External and internal factors are increasingly encouraging research funding bodies to demonstrate the outcomes of their research. Traditional methods of assessing research are still important, but can be merged into broader multi-dimensional categorisations of research benefits. The onus has hitherto been on public sector funding bodies, but in the UK the role of medical charities in funding research is particularly important and the Arthritis Research Campaign, the leading medical charity in its field in the UK, commissioned a study to identify the outcomes from research that it funds. This article describes the methods to be used.A case study approach will enable narratives to be told, illuminating how research funded in the early 1990s was (or was not) translated into practice. Each study will be organised using a common structure, which, with careful selection of cases, should enable cross-case analysis to illustrate the strengths of different modes and categories of research. Three main interdependent methods will be used: documentary and literature review; semi-structured interviews; and bibliometric analysis. The evaluative framework for organising the studies was previously used for assessing the benefits from health services research. Here, it has been specifically amended for a medical charity that funds a wide range of research and is concerned to develop the careers of researchers. It was further refined in three pilot studies. The framework has two main elements. First, a multi-dimensional categorisation of benefits going from the knowledge produced in peer reviewed journal articles through to the health and potential economic gain. The second element is a logic model, which, with various stages, should provide a way of organising the studies. The stock of knowledge is important: much research, especially basic, will feed into it and influence further research rather than directly lead to health gains. The cross-case analysis will look for factors associated with outcomes.The pilots confirmed the applicability of the methods for a full study which should assist the Arthritis Research Campaign to demonstrate the outcomes from its funding, and provide it with evidence to inform its own policies. Health research funding bodies are under increasing pressure to demonstrate the outcomes, or benefits, of the research that they fund -6. TradiThe onus hitherto has been on public sector funding bodies. There is a general recognition in the UK, however, of the importance of the role of the medical charities: they fund approximately one third of UK medical research – a level 'unparalleled elsewhere in the world and nor is it found in other areas of science' . TherefoThe purpose of this paper is to set out the aims of the study and the methods being adopted. In particular, it will show how an existing generic approach to the assessment of benefits from health research ,12 has bAttention is being drawn to the increasing scale of the burden of musculoskeletal conditions, and the associated costs, in various ways including through the establishment of the Bone and Joint Decade 2000–2010 and the recent collaborative report with the World Health Organization (WHO) . At the ARC is the leading medical charity in this field in the UK and one of the largest collection-based medical charities in the UK. The Research Outputs Database (ROD) records the funding acknowledgements on all UK biomedical papers contained on the citations indices of the Institute for Scientific Information . AnalysiWithin the general climate of an increased emphasis on the outcomes from research, four main objectives were specified for the particular study described in this article:• Review and document outcomes for ARC research grants• Illustrate the strengths and weaknesses of different modes of research funding• Identify factors associated with translation of research, and attempt to develop 'early indicators' of likely successful translation• Identify 'good news stories' and vignettes of the research process for use by ARC in public engagement and fund raising activities.Traditional methods of peer review have long been favoured by medical research funding bodies for evaluating research, but bibliometric methods have had a variable history. An early move by the National Institutes of Health (NIH) to establish a publications' database was cancelled by the 1980s 'as too expensive for the management information it produced' . The MRCThe Economic and Social Research Council (ESRC) in the UK commissioned a project to identify the impact of their research on non-academic audiences. It involved tracing the activity of the participating researchers after their projects ended and mapping the networks of researchers and relevant non-academic users and potential beneficiaries . The stuCase studies will enable narratives or stories to be told to illuminate how the research funded in the early 1990s was translated (or not) into practice; each case, therefore, could potentially provide an illustrative example of the outcomes from ARC research. Furthermore, the planned 16 case studies will be based on a variety of modes of ARC research funding and types of research. They will also be organised using a common structure. This should enable cross-case study analysis to demonstrate (via illustrative case studies) the strengths and weaknesses of different modes of funding and categories of research. It should also facilitate the identification of factors associated with the translation of research, perhaps through various phases, into policies, products, and clinical practice that produce a health gain.The evaluation framework described below was developed in a way that incorporates previous experience and knowledge on these issues ,24,26,28In deciding the time window to use for selecting case studies, a compromise usually has to be made between the quality of records/likely ability of researchers to recall their activities and the selection of grants whose outputs have had sufficiently