probability that some trees will capture interactions among variables with no strong main effect. Unlike variable selection methods, interactions among predictors do not need to be explicitly specified in order to be utilized by a forest of trees. Instead, any interactions between variables serve to increase the importance of the individual interacting variables, making them more likely to be given high importance relative to other variables. Thus, random forests appear to be particularly well-suited to address a primary problem posed by large scale association studies. In preliminary studies, we have shown the potential of random forests in the context of linkage analysis . Other iTo fully understand the basis of complex disease, it is important to identify the critical genetic factors involved, and to understand the complex relationships between genotypes, environment, and phenotypes. The few successes to date in identifying genes for complex disease suggest that despite carefully collected large samples, novel approaches are needed in the pursuit to dissect the multiple and varying factors that lead to complex human traits. Ultimately, the challenge in identifying polymorphisms that modulate the risk of complex disease is to find methods that can seamlessly handle large numbers of predictors, capitalize on and identify interactions, and tease apart the multiple heterogeneous etiologies. Here, we explore the use of the Random Forest methodology ,29 as a λs) fixed at 2.0 and population disease prevalence Kp equal to 0.10. These values are consistent with or lower than estimates from known complex genetic traits, such as Alzheimer disease, where estimates of cumulative prevalence in siblings of affected range from 30–40%, compared to a population prevalence of 10% at age 80 [λs = 2 and Kp = .10 for models H4M4 and H8M2, and 32 loci are responsible for models H8M4 and H16M2. Table We simulated complex diseases with sibling recurrence risk ratio for the disease , independent of disease status, with allele frequencies distributed equally across the range .01–.99. To simulate the results of an association study, in which we do not expect to be lucky enough to genotype all polymorphisms related to a trait, we included only a subset of the total number of rSNPs in each analysis. We denote the analysis design using the shorthand KkSsNn, where K = k is the total number of rSNPs genotyped in the analysis, S = s is the number of SNPs within each interaction system genotyped, and N = n is the total number of SNPs genotyped in the design. Thus, N-K is the total number of nSNPs in the analysis. For example, suppose the genetic model is H8M4, and the design is K4S2N100. Then out of the total of 8 × 4 = 32 rSNPs that contribute to the trait, four are genotyped: two interacting SNPs from within each of two heterogeneity systems. Six heterogeneity systems are not represented at all in the analysis. In addition to the four genotyped rSNPs, 100-4 = 96 total nSNPs are also genotyped in the design.IT) and standardized (ZT) variable importance scores (see Methods section for definitions of the scores). Little is known about the properties of importance indices under different distributions of the predictor variables. We use simulation to investigate their properties in the context of discrete predictors such as genetic polymorphisms conferring susceptibility to a complex trait.Random forests version 5 software  producesIT and ZT are highly correlated; the average correlation coefficient over 100 replicate data sets ranged from .93 (H8M4) to >.99 (H2M2 and H4M2)  to ranking by Fisher Exact test p-value using K4S2N100 and K4S2N1000 analysis designs, where two SNPs from each of the first two interaction systems are in the analysis. Figure T criterion ranks the four rSNPs as the most significant SNPs more often than the univariate Fisher Exact test association p-value under all genetic models. The difference between the random forest and association p-value ranking is less extreme for N1000. For the H8M4 genetic model, the results do not suggest that one ranking system is better than the other overall. Figure T contain all of the rSNPs. Thus, for a given probability of retaining all of the rSNPs, more SNPs can be eliminated using the ZT criterion than the Fisher exact test p-value. For example, for model H16M2, only 15 SNPs must be retained to have 80% probability that the 4 rSNPs are in the retained set, while 44 SNPs must be retained if the p-value criterion is used. The difference is less dramatic for H8M4: 37 SNPs give 80% probability that the four genotyped rSNPs are in the retained set for the ZT criterion, compared to 43 for the p-value criterion. For N1000, the advantage of the ZT criterion is clear for the H8M2 and H16M2 models. For H4M4, the advantage of ZT is minor, while for H8M4, ranking by ZT appears to give poorer results than the p-value criterion for the higher cutoff values of N. A second interpretation of Figure T criterion than for the univariate p-value criterion for all but the H8M4 model with 1000 total SNPs.We next compared the ranking of rSNPs by importance score  and a constant 96 nSNPs, we looked at the effect of increasing S, the number of rSNPs from each interaction system that were genotyped. Thus, for K8S1, along with 96 nSNPs, one SNP from each of the first 8 systems was included in the analysis, while for K8S4, all four SNPs in the first two systems were included in the analysis. For K8S3, three SNPs from the first two systems, and one from the third were included. Assuming that the random forest analysis was taking advantage of the interactions among the rSNPs, and