The causes of PBIS are well known, consisting of various decisions about reporting that are influenced by the data. But its impact is not generally appreciated, and very little attention is devoted to it. What attention there is consists largely of rules for statistical analysis that are impractical and do not actually reduce the bias in reported estimates. PBIS cannot be reduced by statistical tools because it is not fundamentally a problem of statistics, but rather of non-statistical choices and plain language interpretations. PBIS should be recognized as a phenomenon worthy of study – it is extremely common and probably has a huge impact on results reported in the literature – and there should be greater systematic efforts to identify and reduce it. The paper presents examples, including results of a recent HIV vaccine trial, that show how easily PBIS can have a large impact on reported results, as well as how there can be no simple answer to it.PBIS is a major problem, worthy of substantially more attention than it receives. There are ways to reduce the bias, but they are very seldom employed because they are largely unrecognized. A study is more likely to appear in the literature, and thus be indexed and publicly available, if it shows a strong, statistically significant, or otherwise "better" result. A study that does not show such results has a greater chance of remaining hidden in a file drawer, either because the author (or funder) does not think the result is worth mentioning, or because journals are less interested in publishing studies that find "nothing". This form of publication bias, wherein readers have access to only a biased sample of the full distribution of results, is well studied, particularly in the literature on meta-analysis and other systematic reviews where the problem is most apparent (a primer on the topic can be found at the Cochrane Collaboration webpage ). Even wwhich results to publish produces bias. As with traditional publication bias, the tendency is to analyze data and choose to present results which are statistically significant, further from the null, or closer to what the researchers believe is the true value. The implications for the literature as a whole, though, are much the same as the file drawer bias. I label this "publication bias in situ" (PBIS) because the biased reporting of study findings exists within each individual research report . Despite the similarities between the file drawer bias and PBIS, there are fundamental differences. In particular, the bias from some studies having no published findings exists only at the level of the whole literature (no particular study can be said to be biased), while PBIS exists within the results reported from a single study (and thus exists in the literature as a whole by aggregation). More practically, PBIS is substantially more difficult to even identify, let alone correct.Far less attention is paid to the bias that occurs when some results from a study are published, but the choice of This paper defines and describes PBIS and identifies some of the choices that create it. The purpose of the paper is not to make novel technical or statistical claims. Indeed, there is probably no single statistical observation here that will not be clear to a skilled data analyst . The literature includes many observations about issues relating to PBIS. Yet the challenge to the validity of the entire health science literature posed by PBIS – arguably a greater issue than conflict of interest, traditional publication bias, or any other commonly discussed threat to the integrity of the literature – has not received the attention it deserves.ex ante protocols were stated and followed, and is thus put on par with many protocol violations that could be considered mere technicalities. For example, a few years ago, a highly-publicized meta-analysis [In the literature about meta-analysis and systematic reviews there is substantial attention to file-drawer publication bias . But there is considerably less attention to possible picking and choosing which study results to report or statistical methods to use. To the extent that this is considered, it is often bundled with questions about whether analysis  called iThe literature and pedagogy related to observational inference, where the potential for PBIS is considerably greater than for well-designed experiments, seems to pay even less attention. Epidemiology textbooks typically discuss some of the methods that can reduce PBIS , but say little or nothing about the possible bias. Indeed, many study and data-analysis methodologies (some of which are noted below) that are typically taught in classes or by apprenticeship seem designed to create PBIS. Despite this, highly-trained experts summarize claims reported in the literature without mention of the likely bias, indicating an unawareness of the major implications of PBIS.In a notable exception, Hahn and colleagues ,4 discusIt is not clear how common PBIS is or how large the resulting bias, but a few efforts to find it suggest that the potential is great enough that it deserves much more attention.  Only by systematically addressing the problem are we likely to substantially affect it. Moreover, as will be discussed, statistical and research methods that ostensibly address some of the sources of PBIS are unsatisfactory, and a systematic attempt should be made to find better solutions.Reviewers (systematic or otherwise) of the literature can only see what researchers choose to report and highlight in their publications, and that choice can be biased in a number of ways. Researchers have great freedom in deciding exactly what to analyze, how to analyze it, and what to report. All research results are derived from data that can be used to measure many associations. Even the most narrowly focused clinical trial can be analyzed with the endpoint defined in different ways, stratified by age, etc. As with traditionally defined publication bias, the analyses that are deemed unworthy of publication are largely invisible to the scientific and policy community.Among the dimensions of freedom researchers have in deciding what to analyze and report are three choices illustrated by examples in this paper:(1) Which exposures and outcomes to consider in datasets with many variables.(2) Which functional forms to use to represent variables .(3) Whether to conduct separate analyses by subgroup, and which subgroup results to emphasize.some question), there is no clearly wrong choice, and thus no clear way of concluding that a particular choice was biased. Fortunately, as should become apparent from the following analysis, PBIS results less from the choices made and more from what the choices are based on (which can often be inferred) and, to an even greater extent, the generally overlooked issue of how the results are presented (which can be easily observed).Making such choices is a legitimate part of research. Indeed, the choices must be made. But when those choices are primarily driven by what produces stronger (or otherwise "better") results, bias is created. This creates a difficult challenge: It is easy to recognize traditional publication bias . But since there is no clearly correct option for any of the above choices , creating a bias from what would be unbiased random variation. Systematic errors  and methods for correcting for them create many additional opportunities for PBIS; however, for simplicity, systematic errors are ignored in this paper.Choice (1) in the above list has probably received the most attention in previous literature . Despite the disproportionate attention, this choice is probably not the major source of PBIS, but it provides a familiar starting point.Many epidemiologic datasets are characterized by thousands or even millions of possible combinations of exposures, endpoints, and covariates. It is frequently assumed that statistical science tells us the "right" way to deal with this challenge, but current practice (not to mention the confusion of students coming out of epidemiology and biostatistics classes) makes clear that there is substantial disagreement among viewpoints about how to apply statistical methods when dealing with multiple hypotheses or measurements. Further consideration makes it clear that statistical rules cannot actually provide clear answers.At one extreme are viewpoints such as, "We must correct measures of statistical certainty  or confidence interval widths) whenever multiple comparisons are made using the same dataset" and even, "statistical analysis can only be legitimate for a short list of pre-specified hypotheses." At the other extreme are viewpoints such as, "regardless of how many comparisons are examined, each can be considered and statistically tested as if it were the only one," and, "it does not matter at all if a hypothesis was proposed after looking at the data." The impasse in this debate seems to stem from both sides attacking straw men, without recognizing that each side has a stronger case some of the time. This can be illustrated with examples.Helicobactor pylori infection contains data that is later used to look at the relationship of household crowding and performance in school. It is difficult to understand why we would make an adjustment when doing the second analysis because we have already done the first . A logical extension of that argument would be to consider the dataset that contains all quantitative human knowledge , and declare that we have to adjust for every statistical analysis ever done, effectively precluding further statistical analysis.A cohort dataset originally used to report the relationship of drinking water source and the occurrence of H. pylori infection. The dataset contains dozens of different measures of food and nutrient intake, as is usually the case for nutrition data. This, plus multiple diagnostic tests for H. pylori which are sometimes discordant, creates a large number of statistical comparisons, any of which could be described as supporting the plain language claim, "poor nutrition affects H. pylori status." A typical approach is to find individual comparisons that support the hypothesis, presenting only these comparisons with statistical tests as if each were the only analysis conducted. The claim about the relationship between the particular measure of nutritional status and the particular measure of H. pylori status is accurate, as are the test statistics reported for that association. But the plain language conclusion (which would probably be drawn) was very likely to be supported by some relationship in the data by chance alone, even in the absence of any true underlying association. This fact is obscured by the reported unadjusted tests statistics or confidence intervals.Researchers investigate the hypothesis that poor nutrition increases the risk of in situ within a single published article, making it a bias of much greater magnitude than the file-drawer effect).As the second example illustrates, unrestricted picking and choosing of comparisons leads to publication bias. A lot of associations that were not deemed worthy of reporting never appeared in the literature, while the few that were "interesting" did. This problem is well known , correcting for multiple comparisons does not affect the reported biased estimates of effect size; changing test statistics and confidence interval widths does nothing to reduce the bias. This alone shows that the standard statistical corrections for multiple tests do nothing to solve the problem. The other standard method for trying to reduce PBIS, rules that limit analyses to pre-specified hypotheses and protocols, will throw away a lot of potentially valuable findings and is nearly impossible to operationalize because detailed protocols require advanced knowledge that may not exist and can never be specified unambiguously.The solutions offered by statistical rules – corrections for multiple hypothesis testing or restricting analysis to Frequentist statistical theory cannot offer a solution to this problem because PBIS, like the file-drawer problem, is not a matter of statistics. The second example illustrates where the problem primarily lies: in the plain-language reporting of results. The statistics that describe the relationship between a particular exposure and outcome measure could be exactly right, but the claim about good nutrition (as a generic concept) and infection status (as if we had a gold standard measure), which will likely be emphasized in the paper and its title (and press releases) and will likely stick readers' minds, might be misleading. Consider how the result would be interpreted if there were a table reporting every analyzed comparison, most of which showed little or no association. Most scientifically literate readers would realize the result was not so convincing, even though