long to develop . The latWithin a case study approach it is unlikely that the selection of cases will follow a straight-forward sampling logic in which those selected are assumed to be representative of a larger group . NeverthAs a first step, we shall identify all publications in the relevant period from the principal investigators awarded ARC funds. Then, the researchers will be classified according to the journal impact factors (see below) of the journals in which their articles appear. The aim will be to draw up shortlists of possible researchers to include in the study: those in the top decile and those the middle of the range, with the final selection made on the basis of advice from ARC's Development Committee.For case studies it is appropriate to use multiple sources of evidence converging on the same issues  and adopWe will read key project documents including the original research grant proposals, referees' reports and end of project reports. On the basis of the end of year reports from researchers, and the interviews (see below), we will also identify and read the core publications attributed to the research grant and any subsequent publications such as key citing papers, relevant clinical guidelines etc.There will be about three interviews per case study. They will be based on a semi-structured interview schedule informed by the evaluation framework described below. They will, therefore, explore the origins of the research and the primary outputs such as the publications. In this way the initial list of publications identified as being related to the project will be refined. Furthermore, there will be a full exploration not only of the contribution to research training and career development, but also of any translation of the research findings into product development, policy and practice. In each case study the initial interviews will be with members of the relevant research team. Then snowballing techniques will be used to identify the people who might be able to provide most information about how the research has influenced subsequent research or been translated into product development, policy and practice.Bibliometric approaches can play a useful role in the analysis of the research funded by specific biomedical research-funding bodies ,31. In tIn every case a draft copy of the case study report will be sent to the principal investigator for comment. Such a step is an important part of the validation process and not just a matter of professional courtesy .There are two elements in the evaluation framework adopted to organise the case studies being conducted in the assessment of the outcomes from ARC-funded research. Building on the framework developed by Buxton and Hanney ,12, the There are many steps involved in assessing outcomes from research. One of the key advantages in taking a detailed approach, such as that described below, is that it enables the issue of the counter-factual to be addressed. In other words, what would the world have looked like without the specific research being examined?The multi-dimensional category of payback provides the evaluation criteria for the outputs and outcomes from ARC funding. The 5 main categories are:a) Knowledge productionb) Research targeting, capacity building and absorptionc) Informing policy and product developmentd) Health benefitse) Broader economic benefits.Each can be considered in turn, with various sub-categories explored and possible measures described.The knowledge produced by research is the first output and is contained in various publications and patent applications. Any type of publication can be considered, but it is generally thought that peer reviewed articles are the most important and, at least for biomedical research in industrialised countries, it is thought reasonable to assume that the overall output of research publications is fairly represented by peer-reviewed papers in international journals . In addiCitation analysis can be applied to assess the impact the specific article is having within the research community ,34. PrevA journal's 'impact factor' is based on the average number of times an article in the journal is cited; it can provide a short-hand version of citation analysis by giving some indication of the importance of the journal in which an article appears. The use of impact factors in analysis of biomedical research has been criticised  but, proParticularly when considering research that might be aimed at potential users outside the research community, it is often desirable to use a range of publication outlets including those journals with the highest readership among the groups at whom the research is targeted. In some fields these might well be journals that do not have an impact factor but are, nevertheless, significant as vehicles for dissemination of the knowledge produced -40.The better targeting of future research is frequently a key benefit from research, especially from research that is more basic and/or methodologically oriented. An indication of this comes from citation analysis. The enhanced targeting can be of the research conducted both by others and by the original researcher(s). Where follow-on research, especially by members of the original research team, is clearly associated with the original research it can be useful to obtain information on the source and amount of such funding . As is dResearch training can be provided both as a result of the employment of staff on research projects and programmes, and through explicit funding for research training and career development . One meaResearch can be used to inform policymaking in a wide range of circumstances and the key issue is that policymaking involves those in positions of authority making choices that have a special status within the group to which they apply . PolicymThe position of systematic reviews is a little complex. They are themselves a form of research, but inclusion of a study in a systematic review is a form of secondary output and might lead on to further