that this was responsible for the improved performance of the random forests over the univariate tests, we expected the Fisher p-values and random forest importance ZT to perform similarly when only a single rSNP was genotyped from each system, and the random forests to perform increasingly better than the univariate Fisher tests as S increased from 1 to 4. Figures T, the S = 1 analyses for each K were similar to the Fisher results, while for each increase in S, the proportion of replicates for which the N most significant SNPs were rSNPs increases  had a more substantial improvement in ranking using ZT ranks interacting rSNPs higher than the univariate test, but also that the differences in rank correspond to differences in magnitude of ZT that are meaningful. In other words, we want to know how much "better" in terms of ZT  the rSNPs are than the nSNPs. Toward this goal, we computed the difference between the importance ZT of the top ranked rSNP and the top ranked nSNP:Beyond simply ranking SNPs, we may wish to use the magnitude of the difference in importance or p-value to determine which subset of top-ranked SNPs should be prioritized for further study. Thus, particularly for the cases where the rSNPs are among the top-ranked SNPs, we want to determine not just that Zmax(ZT) = maxrSNP(ZT) - maxnSNP(ZT),Das well as the lowest ranked rSNP and the top ranked nSNP:min(ZT) = minrSNP(ZT) - maxnSNP(ZT).Dmin(ZT) is positive when the lowest ranked rSNP is larger than the highest ranked nSNP, and negative when the lowest ranked rSNP is smaller than the highest ranked nSNP.Thus, Dmax(-log p) and Dmin(-log p), for the -log10 transformed Fisher Exact test p-values. In Figure T that is significantly higher than the highest ranking nSNP for both N100 and N1000, while the difference in -log10(p) is not significantly different from 0 for N100, and is significantly less than 0 for N1000. These plots illustrate that the positive differences are typically more extreme for ZT than for -log p, and that the negative differences are less extreme.We computed the analogous quantities, Da priori, such as the underlying genetic model and the ratio of associated risk SNPs to noise SNPs in an analysis.A key advantage of the random forest approach is that the investigator does not have to propose a model of any kind. This is important in an initial genome-wide or candidate region association study, where little is known about the genetic architecture of the trait. If interactions among SNPs exist, they will be exploited within the trees, and the variable importance scores will reflect the interactions. Therefore, we expect that when unknown interactions between true risk SNPs exist, the random forest approach to screening large numbers of SNPs will outperform a univariate ranking method in finding the risk SNPs among a large number of irrelevant SNPs. Our genetic models for simulation feature both multiplicative interaction and genetic heterogeneity. The multiplicative interaction results in a marginal effect in the population, the size of which is dependent on many factors, including the amount of heterogeneity. Thus, we have highlighted models in which univariate tests still have power, and shown that the random forest analysis can outperform these tests for selecting subsets of SNPs for further study. For models with genetic heterogeneity and interactions resulting in no main effect, similar to the models described by Ritchie et al. , the perOur results from analyses with four risk SNPs among 1000 SNPs suggest that even when a high proportion of the analyzed SNPs are unassociated, a random forest can rank interacting SNPs considerably higher than a univariate test, and that the proportional difference in importance between the risk SNPs and the best of the noise SNPs can be larger on average for a random forest. In our scale-up from 100 to 1000 total SNPs, we kept the number of risk SNPs constant. In practice, as we increase the number of SNPs genotyped, we expect that we will also increase the number of risk SNPs (or SNPs in linkage disequilibrium with risk SNPs) that are captured in an analysis. Thus, as a larger and large proportion of the genome or candidate region is captured by a scan, the more likely we will be to have all or most of sets of SNPs that interact, and thus the more likely we are to be in situations where random forest screening will outperform univariate screening of SNP data.It is important to consider the tuning parameters for such analyses. Consistent with the recommendations made by Breiman and Cutler ,29, the We have examined the use of random forests in the context of association studies for complex disease with uncorrelated SNP predictors. Random forests can also be used when predictors are correlated, as is the case with multiple SNPs in linkage disequilibrium within a small genetic region. For any analysis procedure, the more highly correlated variables are, the more they can serve as surrogates for each other, weakening the evidence for association for any one of the correlated variables to the outcome. In a random forest analysis, limited simulations suggest that correlated variables lead to diminished variable importance for each correlated risk SNP (data not shown). One way to limit the problems presented by SNPs in linkage disequilibrium is to use haplotypes instead of SNPs as predictor variables in a random forest. Future challenges include quantifying more completely the effect of linkage disequilibrium among SNPs submitted to a random forest analysis, and developing random forests in the context of haplotypes.With the increasing size of association