those same readers seldom think to object when – as is typical – only one or a few results are reported. By contrast, if the researchers in the first example reported the result of the previous study, it would be unlikely to change most readers' assessments.ex ante hypothesis or having "used up" the α), but without creating the PBIS that would result otherwise. Indeed, this appears to summarize the most obvious generic rule to reduce PBIS : publish everything.This suggests the simplest partial solution to the problem. By reporting a table of results from other comparisons considered, researchers could report their interesting result , and can include dozens or thousands of alternative analyses in appendices or links to data or software that allow the reader to review still more results. Of course, this opportunity is beneficial only if authors choose to take advantage of it (or editors and reviewers demand that they do).The implications of choice (2), the functional form for variables, can be clearly illustrated with a simple example. Consider a study with an exposure variable measured as 10 ordered categories . Assume researchers wish to analyze the association of a disease endpoint and a dichotomous definition of exposure. If there is no clear cutpoint for defining exposure, there are many options. There are 9 cutpoints that divide the observations into two categories, defining those above the cutpoint to be exposed and those below unexposed. Other options include comparing a group of highest categories to a group of lowest categories, leaving out the middle, such as 8–10 versus 1–3, yielding an additional 36 possibilities.How will the researchers choose a definition of exposure? A typical procedure is to let the data inform the choice: The cutpoint that provides the clearest contrast between the exposed and unexposed is judged to be the right one, the "most sensitive" to the presumed effect. It should be immediately obvious that this procedure will bias the result away from the null.To illustrate this, consider a case-control study with 200 subjects . Calculations for this example and others are based on Monte Carlo simulation of different realizations of the data based on the assumed underlying relationship. All simulations were performed using Crystal Ball .Assume that each of the 10 exposure categories is equally likely for cases and non-cases. If the researchers consider only the 9 cutpoints that dichotomize the data and choose the cutpoint for "exposed" to get the largest odds ratio (OR), the median result will be 1.5. Since the exposure and disease are not associated, this is clearly an upward bias. For those inclined to focus on statistical significance, the chance of observing a significant positive association at a one-tailed significance level of .025 is 13%. Even if researchers do not analyze their data in every possible way and report the strongest association, any decision to report results that is based on associations in the data  will create bias. Although this observation should be obvious to anyone with an understanding of statistics, letting the data have some influence on the choice is probably more the rule than the exception among researchers. It is often defended on the grounds that there was no way to know what the "right" cutpoint was before doing the study, and the study data is the only existing answer to the question. This is a legitimate point, but it does not reduce the resulting bias. Less scrupulous researchers – who are trying to support a preferred answer to further a policy agenda or advance their careers – need make no such explanation and can intentionally choose the extreme results.As with the file-drawer effect, results in the literature will tend to show effects greater than the true value. Most important, whichever analysis is reported, the plain-language result will be "we found an association between the exposure and the disease," and so collections of studies that each report an exposure-disease comparison with a greater-than-average association, and will seem to be confirming the same result, even though the comparisons are not the same.Extending this example to illustrate how PBIS can compare to traditional publication bias, assume now that there is a positive association between the exposure and disease. In particular, non-cases are still equally likely to be in each of the ten categories, while cases have respective probabilities for each category of . These values were chosen so that the true OR is similar, whichever of the 9 cutpoints is chosen . True ORs round to 1.5 for all cutpoints.Consider a collection of studies of varying sizes, with fewer larger studies, as we would typically see in the literature, specifically 100 randomly generated studies  of random size . Note that to avoid committing the very type of transgression discussed in this paper – repeating an analysis until "good" results are found – the reported results are from the first and only run of the simulation.For a single definition of "exposed" values >5), a typical result appears in Figure , a typicCompare this to the results for the same 100 studies where the cutpoint is chosen based on the largest OR Figure . The disUnbiased random errors, when combined with picking and choosing functional forms, lead to biases in reported results. A solution to this problem is much less obvious than its existence.The commonly proposed solution of only reporting results for pre-specified functional forms is not satisfactory, because it is difficult to enforce  and it forces us to ignore real unpredicted findings. Sticking to pre-specified analyses is especially unrealistic in research studies that collect data on many different risk factors and outcomes. Simply labeling all results that were not pre-specified with the caveat, "hypothesis generating," accomplishes nothing. If such results were actually treated as not yet "real", the problems of determining exactly which results were pre-specified and the loss of important serendipitous findings are reintroduced. Of course, results with the caveat are very seldom treated as less real than any others in the literature. Moreover, the "generated" hypotheses will never be retested in exactly the form reported, so the label is simply disingenuous. In sum, the proposed solutions to this type of PBIS are no more realistic or satisfactory as solutions than trying to eliminate traditional publication bias by requiring that all studies be adequately powered.A better family of solutions would be to establish a standard of reporting results for alternative variable definitions (perhaps in online appendices). Not only does this directly reduce PBIS by publishing more results, but it provides readers with a choice of results if they prefer different definitions (information that would be discarded by a pre-specified hypothesis rule). If results for every cutpoint from the 100 trials in the example were reported, the results, as pictured in Figure In many cases there will not be an obvious short list of variable definitions, but some alternative definitions should be obvious and others could be found in previous literature. A simple, but very useful, improvement would be a standard practice of reporting the closest possible replication of previous published analyses using data from the new study. This would directly address the problem of PBIS resulting from data-driven picking and choosing of functional forms . Repeating whatever analyses that previous researchers happened to choose is somewhat arbitrary, but each round of new research can also add a new preferred functional form. The key is that results based on previously published functional forms cannot be data driven and, unlike the standard practice of new studies that make different comparisons but describe them with the same plain language, would actually replicate (or fail to replicate) previous results.New York Times articles actually contained more complete study results. VaxGen tried to salvage some hope for the drug by pointing out the results for non-whites, suggesting that maybe it held promise for some populations [In 2003, VaxGen Corporation  released results of a large HIV vaccine trial in the United States, one of the highest-profile clinical trials of the year. The disappointing result showed a trivial reduction in incidence among the treatment group compared to the placebo group. But the three non-white racial groups  each showed a substantial reduction in incidence Table . VaxGen ulations . VaxGen'ulations -12.Given the failure to publish the results in a scientific journal, some might argue that VaxGen was guilty of traditional publication bias – not publishing unfavorable results about its products – a charge that is currently being leveled at many drug companies. However, the company actively released fairly complete study results and accompanying analysis to the press, and the results appeared in forums that are more widely read than all scientific journals combined, so the results were clearly not buried in a file drawer.But despite reporting their results, VaxGen was biasing what they published. Singling out of the result for non-whites is a clear case of PBIS. VaxGen did not give equal emphasis to the apparently harmful effect of the vaccine for whites. The company's subsequent report that the different results for whites and non-whites could not be due to chance  does not4-1 = 15 different hypotheses. To adjust for 15 implicit hypotheses makes it very unlikely that any will pass the statistical test, including the population as a whole, even for substantial associations.Setting aside some controversy that erupted about systematic errors in the data, what can we say about the results from the perspective of PBIS and chance alone? To look at the result for non-whites, with its one-tailed p-value of .002, ignoring the fact that the subgroup definition was clearly data-driven, would overstate the finding, as suggested in the preceding examples. But a naive correction for multiple hypothesis testing would make the opposite error. Setting aside the possibility that other covariates would have been used to stratify the data had they produced subgroups with positive findings, the combination of the 4 racial groups implies a test of 2An alternative is to ask the question, "if the vaccine has no effect, what are the chances of seeing, in any racial group or combination thereof, a result at least as strong as the observed 63% reduction?". Phrased that broadly, simulation shows the answer is about 20%. However, most of the 20% comes from the unstable results for the two smallest groups, Asians and "other". Restricting the analysis to combinations of racial groups that contain black, with or without Asians or other, the probability is only 2.1% .So, what is the right answer? We must return to the observation that there is never a single Right Answer from a study; the quality of an answer always depends on what question was being asked. Did VaxGen find a successful vaccine? Clearly not, as the relative risk for the whole population shows. Should the result for non-whites be considered unlikely to be due to chance ? It depends on whether you consider it the answer to the question "does the vaccine show a result for non-whites?", in which case the answer is 'yes' , or "does the vaccine show a result for any racial group?" in which case the answer is 'it is fairly likely we would see such a result due to chance alone.' It is worth noting how this illustrates a popular fallacy in data analysis: Frequentist hypothesis testing is not the objective exercise that some think it to be; it depends on subjective decisions about what to test.ex ante hypothesis that the vaccine would work only in non-whites or because the multiple-hypothesis correction for hundreds of possible racial and other subgroups makes it non-significant. A general rule requiring us to ignore interesting but surprising findings is a huge waste of information. Requiring a data-driven subgroup analysis to be biologically plausible before reporting it offers no solution, since we can usually construct a story to explain whatever associations appear in the data .We might decide to infer that VaxGen would have emphasized the results from any racial subgroup that showed a positive result , and thex ante plan to analyze racial and other subgroups [ex ante, as if that should change our interpretation of the result, seems particularly absurd.To offer the "hypothesis generating" caveat would make little difference, scientifically or in the securities market. It is unrealistic to suggest that this "generated" hypothesis will be re-examined given the overall disappointing result. Two studies in Thailand  that suggests there is a beneficial effect for some people should equally emphasize any apparent harmful effects for other people (and vice versa). The fact that one result is statistically significant and the other is not should be of no consequence. Indeed, selecting which results to report based on statistical significance guarantees there will be publication bias . The reporting of the multiple subgroups results should be accompanied by statistics similar to those calculated here, instead of standard test statistics, so that readers know the probability that an estimated effect (or test statistic) at least as great as the one found would result from chance alone for any of the subgroup analyses. Such information will allow readers  to focus on what they consider to be the answer to their own questions.The opportunities for PBIS, along with the almost universal failure to report research results in ways that avoid it, create the possibility that biased study results are very prevalent in the health science literature. Some of the causes of PBIS are well understood, but the enormity of its implications is largely ignored.PBIS can produce very misleading results, leading to widespread misperceptions and misguided policies. The examples presented here show just a few of the many ways that PBIS can result from random error and researchers'  choices. Neither the problem nor the solution lies in the mathematics of data analysis, so answers will not be found by appealing to statistical theory. The critical issue is the completeness of reporting and the plain-language interpretation of results.The simple solutions offered by statisticians are not satisfying or even realistic. All they really let us do is observe that in almost every research report, "the rules" have been violated. This is not helpful. Rather than a right-vs.