use.At a similar level, although involving very different processes, research can also be used to inform product development . InformiBenefits in terms of health gains might be viewed as the 'real' payback or outcomes from health research. Greater effectiveness of health-care resulting from research-informed drugs or procedures should lead to increased health. Various measures of health gain exist, but for arthritis the emphasis, in most cases, is likely to be on those that assess reduction in pain or disability, and increase in mobility. While the benefits from arthritis research will not generally be measured in terms of life years gained, in some circumstances they might be captured by using Quality Adjusted Life Years (QALYs). This is often seen, in countries such as the UK, as a more appropriate approach than using Disability Adjusted Life Years (DALYs) . There hThis category of benefits can be thought of as going wider than health gain, and some aspects can be seen as benefits to the health sector more generally. Cost savings in the provision of health-care may result from research-informed changes in the organisation of services or in the particular therapies delivered. It might be necessary to consider various issues here. These include whether potential savings have in practice been realised – either as cash savings or as the release of resources for other valuable uses . FurtherA range of benefits can accrue to the national economy from the commercial exploitation of research. These can take the form of employment and profits resulting from the manufacture and sale of drugs and devices . The natWhilst there is a danger of double counting, it is probably also important to adopt a human capital approach and focus on the value of production gained from having a healthy workforce. This can be measured by examining the reduction in days off work. Typically, in a human capital approach, potential future earnings are calculated for people who, as a result of advances in medical research, can continue to contribute to national production ,15,48. TThe second element of the evaluation framework is the logic model. Its various stages are shown on Figure Stage 0: Topic/issue identificationInterface A: Project specification and selectionStage 1: Inputs to researchStage 2: Research processesStage 3: Primary outputs from researchInterface B: DisseminationStage 4: Secondary outputs – policymaking and product developmentStage 5: Adoption by practitioners and publicStage 6: Final outcomesWhile it is not possible totally to tie the categories of benefits to certain stages of the model, it is possible to identify broad correlations: categories a) and b) (knowledge and research benefits respectively) are together considered to be the primary outputs from research; category c) (informing policy and product development) relates to the secondary outputs; and categories d) and e)  are the final outcomes. This approach can be incorporated into the analysis of each stage in turn as is set out below, where a few examples, drawn from the pilot studies, are used to illustrate how the framework seemed to be working in practice but could be refined in certain ways.The topic or issue identification stage covers the generation of the original ideas for the research. Its nature can vary considerably depending on whether the main driving force is internally generated by the researcher, or externally generated . Most ARThe nature of the activities at Interface A will vary depending on the type of issue identification. Where the topics are externally generated, there are potential difficulties in ensuring both that the research community is actively engaged with the priorities that have been identified and that the project specification meets the needs as identified . Where tIt can be important to consider not only the financial inputs, including any beyond the specific ARC funding, but also the experience of the research team and the knowledge base on which they built. Part of the idea behind examining any other funding brought in to support ARC research is again to see how far ARC funding is helping to facilitate the funding of arthritis research by general funders of health research: is ARC funding studies that produce findings that others believe are worth further investigation? The pilot studies confirmed that the complexities of identifying the exact funding streams behind any piece of research were best addressed by using a case study approach involving initial documentary review and following up issues in interviews. The pilots involved a case where other contributory funding contributed to what was clearly an ARC project, and therefore little attempt was made to portion out credit for outcomes to any funder other than ARC. In another case, however, the research was part of a stream of ARC-funded work and an effort was made to try to draw boundaries around what would be appropriate to include in the case study.Consideration can be given to how appropriate the proposed methods for a study turned out to be, and whether any difficulties were encountered. In some cases it could be relevant to explore how far potential users were involved at this stage. It is possible that difficulties identified at this stage could explain later problems with translation or uptake of the research findings.Knowledge production, as represented by the various types of publications, is a major primary output from the research. Various ways of measuring this were discussed above. The pilots also showed that the interviews used to refine the lists of publications from the specific funding in question, could also sometimes help to identify where non-conventional sources were being used as outlets for publications. Most of the primary outputs will feed into the stock of knowledge.The research benefits in terms of targeting future research represent either feedbacks to further research conducted by team members, or findings that feed into the stock of knowledge and help target future