studies, two-stage analyses, in which in the first stage a subset of the loci are retained for further analyses, are becoming more common. The most frequently voiced concern for these analyses is that variables that interact to increase disease risk but have minimal main effects in the population will be missed. Random forest analyses address this concern by presenting a summary importance of each SNP that takes into account its interactions with other SNPs. Current implementations of random forests can accommodate up to one thousand of SNPs in one analysis with the computation of importance. Further, there is no reason to restrict the input variables to SNPs. Potential environmental covariates can also be easily accommodated, allowing for SNPs with no strong main effect, but environmental interactions, to be distinguished from unassociated SNPs. We have shown that when unknown interactions among SNPs exist in a data set consisting of hundreds to thousands of SNPs, random forest analysis can be significantly more efficient than standard univariate screening methods in ranking the true disease-associated SNPs highly. After identifying the top-ranked SNPs and other variables, and weeding out those unlikely to be associated with the phenotype, more thorough statistical analyses, including model building procedures, can be performed.Xi represent the vector of predictor variable values, yi its true class, Vj(Xi) the vote of tree j and tij an indicator taking value 1 when individual i is out-of-bag for tree j and 0 otherwise. Let X = ,..., XN) represent the vector of predictor variables with the value of variable A randomly permuted among the out-of-bag observations for tree j, and X(A) the collection of X for all trees where N is the total number of individuals in the sample. Letting 1(C) denote the indicator function taking value 1 when the condition C is true and 0 otherwise, the importance index averages over the trees of the forest, and is defined as:Rather than selecting variables for modeling, a random forest uses all available covariates to predict response. Here, we use measures of variable importance to determine which covariates  or sets of covariates are important in the prediction. Breiman  proposedNj represents the number of out-of-bag individuals for tree j and T is the total number of trees.where IT, . The standardized index is defined as:The importance index can be standardized by dividing by a standard error derived from the between-tree variance of the raw index  represents the tree to tree variance of IT, rather than the variance of IT due to the sampling of the individuals from a population: the magnitude of ZT increases with the number of trees in the forest, and the number of trees is limited only by computing time. Thus, this standardized index cannot be treated as a Z-score in the traditional sense.The variance 0, q1, q2) represent the penetrance factors for 0, 1, or 2 risk alleles for an individual locus in a given model. Let G = {g11, g12, . ., gHM} be the multilocus genotype for an individual, where ghm indicates the individual's genotype at locus m  of heterogeneity system h . Then the penetrance for genotype G is defined as:For simplicity, assume each locus has the same effect, and let (qPG = 1 - (1 - q0q1)2 = 2q0q1 - (q0q1)2.For example, for model H2M2, an individual with genotype G = {0101} would have penetrance 0, q1, q2) and risk allele frequencies, as well as other features of our genetic models, are listed in Table λs and Kp, there is a unique allele frequency when we make the assumption that each SNP subunit has equal effect (the given penetrance factor vector) in the population. We chose penetrance factors such that the risk alleles at each locus for the H2M2, H4M2, H8M2, and H16M2 models are approximately additive in effect on the penetrance factor scale. For H4M4 and H8M4, we chose penetrance factor vectors such that the risk alleles show a moderate degree of dominance.The penetrance factors q0, q, q2 and The marginal genotype relative risks (GRRs) listed in Table All analyses were performed on 100 replicate data sets of 500 cases and 500 controls. We treated the SNPs as ordinal predictors. Random forests have one primary tuning parameter: "mtry" the number of randomly picked covariates to choose among for each split. The Random Forest v5 manual  recommenIT, ZT,), which can be used to rank order the importance of SNPs much as p-values from association tests are used. Using either method, the ranking of the SNPs in an analysis can be used to prioritize which sets of SNPs (or genome regions) should be followed up with further genotyping and/or additional analyses. We use the convention that a rank of 1 is the highest ranking SNP.The random forest analysis produces the raw and standardized importance indices (We compare the ranking of the raw and standardized importance measures, and further compare these with the rank based on p-values from a test of association, the Fisher Exact test, to determine whether the random forest can better discriminate susceptibility SNPs from SNPs unrelated to disease status when there is interaction and heterogeneity among and between SNPs.KLL conceived of and led the design of the study, coordinated all phases of simulation and analysis, and drafted the manuscript. LBH and JS automated the simulation and random forest analysis procedures, and participated in the design and analysis of study results. PVE added critical insight to the development of the genetic models and participated in the interpretation of study results. All authors provided comments on a draft manuscript and read and approved the final manuscript.