-wrong view of proper use of statistics that would condemn most of the literature as invalid, we need a realistic way of addressing this problem. The solutions, like the solutions for traditional publication bias, will generally consist of doing a more complete job of reporting what can be reported.HIV = human immunodeficiency virusOR = odds ratioin situPBIS = publication bias None declaredSingle authorThe pre-publication history for this paper can be accessed here:


Why haven't we developed an HIV vaccine yet? And will the new roadmap from the Global HIV/AIDS Vaccine Enterprise help our efforts towards vaccine development? Ho addresses these crucial questions PLoS Medicine, is a clear and cogent document describing how major funders and stakeholders in HIV vaccine development should move forward in a collaborative fashion [The scientific strategic plan of the Global HIV/AIDS Vaccine Enterprise, published in this month's  fashion . There iWhy is it that we still do not have a protective vaccine against HIV 22 years after its initial identification? Many possible explanations come to mind.In the natural course of HIV infection, the virus wins 99% of the time, showing that specific immunity in an infected person is unable to completely clear the virus. We have also known for over a decade that primary HIV isolates are relatively resistant to antibody neutralization, probably because of a “protective shield” on the viral envelope glycoproteins, consisting of variable loop sequences and extensive N-linked glycosylations. Another explanation is the extreme plasticity of HIV that allows new viral variants to evade immune recognition in the same way that they escape from drugs. Moreover, superinfection by a second viral strain has been documented in a number of individuals who have already mounted immune responses to the initial HIV infection. Yet another problem is that the AIDS research community has yet to uncover the correlates of immune protection in vivo. Lastly, proven vaccine approaches from the past have either failed (whole killed virus and subunit vaccines) or faced seemingly insurmountable regulatory hurdles (live attenuated virus vaccine).Given these daunting obstacles, why have so many continued in the long struggle to develop an HIV vaccine? The answer must lie, in part, in the noble cause at hand. Yet there are also some encouraging clinical and experimental observations . Rare paThe scientific strategic plan of the Enterprise is spot-on in identifying the major roadblocks in HIV vaccine development, as well as in establishing the key scientific priorities as we see them today . It righOne might ask, however, whether there are potential downsides to the plan. In the name of continuing this important dialogue, I would like to offer one general concern. Arguably, the reason for the lack of an effective HIV vaccine today is rooted in the basic problems posed by the virus itself. What we need foremost are new scientific solutions, although a prim and proper “process and structure” in our approach will be helpful. The needed breakthroughs to develop a vaccine will likely emerge from the creativity of scientists doing fundamental research that is free of preconceived biases. It is my contention that great new ideas are as likely to come from curiosity-driven basic studies as from the mission-oriented approach that is represented by the new proposal. Therefore, the leadership of the Enterprise must safeguard against the kind of “group think” that is so pervasive in large collaborative endeavors of this nature. The views of a small number of researchers, no matter how smart or accomplished, must not supersede the collective wisdom of the scientific community at large.No doubt important contributions will be made by scientists working outside of the Enterprise. Measures should be taken to ensure that their views and approaches, even if deemed unconventional, are not stifled by the newly established system. Likewise, their research support should not be compromised because the creation of the Enterprise concentrates the funding into the hands of a relatively small number of designated scientists. To me this is a serious risk given the current “flat funding” at the National Institutes of Health.The authors of the “The Global HIV/AIDS Vaccine Enterprise: Scientific Strategic Plan” have laid out a timely and insightful plan to address perhaps the greatest public-health need of the millennium. This document and its later revisions will serve as useful guideposts for the AIDS vaccine development effort for years to come. To be successful in this mission, our research community will ultimately need a specific “scientific blueprint” for making an HIV vaccine. That day will come only after we get another shot in the arm, infusing us with new knowledge and know-how. Is there any doubt that we need to redouble our investment in basic research on the challenges posed by HIV?


Anal smears are increasingly being used as a screening test for anal squamous intraepithelial lesions (ASILs). This study was undertaken to assess the usefulness and limitations of anal smears in screening for ASILs.The cytomorphological features of 200 consecutive anal smears collected in liquid medium from 198 patients were studied and findings were correlated with results of surgical biopsies and/or repeat smears that became available for 71 patients within six months.Adequate cellularity was defined as an average of 6 or more nucleated squamous cells/hpf. A glandular/transitional component was not required for adequacy. Dysplastic cells, atypical parakeratotic cells and bi/multinucleated cells were frequent findings in ASIL while koilocytes were infrequent. Smears from LSIL cases most frequently showed mildly dysplastic and bi/multinucleate squamous cells followed by parakeratotic cells (PK), atypical parakeratotic cells (APK), and koilocytes. HSIL smears contained squamous cells with features of moderate/severe dysplasia and many APKs. Features of LSIL were also found in most HSIL smears.In this study liquid based anal smears had a high sensitivity (98%) for detection of ASIL but a low specificity (50%) for predicting the severity of the abnormality in subsequent biopsy. Patients with cytologic diagnoses of ASC-US and LSIL had a significant risk (46–56%) of HSIL at biopsy. We suggest that all patients with a diagnosis of ASC-US and above be recommended for high resolution anoscopy with biopsy. For corresponding Editorial, please see Leiman, 2005 The incidence of anal squamous carcinoma and its precursor lesions has increased in recent years particularly among men having sex with men (MSM) . Prior tAnal and cervical lesions share many histological and pathological characteristics including the implication of human papilloma virus (HPV) in the pathogenesis of precursor squamous intraepithelial lesions and invasive cancer . Just asAfter approval from the IRB, 200 consecutive anal smears submitted from 198 patients were retrieved from the files of the pathology department at Cedars-Sinai Medical Center. The samples had all been collected from the anal canal using the Rovers endocervex brush , the Digene cervical sampler brush , or the brush from SurePath sample collection kit  , parakeratotic cells (PKs), atypical parakeratotic cells (APKs), koilocytes, binucleated and/or multinucleated squamous cells (B/MSCs), and dysplastic cells. The number of cells exhibiting each of these morphologic features was recorded as none, rare (no more than 2 cells/smear), and present (3 or more cells/smear). For this study cellularity was defined as the average number of nucleated squamous cells per 40x high power field (nsc/hpf) calculated by counting 10 hpfs. All of the anal smears had been reported using a modified Bethesda 2001 System terminology recommended for cervical smears . After dRevised cytologic diagnoses were correlated with concurrent and/or follow up tissue biopsies or with repeat anal smears all obtained within six months. Statistical analyses were performed using the Fishers Test. A two sided p value of 0.05 was considered as significant .For purposes of this study we required an average of at least 6 nsc/hpf for cellularity to be considered adequate. This was based on the observation that only smears averaging 6 or more nsc/hpf included abnormal cytologic diagnoses ranging from ASC-US through HSIL whereas smears averaging 5 or fewer nsc/hpf were either NIL or ASC-US. 91% (181) of the 200 smears contained an average of 6 or more nsc/hpf. Of the 19 cases that averaged fewer than 6 nsc/hpf, 17 were designated as unsatisfactory and excluded from the study while two that contained atypical squamous cells were reported as ASC-US and included in the subsequent morphologic analysis. Of note, all of the 7 smears with HSIL and 16/17 smears with ASC-H were cellular with an average of 8 or more nsc/hpf.Anucleated squamous cells were present in smears that were NIL and in smears with diagnoses ranging from ASC-US to HSIL. There were numerous anucleated squamous cells in 7 smears that were reported as unsatisfactory. Among the abnormal smears, neither the presence nor number of anucleated squamous cells correlated with cytologic diagnosis.Three or more groups of G/TZ were present in 56% (103) of the smears while rare G/TZ were seen in an additional 18% (33) smears. Of these smears 68% (93/136) had an abnormal cytologic diagnosis . In comparison 26% (47) of smears contained no G/TZ of which 68% (32) were reported as abnormal  suggesting that the presence of glandular cells did not facilitate abnormal diagnoses. Again all of the 7 smears with HSIL and 12/17 smears with ASC-H contained 3 or more groups of glandular cells, while two smears with ASC-H contained only rare glandular cells.Parakeratotic cells were observed in 71% (130) of the smears in the study. Parakeratotic cells were observed in negative  as well as in abnormal  cases. In negative cases rare parakeratotic cells were observed more often  whereas in smears with epithelial abnormalities presence of rare parakeratotic cells and frequent (>3) parakeratotic cells were about evenly distributed .APKs were found in 40% (74) of the 183 smears. The number of cases showing APKs increased with the severity of the dysplasia. There were 3 or more APKs in 22% (41) of the smears constituting 7% of ASC-US, 41% of LSIL, 53% of ASC-H and 71% of HSIL cases. Rare APKs were found in 18% (33) of the smears that were interpreted as ASC-US or above. APKs were not found in any of the negative smears and were seen in 72% of the SIL smears.Classical koilocytes were infrequent (17%) in all diagnostic categories Figure . Three oB/MSCs were observed more frequently in abnormal smears – 81% (101/125) as compared to 33% (19/58) of the NIL smears  of the smears diagnosed as LSIL contained 3 or more squamous cells with features of mild dysplasia  of the 183 smears constituting 39% (181) of the patients in the study. As shown in Table ASIL presents unique challenges in diagnosis and clinical management. By decreasing deaths from opportunistic infections, widespread use of highly active antiretroviral agents and other therapies