research of others. An example from one pilot study showed not only how the principal investigator used her project to inform her own further work, but was also able to contribute to a much larger collaborative project. Interviews in another study showed that the research had informed considerable further work in industry, but as yet this had not led to any product development. Under the framework being used, it is possible to give that ARC-funded work considerable credit for informing the further research, but record its limited impact at the subsequent stages.Capacity building can also be seen as a primary output. Accounts were given, in pilot study interviews, of the research training and higher degrees that resulted from the research.Dissemination is usually seen as being somewhat more active than the mere production of academic publications containing the knowledge. There are, however, clear overlaps between some activities. Sometimes it is possible to record not just dissemination activities but also the successful transfer of research findings to potential users in the political, industrial, professional environment and wider society. Previous analyses of how to increase the implementation of research findings  will helAs noted above, policymaking and product development activities can result in a wide range of secondary outputs, and various methods are needed to identify research-informed policies. In one case study, a review of a database revealed that one project had been cited in a clinical guideline unbeknown to the research team, whereas in another pilot it took interviews to identify that the research was informing local guidelines and care pathways. The use of the research in systematic reviews was also revealed in various ways in the pilot studies. Where the research seems to have resulted in secondary outputs it is useful to explore the factors that have led to this.In relation to product development, if research findings are incorporated into the process of developing a product, for example a new drug for arthritis, this can be seen as an important secondary output. In the preliminary set of interviews, most people referred to how ARC-funded research had played a key role in the production of anti-TNF therapy for arthritis. In a pilot study, interviews revealed the extent to which industry's attempts to use one stream of research for product development had not, so far, been successful.For the research findings incorporated into secondary outputs to result in final outcomes there usually has to be some behavioural change by practitioners, and/or the public. This may involve take-up of new drugs or procedures as set out in a secondary output such as a guideline from the National Institute for Clinical Excellence (NICE). Sometimes the adoption comes as a direct result of the primary outputs, as when clinicians – often at the cutting edge – decide to implement research findings even prior to the development of clinical guidelines. Either way, it is important to try to establish the adoption or take-up rates and to explore how far the behavioural change can be attributed to the specific research findings, as opposed to other factors such as a more general change in climate of opinion in relation to, for example, the importance of exercise. In one pilot study where interventions based on research filtered into practice, a series of interviews was used to attempt to identify both the precise role of the specific ARC-funded project and possible levels of uptake.The role of the public in responding to informed advice – often research-based – is seen as increasingly important, especially in a field such as arthritis . VariousThe final outcomes are the health and broader economic benefits identified in categories d) and e) above. These are increasingly seen as being the ultimate goal of health research funding, but their precise estimate in practice often remains difficult -7. In onEach of the 16 cases will be written up as a narrative organised according to a common structure based on the various stages of the logic model. Each study should potentially, therefore, provide illumination as to the processes that could lead to outcomes and illustrations of such outcomes. In addition, the common structure of each case should facilitate some cross-case analysis that will not only look for common factors associated with research that has led to outcomes, but also see how far such outcomes are associated with different modes of funding and types of research. Some of this analysis should be based on the previous findings that are embedded into the evaluation framework: for example, basic research might be expected to produce a reasonable number of knowledge outputs but be less likely than clinical or AHP research to inform policies. Some other aspects of the analysis, however, are likely to be exploratory: detailed analysis of factors related to the role of medical charity research in contributing to outcomes appear, as yet, not to be well established.This paper sets out the aims and methods to be adopted in an innovative study to review the outcomes of the research funded by the Arthritis Research Campaign, one of the leading medical charities in the UK. At a time of growing emphasis on both accountability and evidence-based policy making, it is important for research-funding bodies to be able to show the results of their funding and base their policies on analyses of the processes involved in producing outcomes . Based oFinally, one of the challenges for the future will be to operationalise such analysis on a regular, and therefore less resource intensive, manner. It is hoped that the study will also shed light on these practical considerations, and do so in a way that will enable a system to be developed that meets the specific needs of the particular research funding body , in thisThe work was funded by ARC.JG led the design of the study, with all authors making contributions. SH drafted the article with contributions from all authors.


MRI slices of 1.5 mm thickness have been used in both cross sectional and longitudinal studies of osteoarthritis, but is difficult to apply to large studies as most techniques used in measuring knee cartilage volumes require substantial post-image processing. The aim of this study was to determine the optimal sampling of 1.5 mm thick slices of MRI scans to estimate knee cartilage volume in males and females for cross-sectional and longitudinal studies.A total of 150 subjects had a sagittal T1-weighted fat-suppressed MRI scan of the right knee at a partition thickness of 1.5 mm to determine their cartilage volume. Fifty subjects had both baseline and 2-year follow up MRI scans. Lateral, medial tibial and patellar cartilage volumes were calculated with different samples from 1.5 mm thick slices by extracting one in two, one in three, and one in four to compare to cartilage volume and its rate of change. Agreement was assessed by means of intraclass correlation coefficient (ICC) and Bland & Altman plots.Compared to the whole sample of 1.5 mm thick slices, measuring every second to fourth slice led to very little under or over estimation in cartilage volume and its annual change. At all sites and subgroups, measuring every second slice had less than 1% mean difference in cartilage volume and its annual rate of change with all ICCs ≥ 0.98.Sampling alternate 1.5 mm thick MRI slices is sufficient for knee cartilage volume measurement in cross-sectional and longitudinal epidemiological studies with little increase in measurement error. This approach will lead to a substantial decrease in post-scan processing time. Osteoarthritis (OA) is the most common form of arthritis and a leading cause of musculoskeletal disability in most developed countries . The kneThe present study consisted of two datasets; one was part of the Tasmanian Older Adult Cohort Study (TASOAC), an ongoing prospective population-based study aimed at identifying the environmental, genetic, and biochemical factors associated with the development and progression of OA at multiple sites (hand, knee, hip, and spine), which commenced in 2002. Subjects aged between 50 and 79 years were selected randomly from the electoral roll of Southern Tasmania, with an equal number of males and females. Another dataset was a younger adult sample from the Knee Cartilage Volume study (KCV) as previously reported . Both stAn MRI scan of the right knee was performed on all subjects. Knee cartilage volume was determined by means of image processing on an independent work station using the software program Osiris as previously described ,15. Two Then, the volumes of all individual cartilage plates were recalculated based on different sampling intervals from 1.5 mm thick slices by extracting one in two, one in three, and one in four slice areas from the individual data file. These were then summed and the total was multiplied by the corresponding slice distance.Femoral cartilage volume was not assessed in this study as it is strongly correlated with tibial cartilage volume and thus adds little extra information , tibial 2).Weight was measured to the nearest 0.1 kg (with shoes, socks and bulky clothing removed) using a single pair of electronic scales (Seca Delta Model 707) which were calibrated using a known weight at the beginning of each clinic. Height was measured to the nearest 0.1 cm (with shoes and socks removed) using a stadiometer. Body Mass Index (BMI) was calculated as weight (kg) / height  were from the TASOAC study and 50 subjects with longitudinal data  were from the KCV study. Characteristics of the study sample are presented in Table In cross-sectional analysis, compared to the cartilage volume measured using 1.5 mm thickness, decreasing the number of the slices by extracting one in two to one in four led to a very little underestimation in the magnitude of the average cartilage volume at lateral, medial tibial and patellar sites with ICCs of 0.98–1.00 Table . The maxSimilarly, in longitudinal analysis, compared to the cartilage volume change using 1.5 mm thick slices, decreasing the number of the slices by extracting one in two to one in four slices led to very little over or under estimation of the mean changes in cartilage volume at lateral, medial tibial and patellar sites Table . The meaThis study suggests that lateral, medial tibial and patellar cartilage volumes measured from up to one in four 1.5 mm thick slices are quite comparable to those obtained from 1.5 mm thick slices. If the agreement is defined at high levels expected to lead to minimal measurement error, then knee cartilage volume can be measured sufficiently and accurately with one in two slices both cross-sectionally and longitudinally regardless of ROA status and./or reader. This approach will lead to a substantial decrease in