have done much to improve survival of HIV infected individuals. However, because these therapies do not impact the incidence of HPV infections or malignancies in these individuals, the increased life span of HIV+ individuals probably provides the primary explanation for the rapid and continuing increase in HPV associated AIN that these individuals are experiencing -12. WithAnal cytology has been shown to be a cost-effective screening method for detection of ASIL in populations at high risk for anal carcinoma. To datePrior experience with anal smears as documented in the literature,17 reveaThere is a paucity of literature regarding criteria for adequate anal cytology samples. The 2001 Consensus Conference in Bethesda  suggesteThe presence of G/TZ was not a prerequisite for adequacy in our study. Smears with and without G/TZ detected the same percentage (68%) of abnormal cases. Although most HSIL and ASC-H smears contained 3 or more groups of G/TZs, absence of G/TZ did not correlate statistically with undetected AIN II/III lesions. Thus we do not consider the presence of G/TZ as essential for adequacy, a situation analogous to cervical Pap smears ,21,22. IOn review of the morphological features of AIN lesions in cytology smears, we noticed some salient features. Dysplastic cells were the most reliable indicators of ASIL/AIN. Typical koilocytes were infrequent, observed in only 17% of SILs, a finding previously observed by Darragh et al  who repoIn ASC-H and HSIL, high grade squamous cells are usually small, found as single cells or small sheets admixed with mildly dysplastic cells and atypical parakeratotic cells. Careful scrutiny is required to not miss these high grade lesions.Our experience with anal cytology also indicates that other infectious agents are rarely diagnosed in anal smears. Candida was present in one case. Herpes or trichomonads were not seen.To summarize, liquid based anal smears provide a sensitive method for screening populations at increased risk for ASIL but have a low specificity for predicting the severity of the lesion. Patients with cytologic diagnosis of ASC-US and LSIL have a significant risk of having HSIL and should be recommended for high resolution anoscopy with biopsy.The author(s) declare that they have no competing interests.SA participated in the acquisition, analysis and interpretation of data and helped to draft the manuscript.AEW participated in its design, in the acquisition, analysis and interpretation of data and helped to write the manuscript.PT participated in its design, in the acquisition, analysis and interpretation of data and helped to write the manuscript.SB conceived of the study, participated in its design, in the acquisition, analysis and interpretation of data and helped to write the manuscript.All authors read and approved the final manuscript.


The Exploratorium in San Francisco offers museum visitors the opportunity to use and manipulate state-of-the-art microscopes to visualize an array of living specimens The Exploratorium, based in San Francisco, is a “hands on” science museum filled with interactive science and art exhibits, as well as a laboratory for the research and development of innovations in science education. In the summer of 2004, the Exploratorium launched the most ambitious microscope facility ever created for general public use. This initial phase of the project gives visitors the ability to image living specimens, as well as control the instruments themselves. Visitors can select among various specimens, move over them, change the magnification and focus, and, where appropriate, change the lighting to illuminate through the specimen, or use reflected light and fluorescence to dramatically change how it looks. They can image and explore tiny zebrafish embryos from the first stages of development to two-day-old fry with beating hearts and circulating blood cells, as well as a host of other organisms and cells from crawling amoebas to human blood cells.Below the surface, all living things share common features. The primary goals of this facility are to open a door on the wonder of the microscopic world to a diverse range of museum visitors and allow them to explore it, and to allow them to make connections to science and biomedical research. By empowering visitors with the instruments to explore this unfamiliar universe, the Exploratorium seeks to recreate some of the excitement and wonder that the earliest researchers found as they discovered another world all around them .The light microscope falls amongst the greatest inventions of human history. Images from it in the 17th century literally revolutionized our understanding of life, providing first-hand evidence of a previously unseen or unsuspected world of organisms and cells all around us. This knowledge profoundly shaped our view of life, and of our placement in the universe. Robert Hooke used a primitive early microscope to see the walls between cells in a piece of cork , and Anton van Leeuwenhoek's simple scope revealed a previously unknown world of microorganisms living inside his own mouth. Swimming sperm were observed in semen, changing our fundamental understanding of conception. Since that time our world has become populated by marvelously beautiful and intriguing images and movies created by scientists using precise lighting and optics. Most recently, computer-controlled image-capturing techniques and digital technologies capture events and processes too small, slow, or fast for our unaided eyes to see.Van Leeuwenhoek's first microscopes were probably about as powerful as a simple water-drop scope see ; he usedBringing a first-hand, high-quality microscope experience to a wide range of visitors and students has been a major challenge for many science museums and classrooms.In the expanded, frenetic classroom of the science museum exhibition floor, microscopes themselves are too delicate and precise for operation without assistance and supervision: the optics can be easily damaged, all but the most robust of specimens are easily destroyed, and the user interface with lighting, positioning, and focus compose a world unfamiliar to most novice users. Over the years, we had collected our share of failed and broken microscopes in various projects. For these reasons most microscopes in museums have been armored, stripped-down, single-magnification instruments.Problems related to the operation of the microscope hardware, however, represent only some of the challenges created by bringing uninitiated visitors to the microscopic world. The experience has to be repeatable. It has to be engaging. It has to be both simple and complex. It needs to work for individuals and groups. It needs to support investigation as well as provide essential information.An array of research scientists joined us as we began work to redefine and renew our existing presentation on biology, most notably Christian Sardet, a cell biologist with a passion for visualization imagery. During the summer and fall of 1999, we engaged in a series of conversations directed at defining and developing a publicly accessible microscope imaging station, and the more we explored, the more technically feasible the project became.At the same time, research-grade microscopes with high-quality optics underwent a revolution of their own. Many manufacturers planned to introduce fully automated, computer-controllable microscopes in their next models, and the prospects of having an off-the-shelf remotely controllable microscope seemed to solve a major hardware problem. It seemed as if software programs might be used to address many other user interface issues. In 2000, we applied for and received funding from the National Institutes of Health  and from the David and Lucile Packard Foundation to support the development of a microscope imaging facility.Seven months later we obtained the first microscope and its associated imaging equipment. But the software to control the microscopes proved dauntingly problematic. Programs that worked well in the laboratory proved difficult to adapt for our educational uses. Instead, we wrote our own programs to control stage movement (for specimen positioning), focus, specimen selection, magnification, and lighting. Each of these controls needed to be selectable and limited so that visitors might operate the microscope within a defined range.Caenorhabditis elegans attracted attention with its earthworm-like movements. Overall, these biomedically relevant specimens provided a treasure trove of potential educational activities for visitors.On a parallel track, with the generous help of numerous biomedical researchers and their laboratories across the United States, we explored specimens for their visitor attraction and interest. Not surprisingly, we found that familiar structures and organisms provided the best entry points. A zebrafish embryo with a beating heart, circulating blood, and twitching tail movements rated more popular than zebrafish or sea urchin embryos at earlier stages of development. The tiny transparent roundworm Given a range of wonderfully attractive specimens and potential activities, we then aimed to create an educational experience that combined the observable features in the specimens, supporting information, and further activities to be chosen as desired by the visitor. To achieve this, we adopted a multimedia approach . The incVisitors can pick and choose specimens of their choice, refine their selection, change magnification, and engage in suggested activities at their own pace with integrated appropriate information. Individuals and groups engage with the microscope imagery and companion information for relatively long periods of time, ranging from several minutes to upwards of 20 minutes, a long time for a museum exhibit. To expand the use of the microscope imagery beyond that of one or two visitors, we incorporated large plasma-screen presentations in to the Microscope Imaging Station facility.Escherichia coli) and a roundworm (C. elegans) and found that the genetically modified organisms emitted an eerie green glow under certain conditions. Other scientists built on this technique to create a powerful tool that makes hidden structures and processes easier to study. The coupling of vital staining with the green fluorescent protein gene allows scientists to observe events inside developing cells and detect the presence of diseased structures and environmental toxins with extreme sensitivity. This newly created sensitivity has sparked new insights and discoveries, re-revolutionizing the capabilities of the light microscope.In addition to using some of the latest devices in microscope technologies, the Imaging Station also provides a window on revolutionary research techniques. For example, in 1994, Columbia University's Martin Chalfie inserted a gene for a fluorescent jellyfish protein into a bacterium (http://www.exploratorium.edu/imaging_station).At the Imaging Station, visitors can take advantage of this technique to observe specific types of cells—such as the brain cells, sex cells, or muscle cells of roundworms. Visitors can also peer inside the developing embryo of a tropical zebrafish whose circulatory cells have been made visible by the protein made from the transplanted green fluorescent protein gene. In the immediate future, visitors will be able to closely examine living human blood cells and fruit flies that researchers use to study the genetics of a wide range of human disorders. Over the next year, we intend to add a major component on mouse stem cells and the process of differentiation. At the Imaging Station, visitors have access to seamless video footage of events that take place in a fraction of a second or occur slowly over weeks or months . Using tVideo S1Collected at the Microscope Imaging Station Many of the cells and organisms found in these movies are available via visitor-accessible microscopes on a daily basis. At the station, visitor-directed, microscope-based observations form the basis for informal education on basic and biomedically relevant research. Video microscopy and production by Kristina Yu.(5.4 KB MOV).Click here for additional data file.Materials. You'll need the clear plastic part of a CD case; if you can't find one, any thin sheet of rigid, clear plastic will work. Remove any paper or packaging so that you can see through the plastic. What to do. (1) Put the object you want to examine—a bug, a tiny leaf or plant, the small lettering on a coin or newspaper—on a flat surface. (2) Put one large drop of water on the center of the clear plastic. (An eyedropper makes this easy.) (3) Pick up the plastic and hold it horizontally so that the water drop is directly over the object. Move the plastic slowly back and forth to center the object, and up and down to focus. The water will form a curved shape that magnifies the object!