post-scan processing time and make large-scale studies of knee cartilage volume more feasible.Currently, there is no reported information on the number of the slices of MRI scans to measure cartilage volume apart from a recent paper from our own group which had similar findings to this study with different readers and geographic location . In a stIdeally, the more slices used, the more accurate the estimation of the object's volume, as they may contain more information. However, for a completely regular structure, such as a cylinder, the area of a single slice with length gives an exact volume. It is therefore reassuring but not surprising that the current study demonstrates a minimum reduction in the knee cartilage volume and volume change over time as tibial and patellar cartilages have a relatively regular structure. A different interpretation may apply to femoral cartilage and we do not have data on this imaging site.The current study simply examined the effect of decreasing the number of slices on the estimation of knee cartilage volume and volume change while all other variables were kept constant. We did not re-scan the study subjects but simply estimated the cartilage volume by using one in two, one in three, or one in four slices. This has an advantage of allowing us to examine the single effect of sampling intensity in the situation where all other variables such as re-positioning the subject and measurement were kept constant. The effect of these errors on measurement have been well-documented ,31. For The current study has a number of potential limitations. Firstly, which sampling intensity should be used in the MRI scan of knee cartilage depends on the purpose of the measurement. Our results cannot be applied to individual cartilage volume, but only for mean cartilage volume in groups as the individual difference in cartilage volume increases with decreasing sampling intensity. Secondly, decreasing sampling intensity will increase measurement error as the remaining slices focus on different portions of the irregularly shaped cartilage. Depending on what particular surfaces remain, however, the overall volume may be increased or decreased. If this is random, then the mean will remain the same as demonstrated in the current study. Thirdly, the ICC can be influenced by traits in the sample in which it is assessed. Age, sex and BMI have been reported to be associated with knee cartilage volume . These mKnee cartilage volume and its rate of change can be accurately measured with every second 1.5 mm thick MR slice. This approach will lead to a substantial decrease in post-scan processing time and make large-scale studies of knee cartilage volume more feasible.The author(s) declare that they have no competing interests.GZ designed and carried out the study planning, data collection, analysis and interpretation of the analysis, and preparation of the manuscript. CD participated in data collection and critical revision of the manuscript. FC participated in the study planning and critical revision of the manuscript. GJ designed the study, participated in analysis and interpretation of the analysis, and critical revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


SLC11A1 is involved in susceptibility to both autoimmune and infectious disorders. The aim of this study was to evaluate whether SLC11A1 has a role in the susceptibility to inflammatory bowel disease (IBD) by characterizing a promoter polymorphism within the gene and two short tandem repeat (STR) markers in genetic proximity to SLC11A1.Significant evidence suggests that a promoter polymorphism withinthe gene SLC11A1 genotype-phenotype correlations. Polymorphic markers were amplified from germline DNA and typed using gel electrophoresis. Genotype-phenotype correlations were defined using case-control, haplotype, and family-based association studies.The studied population consisted of 484 Caucasians with IBD, 144 population controls, and 348 non-IBD-affected first-degree relatives of IBD patients. IBD subjects were re-categorized at the sub-disease phenotypic level to characterize possible SLC11A1 disease association; most significantly, there was no apparent evidence of SLC11A1 promoter allele association in the studied Crohn's disease population.This study did not provide compelling evidence for SLC11A1 promoter polymorphisms are involved in susceptibility to this form of IBD.Our results therefore refute previous studies that have shown  Inflammatory bowel disease (IBD) encompasses a number of disorders that are characterized by chronic inflammation of the gastrointestinal tract. The three specific forms of the disease known as Crohn disease (CD), ulcerative colitis (UC), or the less well-characterized indeterminate colitis (IC), are diagnosed based on the results of clinical