With chronic infection, hepatitis C virus (HCV) RNA can be detected in B cells and associated with B-cell disorders, but these are not well defined.+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells were quantified by flow cytometry using cryopreserved peripheral blood mononuclear cells of 24 hemophilic patients in each of five age-matched groups .The relationship between HCV infection and lymphocyte subpopulations was evaluated rigorously in 120 asymptomatic hemophilic patients, randomly selected from a prospective cohort study. CD4+ and increased CD8+ T cells. Irrespective of HIV, patients with chronic HCV infection had approximately 25% fewer CD19+ B cells than those without chronic HCV infection.As expected, patients with HIV had significantly reduced CD4These data support the hypothesis that asymptomatic patients with chronic HCV infection have an altered B-lymphocyte population. The natural history of hepatic C virus (HCV) infection may include B-cell diseases. HCV can be detected in peripheral blood mononuclear cells (PBMCs) and may also replicate in these cells -3. Type + T cells  with a lower limit of sensitivity of 200,000 genome equivalents/mL, which is 31,746 international units (IU)/mL. HIV-1 viral load was determined with the HIV Amplicor Monitor .2 until testing, was analyzed by flow cytometry for proportions of CD4+, CD8+, CD19+, and CD56+ cells. In a biological safety cabinet, vials were thawed in a 37°C water bath with agitation. To each, 300 units of DNase I  was added. The contents were transferred to a 15 ml polypropylene tube. Thawed cells were slowly diluted with RPMI-1640 media supplemented with 20% fetal calf serum, mixed frequently, then pelleted at 1000 rpm in a Sorvall RT6000 refrigerated centrifuge. The liquid was decanted. Cells were mixed in residual volume by gentle shaking, washed a second time with 4.0 ml of thawing media, then resuspended in 2.0 ml calcium- and magnesium-free Dulbecco's phosphate buffered saline (PBS) containing 4% heat-inactivated human AB serum to block high affinity Fc receptors. After 10 minutes the cells were pelleted in the centrifuge, then resuspended in 0.6 ml of PBS/2% BSA. 100 μl of cell suspension was added to each of five 12 mm × 75 m polypropylene tubes containing three-color combinations of fluorescently tagged monoclonal antibodies . The lymphocyte gate was defined with light scatter properties and CD45/CD14/CD3. A Coulter XL flow analyzer was used with stops set to collect 5,000 CD3+ lymphocytes in each sample, if possible. Listmode files were analyzed offline using Coulter System II software (version 3.0). Quality control criteria included purity of gated lymphocytes; percentage recovery of lymphocytes in the gate; reproducibility of CD3 between tubes (range <4%); and sum of CD3+, CD3-/CD19+, CD3-/CD56+ (range 90–110%).A vial of 5 million cryopreserved PBMCs, stored in vapor phase LNThe primary analysis was performed on proportions, rather than absolute levels, of each PBMC subset, to reduce variance that results from counting total lymphocytes in peripheral blood . The Kru6 IU/mL) than with HCV only . Among 48 HIV-infected subjects, 6 (25%) with cleared and 7 (29%) with chronic HCV had clinically defined AIDS (P = 0.75) when their PBMCs were collected for this study. HIV viral load did not differ between the nine with cleared and the eight with chronic HCV who were tested on the date PBMCs were isolated (P = 0.89). Ten of 120 subjects were chronically infected with hepatitis B virus, and the prevalence of chronic HBV infection did not differ among 5 groups (P = 0.12). Only two subjects, both HIV-infected but one with cleared HCV infection and one with chronic HCV infection, had developed ascites, a manifestation of hepatic decompensation, at the time that their PBMCs were collected for this study.Of the 120 patients, 78 had hemophilia A (factor VIII deficiency). Because patients with hemophilia A generally required more intensive clotting factor replacement therapy as well as product (Factor VIII concentrate) that was more infectious for HIV than was Factor IX concentrate used for patients with hemophilia B, hemophilia A was significantly more common in the two groups with HIV co-infection (n = 19 with chronic HCV and n = 21 with cleared HCV) than the three groups without HIV co-infection . By design, the five groups had similar ages  or chronic HCV .As expected, subjects with HIV had lower proportions of CD4+ T cells differences mirrored those of CD4+ T cells (Table + T cells whether they had cleared HCV (52.3% vs. 26.0%) or chronic HCV . Regardless of HIV, subjects with cleared and with chronic HCV had similar proportions of CD8+ T cells (P∃0.30).CD8ls Table . Subject+ B cells differed significantly among the five groups . CD19+ B cells also were lower, albeit not significantly, with chronic HCV than with cleared HCV infection in both HIV-uninfected  and HIV-coinfected  groups. CD19+ B-cell levels did not differ between uninfected subjects and those with cleared HCV infection (P = 0.63).Proportions of CD19+ B-cell proportions did not correlate with HCV viral load .CD19+ NK cells did not differ significantly among the five groups /15.5 = 0.26]. We found subjects with chronic HCV without HIV co-infection had a reduction of the same magnitude, albeit of marginal statistical significance. HIV infection was more frequent with hemophilia A than with other coagulation disorders [Among HIV-infected subjects, we found that CD19isorders . OverallInfection of B cells by HCV is controversial. Both B cells and hepatocytes express CD81, a possible receptor for HCV . If HCV + NK cells with HCV infection, but this does not negate possible impairment of NK cell function with HCV infection. In vitro, binding of HCV E2 to CD81 inhibits the functions of cross-linked NK cells [+ NK cell proportion was reduced among our subjects who cleared HCV despite HIV co-infection, an observation that has no ready explanation and may have appeared by chance.We found no differences in proportions of CD56NK cells ,15. CD56+ and CD8+ T cells with HIV infection. These findings, our careful flow cytometry methods, and our rigorous study design, with frequency matching on age and random selection of subjects from a large, well characterized cohort, probably offer a reliable "snapshot" of PBMCs in hemophilic patients infected with HCV alone or co-infected with HIV.Our study is limited by its cross-sectional design and analysis of PBMCs collected long after primary HCV and HIV infections had occurred. We cannot define sequential PBMC changes in relation to critical virologic events, such as spontaneous clearance of HCV. Furthermore, the cells that we detected in cryopreserved PBMCs may not be representative of those in fresh PBMCs. Still, we did observe the expected associations of CD4+ or CD8+ T cells, although this does not negate the likely functional importance of HCV-specific T-cell subpopulations in clearance of HCV [+ B-cell levels. Further studies need to clarify the temporal sequence of B-cell changes with HCV chronicity and associated clinical conditions.We found no association of cleared or chronic HCV infection with altered levels of CD4e of HCV ,17. SubjM. Zhang, J.J. Goedert, T.R. O'Brien, P.S. Rosenberg, C.S. Rabkin, E.A. Engels, D. Whitby ; M.E. Eyster ; B. Konkle ; M. Manco-Johnson ; D. DiMichele, M.W. Hilgartner ; P. Blatt ; L.M. Aledort, S. Seremetes ; K. Hoots ; A.L. Angiolillo, N.L.C. Luban ; A.Cohen, C.S. Manno ; C. Leissinger ; G.C. White II ; M.M. Lederman, S. Purvis, J. Salkowitz ; C.M. Kessler ; A. Karafoulidou, T. Mandalaki ; A. Hatzakis, G. Touloumi ; W. Schramm, F. Rommel ; P. de Moerloose ; S. Eichinger ; K.E. Sherman ; and B.L. Kroner ."The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government."The author(s) declare that they have no competing interests.MZ and JG conceived of the study and drafted the manuscript. MZ performed the random sampling and statistical analyses. JG provided advice on the sampling and statistical analyses, obtained funding, managed the parent cohort, and supervised the virologic testing. TOB provided advice on the design. WK provided advice on and performed the FACS analyses. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by repeated inhalations of finely dispersed organic particles or low molecular weight chemicals. The disease is characterized by an alveolitis sustained by CD8(+) cytotoxic T lymphocytes, granuloma formation, and, whenever antigenic exposition continues, fibrosis. Although it is known that T-cell migration into the lungs is crucial in HP reaction, mechanisms implicated in this process remain undefined.Using flow cytometry, immunohistochemistry, confocal microscopy analysis and chemotaxis assays we evaluated whether CXCL10 and its receptor CXCR3 regulate the trafficking of CD8(+) T cells in HP lung.in vitro migratory capability in response to CXCL10. Alveolar macrophages expressed and secreted, in response to IFN-γ, definite levels of CXCL10 capable of inducing chemotaxis of the CXCR3(+) T-cell line. Interestingly, striking levels of CXCR3 ligands could be demonstrated in the fluid component of the BAL in individuals with HP.Our data demonstrated that lymphocytes infiltrating lung biopsies are CD8 T cells which strongly stain for CXCR3. However, T cells accumulating in the BAL of HP were CXCR3(+)/IFNγ(+) Tc1 cells exhibiting a strong These data indicate that IFN-γ mediates the recruitment of lymphocytes into the lung via production of the chemokine CXCL10, resulting in Tc1-cell alveolitis and granuloma formation. Hypersensitivity pneumonitis (HP) is an interstitial lung disease (ILD) caused by the inhalation of and sensitization to a variety of environmental organic antigens. The immune mediated nature of the disorder is testified to by the characteristic sequel of events taking place in the lung after antigenic inhalation: an acute pulmonary neutrophilia occurs early followed by an interstitial T-cell infiltration of CD8 T-cell showing a limited expression of the T-cell receptor . (Mig/CXCL10), interferon-inducible T-cell α-chemoattractant (I-TAC/CXCL11) bind to the CXCR3 receptor molecule which is expressed by activated T lymphocytes and natural killer cells [Saccharopolyspora rectivirgula (SR) and successive granuloma formation [A number of data point to chemokines as orchestrators of inflammatory disorders which are characterized by a massive accumulation of immunocompetent cells within affected organs, including the lung . Chemokier cells ,8. We haer cells ,10, provin vitro migration of CXCR3(+)T cells. The ligand CXCL10 can be detected in pulmonary macrophages and is released by these cells.Using immunohistochemical studies of tissue sections and a flow cytometry evaluation of cells recovered from the bronchoalveolar lavage (BAL), we studied the role of CXCR3/CXCL10 interactions in the regulation of T-cell migration into the lung of patients with hypersensitivity pneumonitis. We have shown that CXCR3 is expressed by T cells accumulating in the lower respiratory tract of patients with this hypersensitivity disorder. In addition, we found that signalling of CXCR3 with CXCL10 induces the 12 HP patients were included in the study . The majority of the patients had farmer's lung disease (10 patients); 1 patient had bird fancier's lung, 1 patient had mushroom worker's lung. The following criteria for HP diagnosis were used: a) history of exposure to HP antigens, b) a symptomatic acute episode with chills, fever, cough, breathlessness 4 to 8 hours after exposure to specific antigens, c) radiological features (mainly diffuse reticular pattern) and/or a functional pattern of interstitial lung disease, and d) evidence of antibodies against S. rectivirgula in all except one case (bird fancier's lung). Each patient underwent bronchoscopy for transbronchial biopsy (TBB) and BAL analysis. BAL was performed according to the technical recommendations and guidelines for the standardization of BAL procedures [Five healthy controls were selected . They showed normal physical examinations, chest X-rays, lung function tests and BAL cell numbers.+ lymphocytes were removed using high-gradient magnetic separation columns  [Alveolar macrophages (AMs) and T cells were enriched from the BAL cell suspensions by rosetting with neuraminidase-treated SRBC followed by F/H gradient separations and residual CD3Germany) . FollowiThe commercially available conjugated or unconjugated mAbs used belonged to the Becton Dickinson and PharMingen series and included: CD3, CD4, CD8, isotype matched controls. Anti-IL-4 and anti-IFNγ mAbs were purchased from PharMingen . Purified rabbit anti-human CXCL10 polyclonal antibody  and anti-hCXCR3 mAb (R&D Systems Inc) were also used.Open lung biopsies from 8 patients with clinical and histological diagnosis of hypersensitivity pneumonitis were studied by immunohistochemistry for the immunophenotype characterization of inflammatory cells and for CXCR3 and CXCL10 expression.Immunohistochemistry for the characterization of inflammatory infiltrate was carried out using the following antibodies : CD45 (1:20), CD43 (1:40), CD45RO (1:100), CD20 (1:100), CD3 (1:50), CD68 (1:50), CD4 (1:100), and CD8 (1:100). The immunoreaction products were developed using the avidin-biotin-peroxidase complex method. Immunostaining for CXCR3 was performed as previously described. Briefly, after the microwave antigen retrieval procedure and neutralization of endogenous peroxidase activity, the slides were incubated with primary antibody for 1 hr in a humidified chamber at 37°C (anti-hCXCR3 mAb 1:100). Immunoreactivity was detected using biotinylated secondary antibodies incubated for 45 min followed by a 30 min incubation with avidin-peroxidase and visualized by a 7 min incubation with the use of 0.1% 3,3'-diaminobenzidene tetrahydrochloride as the chromogen. Parallel control slides were prepared either lacking primary antibody or lacking primary and secondary antibodies, or stained with normal sera to control for background reactivity. The intensity of antibody staining was classified in three groups: strong, weak, negative.Paraffin sections were prepared for immunofluorescent labelling. Briefly, primary antibodies against CD3 and CXCR3  and secondary antibodies (goat anti-mouse IgG and donkey anti-goat IgG) conjugated with TEXAS red or ALEXA 488 (Sigma) were used. Double labelling using both antibodies on the same section was performed. Primary antibodies and secondary antibodies were incubated for 1 h at room temperature. Nuclear staining was carried out with DAPI (Sigma) in PBS. Slides were stored at 4°C and analysed within 24 h. As a control, the primary antibody was omitted.Immunofluorescence was evaluated with a confocal microscopy , We used an argon laser at 488 nm in combination with a helium neon laser at 543 nm to excite the green (CD3) and red (CXCR3) fluorochromes simultaneously. Emitted fluorescence was detected with a 505–530 nm band pass filter for the green signal and a 560 nm long pass filter for the red signal. Images were analyzed using the Adobe Photoshop 7.0 program.® analyzer (Becton Dickinson), and data were processed using the Macintosh CELLQuest software program (Becton Dickinson). The expression of cytoplasmic cytokine was evaluated following permeabilization of cell membranes using 1:2 diluted PermeaFix  for 40 min. After permeabilization procedures anti-IL-4, anti-IFN-γ and anti-CXCL10 antibodies were added. Since pulmonary cells bear cytoplasmic cytokines in a unimodal expression pattern, indicating that the entire cell population exhibits relatively homogeneous fluorescence, the percentage of positive cells does not represent the most accurate way of enumerating positive cells. Mean fluorescence intensity (MFI) was used to compare the positivity of these specific antigens on different cell populations. To evaluate whether the shift of the positive cell peak was statistically significant, the Kolmogorov-Smirnov test for analysis of histograms was used according to the Macintosh CELLQuest software user's guide (Becton Dickinson).The frequency of BAL cells positive for the above reagents was determined by overlaying the flow cytometry histograms of the samples stained with the different reagents as previously reported . Cells wFor immunofluorescence analysis, control IgG1 and IgG2a and IgG2b were obtained from Becton-Dickinson; control rat antiserum consisted of ascites containing an irrelevant rat IgG2b; control rabbit antiserum consisted of rabbit IgG (purified protein) purchased from Serotec ; goat-anti-rabbit IgG and goat F(ab')2 anti-rat IgG were obtained from Immunotech .Each PCR product was analysed and quantitated by Bio-Rad's Image Analysis System Gel Doc using Quantity One software . Briefly, the images of the gels were acquired from the Gel Doc system densitometer and saved in digitised forms to perform volume analysis. The intensity of each band was differentiated by the intensity of the background, whose value was subtracted from each individual band and the resulting PCR product value was expressed in mm*mm*intensity of the pixels of the specific band in the gel.6/ml) were isolated from the BAL of HP patients, resuspended in RPMI medium and cultured for 24 hr in 24-well plates at 37°C in 5% CO2. In separate experiments AMs were stimulated with IFN-γ (100 U/ml), PMA (10 ng/ml) and LPS . Following the incubation period, supernatants were harvested, filtered through a 0.45 μm Millipore filter and immediately stored at -80°C. At the end of the culture time AM viability was always greater than 95%. Chemotactic activity of supernatants was determined as reported below.To verify the ability of AMs to release CXCL10, AMs . The chamber is made of two sections: different chemotactic stimuli were loaded in the bottom section while cells were added in the top compartment. Polyvinylpyrrolidone-free polycarbonate membranes with 3 to 5 μm pores   and coated with fibronectin were placed between the two chamber parts. Only the bottom face of filters was pretreated with fibronectin; the fibronectin pretreatment maximizes attachment of migrating cells to filters, avoiding the possibility that they may not adhere. Using this procedure in preliminary experiments we demonstrated that only a trivial number of cells may be recovered in the bottoms of the wells. To avoid the shedding of fibronectin, fibronectin-treated filters were extensively washed. In preliminary experiments, fibronectin-treated filters did not induce spontaneous chemotaxis in absence of chemokines.6 cells/ml T cells or CXCR3-/+ cells resuspended in RPMI 1640 were added to the top wells. The chamber was incubated at 37°C with 5% CO2 for 2 hrs. The membranes were then removed, washed with PBS on the upper side, fixed and stained with DiffQuik . Cells were counted in three fields per well at 800× magnification. All assays were performed in triplicate. In blocking experiments, cell suspensions were preincubated before chemotaxis assay for 30 min at 4°C with anti-human CXCR3 mAb at a concentration of 20 μg/ml.To evaluate the migratory properties of pulmonary T lymphocytes rhIP-10/CXCL10 (200 ng/ml) were used. The CXCR3- and CXCR3+ cell lines  were used as negative and positive controls. 30 μl of chemokines or control medium were added to the bottom wells, and 50 μl of 5.0 × 10The CXCR3(-) and CXCR3(+) cell lines were also used to evaluate both the chemotactic activities of macrophage supernatants and the fluid component of BAL samples. Supernatants from cell cultures and the fluid components of BAL were obtained as reported above and used undiluted; different concentrations of CXCL10 were utilized as a positive control. Chemotactic assays were performed as reported above. In blocking experiments, anti-CXCL10 was added to the cell supernatants before chemotaxis assay at a concentration of 20 μg/ml.Data were analysed with the assistance of the Statistical Analysis System. Data are expressed as mean ± SD. Mean values were compared using the ANOVA test. A P value <0.05 was considered as significant.In all cases typical pathological examination showed features with poorly formed non-necrotizing granulomas and widespread thickening of the alveolar walls by a diffuse lymphocytic infiltrate. Pleural lymphoid aggregates were seen in a few cases and pleural lymphoid aggregates were seen in a few samples.Diffuse interstitial lymphocytic infiltrates were characterized by an accumulation of T cells and a few B-lymphocytes. Sub-pleural and peri-bronchiolar nodules consisted mostly of T lymphocytes mainly represented by CD8 cytotoxic T lymphocytes which strongly stained for CXCR3 in all cases Figure  and 1B. Confocal microscopy analysis of lung biopsies confirmed that lymphocyte infiltrates were formed by T cells coexpressing CXCR3  cell line; the CXCR3(-) cell line did not migrate in the presence of supernatants (data not shown). The addition of an anti-CXCL10 neutralizing antibody inhibited chemotactic activities of supernatants. The inhibitory activity shown by the neutralizing antibody was not complete, suggesting that other CXCR3 ligands (CXCL9 and CXCL11) are likely to be present in supernatants.in vivo in the lung microenvironment, the fluid component of BAL obtained from 10 HP patients was evaluated for chemotactic activity on CXCR3(+) cell lines  T cells might play a role not only in the pathogenesis of T-cell alveolitis but also in favouring granuloma formation since T cells surrounding the macrophagic core of the HP granuloma expressed this chemokine receptor. This mechanism is likely to be shared by various ILDs since we and others have previously demonstrated the presence of a significant infiltrate of CXCR3(+) Th1 cells in other ILDs characterized by T cell alveolitis and granuloma formation, such as sarcoidosis and tuberculosis ,14.Saccharopolyspora rectivirgula (SR)  [in vivo role of IP-10/CXL10 in preventing or favouring pulmonary fibrosis in HP before proposing this strategy.Our data provide definitive confirmation of the recent findings obtained in an animal model of IFN-γ-knockout (GKO) mice exposed to the particulate antigen atients) . While WThe relationship between CXCL10 release and the local production of other chemokines involved in HP immune response is another important aspect that deserves further investigation. In a murine model it has recently been shown that monocyte chemotactic protein-1 (MCP-1/CCL2) is increased in the BAL of mice challenged with SR . Furtherin vivo in HP lungs.Thus, our findings and the data of Pardo et al.  suggest Our findings clearly indicate the effects of CXCR3/CXCL10 interactions on hypersensitivity reaction to SR antigens. Considering the importance of CD8 T cells in mediating granuloma formation and lung damage, further studies are needed in animal models to explore the therapeutic potential of CXCR3 antagonists with the ultimate goal of offering new clues for immune intervention in subjects continuously exposed to inhaled antigens and thus at risk of developing HP-related lung fibrosis.AM: alveolar macrophage; BAL: bronchoalveolar lavage; CXCR3: Receptor 3 for CXC chemokines; HP: Hypersensitivity pneumonitis; IP10/CXCL10: IFN-γ-inducible protein-10; I-TAC/CXCL11: interferon-inducible T-cell αlfa-chemoattractant; Mig/CXCL10: monokine induced by IFN-γ; TBB: transbronchial biopsy.CA: planned the experimental design, coordinated the research group and drafted the manuscriptFC: participated in the study design and in immuohiostochemical analysisVP: participated in the study design and in the bronchoalveolar lavage executionGM: participated in the study design and in the clinical evaluation of the patientsMF: participated in the study design and performed migration assays studiesMM: participated in the study design and performed cellular studiesAC: participated in the study design and performed flow cytometry studiesIB: participated in the study design and performed flow cytometry studiesRZ: participated in the study design and helped the draft of the manuscriptLT: participated in the study design and helped the draft of the manuscriptGS: participated in the study design and coordination of the research group


Saccharomyces cerevisiae, commonly known as baker's yeast, measures just 2 microns—it takes about 4 billion to fill a teaspoon. But as a eukaryote (its cells have nuclei), its genes function in much the same way a human's do. For a gene to function, its DNA sequence must first be transcribed into RNA , whose sequence can then be translated into a specific string of amino acids to form the unique protein that the gene encodes.The single-celled The population of mRNA transcripts in each cell (its “transcriptome”) is dynamic—the genome uses its vocabulary of genes to write an ever-evolving script for the cell as its life unfolds and its environment changes. By binding to specific sequences of DNA, proteins called transcription factors process signals from the cell's sensory and information-processing systems to control which genes are transcribed in each cell, under what conditions, and at what rate. While the steps and regulatory programs that govern gene expression at this level are reasonably well known, much less is known about the orchestration of the later steps in the gene expression program—where in the cell each mRNA molecule goes when it leaves the nucleus, at what rate and under what conditions it is translated into protein, and how long it survives.RNA-binding proteins (RBPs) have been implicated in diverse aspects of post-transcriptional gene regulation. Hundreds of RBPs are encoded in the eukaryotic genome, but because few have been studied in detail and few of their mRNA targets are known, the nature and extent of an RBP-mediated post-transcriptional program has been obscure. Now a systemic analysis of a specific family of RBPs and their mRNA targets in yeast by André Gerber, Daniel Herschlag, and Patrick Brown, of Stanford University, suggests that such a program may exert detailed control over the life history of every mRNA. By selectively binding and regulating specific classes of mRNAs, RBPs may provide a mechanism to coordinate the collective fate of these transcripts and serve as an integral part of the global transcriptome.Gerber, Herschlag, and Brown focused on the binding targets of a family of RBPs called Pumilio-Fbf (Puf) proteins, which are defined by the presence and configuration of an amino acid domain that mediates RNA-binding. Little is known about the physiological function of the five yeast Puf proteins the researchers studied here . After using “affinity tags” to snag each of the five Puf proteins from yeast cells, together with their bound mRNA targets, the researchers identified the associated mRNAs with microarray analysis. They found more than 700 mRNAs bound by at least one Puf protein, with each Puf RBP targeting a distinct group of mRNAs. The group of mRNAs associated with each Puf protein turned out to encode proteins with strikingly similar functions and locations in the cell. Many of the mRNA sets encode proteins that reside in the same cellular location, are part of the same protein complexes, or act in the same signaling pathway. Some Puf proteins target mRNAs that encode membrane proteins while others preferentially bind to mRNAs that encode proteins involved in cell division. The most pronounced bias occurs with Puf3p, which overwhelmingly binds mRNAs that encode proteins destined for the mitochondria, the cell's power generators.This selective tagging of functionally related mRNAs by specific RBPs suggests a mechanism for coordinated global control of gene expression at the post-transcriptional level. Just as transcription factors regulate transcription by binding to specific DNA sequences, RBPs may mediate regulation of the subcellular localization, translation, and degradation of the set of specific mRNAs they target. Noting the striking themes in the subcellular localization of the proteins encoded by the mRNAs bound by each Puf protein, Gerber, Herschlag, and Brown propose that RBPs may play important roles in the subcellular localization and efficient assembly of protein complexes and functional systems by ensuring that the location in the cell at which mRNAs are translated “is not left to chance.” Since the number of RBPs encoded in eukaryotic genomes approaches that of transcription factors, the regulatory program that controls the post-transcriptional fate of mRNAs—their localization, translation, and survival—may prove to be nearly as diverse and complex as the regulation of transcription itself.


A mutation in the abnormal limb mutant 5 (ALI5) mouse in the region coding for the hydrophobic ridge loop 3 (HRL3) of the phospholipaseCγ2 (PLCγ-2) gene, corresponding to human PLCγ-2 exon 27, leads to acute and chronic inflammation and granulomatosis. For that reason, we screened exons 11, 12 and 13 coding for the hydrophobic ridge loop 1 and 2  and exon 27 of the PLCγ-2 protein by single strand conformation polymorphism (SSCP), sequencing and PCR/ restriction fragment length polymorphism (RFLP) analyses. In addition, we screened indirectly for disease association via 4 microsatellites with pooled DNA in the PLCγ-2 gene.Wegener Granulomatosis (WG) is a multifactorial disease of yet unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis. Analyses of candidate genes revealed several associations, Although a few polymorphisms in these distinct exons were observed, significant differences in allele frequencies were not identified between WG patients and respective controls. In addition, the microsatellite analyses did not reveal a significant difference between our patient and control cohort.PLCγ-2 gene in the pathogenesis of WG in our case-control study.This report does not reveal any hints for an involvement of the  Staphylococcus aureus is a risk factor for disease exacerbation in WG ]]StaphyloPLCγ-2, was proposed on the basis of a novel animal model system. A mutation in the abnormal limb mutant 5 (ALI5) mouse + status Pooling of DNA was performed as reported before . PatientIn this study 3 intragenic microsatellites as well as one in the immediate vicinity of the gene were included  and 50 ng DNA.For PCR we used the 'tailed primer PCR' as described before . For ampElectrophoreses were run using ABI standard protocols. Raw data were analyzed by the Genotyper software (ABI) producing a marker-specific allele image profile )7]). AIPStatistics for comparisons of allele frequencies of patients and controls was performed as described before [ and 7]]]7]]. CasExons 11, 12, 13 and 27 were analysed by the SSCP method. PCR was performed using oligonucleotides reported before (, exon 27PJ and SW carried out the molecular genetic studies, performed the statistical analysis and drafted the manuscript. PY participated in study design and helped to draft the manuscript. EC and WLG provided the samples and performed diagnostics of the patient group. JTE conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Metallothionein (MT) protein expression deficiency has been implicated in carcinogenesis while MT over expression in tumors is indicative of tumor resistance to anti-cancer treatment. The purpose of the study was to examine the expression of MT expression in human renal cell carcinoma (RCC) and to correlate MT positivity, the pattern and extent of MT expression with tumor histologic cell type and nuclear grade, pathologic stage and patients' survival.The immunohistochemical expression of MT was determined in 43 formalin-fixed and paraffin-embedded RCC specimens, using a mouse monoclonal antibody that reacts with both human MT-I and MT-II. Correlation was sought between immunohistochemical  and clinico-pathological data .Positive MT staining was present in 21 cases (49%), being mild/moderate and intense in 8 and 13 cases, respectively. The pattern was cytoplasmic in 7 cases and was both cytoplasmic and nuclear in 14 cases. MT expression in a percentage of up to 25% of tumor cells (negative MT staining included) was observed in 31 cases, in a percentage 25–50% of tumor cells in 7 cases, and in a percentage of 50–75% of tumor cells in 5 cases. There was no significant correlation of MT intensity of staining to histological type, stage and patients' survival, while it was inversely correlated to higher tumor nuclear grade. MT extent of staining did not correlate with histological type, nuclear grade, and pathologic stage while a statistically significant association was found with patients' survival.The inverse correlation between MT staining intensity and tumor nuclear grade in RCC suggests a role of MT in tumor differentiation process. Since extent of MT expression is inversely correlated with survival it may be possibly used as a clinical prognostic parameter. Metallothioneins (MTs) were firstly discovered by Margoses and Valle in 1957  documentMT-I and MT-II isoforms are usually expressed in low levels, but are inducible by a variety of metal ions, hormones, inflammatory cytokines and xenobiotics -12. InduIn organs such as kidney, colon and liver, normally implicated in metal ions homeostasis, MT protein is apparently expressed. It would be of great scientific importance to elucidate the pattern of MT expression in tumors developed from these organs, since it could not only delineate the role of MT in carcinogenic transformation but could also provide prognostic information for patients' outcome. The aim of the present study was to examine the expression of MT in human renal cell carcinoma (RCC) and to correlate the MT positivity, the pattern and extent of MT expression with tumor histological cell type and nuclear grade, pathologic stage and patients' survival.Forty three consecutive patients, 31 men and 12 women, who underwent nephrectomy for RCC comprised the group of our study. Their age ranged from 33 to 85 years (mean age 59.6 ± 11.1). Tumors were histologically classified as clear cell type in 32 cases, papillary type in 2 cases, chromophobe cell type in 4 cases and sarcomatoid type in 5 cases. The 8, 2, 16, 6 and 9 tumors were pathologically staged as T1 or T2N0M0, T2N+M0, T3N0M0, T3N+M0 and T4N+M+, respectively as per the TNM classification . The graSections of 5 μm thickness were deparaffinized in xylene and rehydrated in graded alcohol series. To remove the endogenous peroxidase activity, sections were treated with freshly prepared 0.3% (v/v) hydrogen peroxide in methanol in dark, for 30 min, at room temperature. Non-specific antibody binding was then blocked using normal rabbit serum  diluted 1:5 in phosphate buffered saline (PBS), for 20 minute. A mouse (IgG1k) monoclonal antibody that reacts with both human MT-I and -II isoforms  was used in this study. The sections were then incubated for 1 hour, at room temperature, with the primary antibody diluted 1:50 in PBS. After three washes with PBS, sections were incubated for 30 minute at room temperature with rabbit, peroxidase conjugated, anti-mouse serum (Dakopatts) diluted 1:200 in PBS and rinsed three times with PBS. Sections were then incubated with swine, peroxidase conjugated, anti-rabbit serum (Dakopatts) diluted 1:100 in PBS and rinsed three more times with PBS. The resultant immune peroxidase complexes were developed in 0.5% (v/v) 3,3'-diaminobenzidine hydrochloride  in PBS containing 0.03% (v/v) hydrogen peroxide. Sections were counterstained with Harris' hematoxylin and mounted in gelatin (Sigma). Control slides included in MT immunostaining procedure consisted of specific tissues previously shown to express MT (lung cancer) as positive controls, whereas the primary antibody was replaced by PBS in the case of negative controls.The stained sections were independently assessed by the pathologists  without prior knowledge of the clinical data as previously described . SpecimeCorrelation between immunohistochemical data (MT intensity and extent of staining) and clinicopathological data  was assessed using the Chi-square test. The association of intensity and extent of MT immunohistochemical staining with survival was determined by comparing Kaplan-Meier survival curves constructed for different patient groups, and were compared using the log-rank test.Tubular cells but not glomeruli and interstitial cells of normal autologous renal tissue stained positive (both in the nucleus and the cytoplasm) for MT, although the intensity and extent varied significantly. Positive MT expression was prominent in 21 out of 43 cases (49%), while 22 out of 43 ones (51%) were MT negative. As far as the intensity of staining is concerned, low/moderate intensity was observed in 8 cases, while intense staining was evident in 13 cases. The pattern of positive MT immunostaining observed was either cytoplasmic  or cytoplasmic and nuclear . In certain cases of clear cell RCC membranous staining was also observed. Nuclear pattern of staining only, was not observed in any of the RCC cases examined . No statistically significant differences were also observed between MT intensity of staining and stage , or patients' survival  in the cases of RCC examined . However, no such correlation was found in the case of clear cell RCC .There was no significant difference between MT intensity of staining and the histological types of RCC cases examined , stage  and grade  of RCC cases examined. Statistically significant inverse correlation was found between MT extent of staining and patients' survival  (Figure Non statistical correlation was found among MT extent of staining and histological types (χMT expression has been observed in different types of human tumors ,22, inclet al [et al [Developing and adult kidneys consistently express MT-I and MT-II mRNA  and the et al , using tet al  used anol [et al  who usedet al [The cytoplasm and nucleus of normal and malignant cells may both express MT but there is no conclusive data on the functional significance of their subcellular distribution. The majority of the cases in our study expressed MT in both cytoplasm and nucleus, the expression being cytoplasmic only in one third of the cases. Analogous pattern has been reported in other studies -25 whileet al  pointed et al . The funet al , as MT iIn certain tumors MT over expression has been associated with unfavorable prognostic characteristics such as advanced stage and poor differentiation . In our MT concentrations might also prove useful in predicting the efficacy of a particular cancer treatment protocols. Several types of transformed cells enriched for MT have been shown to exhibit greater resistance to chemotherapeutic agents . In thisThe current data on the expression of MT in RCC cases examined emphasize the necessity to investigate larger numbers of patients with RCC comparing the staining profile of different MT isoforms with other clinico-pathological parameters and survival status of patients. Currently, it is unknown whether the presence of MT in renal carcinoma cells is related to the induction or inhibition of apoptosis or plays an active role in cell proliferation. Since cytokines may also induce MT expression and immunotherapy is the only, albeit with limited efficacy, available treatment for RCC, the intensity and extent of MT immunostaining should be studied in correlation to the immune status of RCC patients before and after immunotherapy. Thus, it will be possible to elucidate the potential role of MT in renal cell carcinogenesis, as well as its clinical usefulness as a tumor marker and as a tool for selecting patients for adjuvant immunotherapy.