observations, histology, radiology, and to a lesser extent, serology. Prominent clinical features include profuse diarrhea, abdominal pain, and increased colorectal cancer risk. IBD affects an estimated one million Americans , with UCSLC11A1 in the susceptibility to IBD. SLC11A1 is a proton-coupled bivalent metal antiporter that is crucial in early macrophage activation [SLC11A1 encodes a highly hydrophobic 550 amino acid membrane protein and is located on the long arm of chromosome 2 (2q35), a region that has not been identified as being within an IBD susceptibility locus. Genetic studies have shown that different alleles of a (GT)n dinucleotide repeat polymorphism located within the promoter region of SLC11A1 cause susceptibility to different human diseases. Allele 2 of this polymorphism is associated with susceptibility to intracellular pathogen infection [The aim of this study was to investigate the role of the gene tivation . SLC11A1nfection , and allsome 2 2q5, a regiSLC11A1 are associated with CD [n promoter polymorphism, however, was not examined in that study. A recent study by Kojima et al [SLC11A1 has been implicated in susceptibility to IBD. Previous work from this laboratory  has show with CD . The escribed . These mThis prospective study was approved by the University of Louisville Institutional Review Board. Written informed consent was obtained from all subjects. Patients were derived from a University-based colon and rectal surgery practice. Initial IBD diagnoses were determined through radiological, endoscopic, and/or histopathological studies. Histology was available in all cases.The study population consisted of a total of 628 Caucasians, including 254 unrelated individuals with CD (63% women), 165 with UC (53% women), 65 with IC 70% women), and 144 population controls (74% women). Demographic data for the IBD population is shown in Table 0% women,A total of 348 non-affected first-degree relatives of IBD patients were available for the study. There were 63 CD families (29 triads, 30 discordant sibling pairs, 4 triad/discordant sibling pair combinations), 43 UC families (11 triads, 24 discordant sibling pairs, 8 triad/discordant sibling pair combinations), and 14 IC families (5 triads, 5 discordant sibling pairs, 4 triad/discordant sibling pair combinations). In addition, there were 17 CD families, 18 UC families, and 9 IC families who could not be classified as either a triad or discordant sibling pairs.SLC11A1 promoter polymorphism was performed using 100 ng template DNA, 2.5 mM MgCl2, 50 mM KCl, 10 mM of tris/HCl, 0.2 μM of each dNTP, 200 pmol of each primer, and 1 unit of Taq polymerase (Promega, Madison, WI). Primer sequences were as follows: 5'GACATGAAGACTCGCATTAG3' & 5'TCAAGTCTCCACCAGCCTAGT3'. The product was amplified using the following conditions: 94°C for 10 min, followed by 25 cycles of 94°C for 30 sec, 55°C for 75 sec, and 72°C for 20 sec. A final extension step was run at 72°C for 6 min. Genotyping was performed using gel electrophoresis (Spreadex EL 300 S-100 gel [Elchrom Scientific, Lake Park, FL]). PCR amplification of the STR markers D2S434 and D2S1323 was performed as described previously [STRs were amplified from genomic DNA extracted from peripheral leukocytes. Polymerase chain reaction (PCR) primers were custom-synthesized (Proligo, La Jolla, CA). PCR amplification of the o = 1, where πo is the proportion of tests in which the null hypothesis is true.Case-control analyses were performed using STR genotype-allele frequencies in cohorts subdivided on the variables outlined in Table only performed in those disease groups possessing a global test q-value <0.05. Therefore, 2 × 2 contingency tables were only constructed for genotypes with a global test q-value meeting this condition. Disease versus control comparisons were performed using Fisher's exact test and corrected for multiple testing using the q-value method as described above.To limit the number of individual disease-control genotype-allele comparisons, further analyses were Since IBD affecting the colon is the primary focus of our research, the L2 colonic CD and L3 ileocolonic CD groups were combined, enabling maximization of statistical power. We argue that this approach will not impair the validity of statistical analyses given that the Vienna Classification is somewhat arbitrary and that group genetic homogeneity may be increased by combining all those cases with colonic IBD. Furthermore, owing to the relatively low numbers of individuals with CD proximal to the terminal ileum (L4 group, n = 32), this disease subgroup was excluded from case-control analyses.Family-based tests used included the pedigree disequilibrium test (PDT) ,12 and aSLC11A1 promoter polymorphism STR D2S434 and STR D2S1323 were named numerically according to molecular weight, with "1" representing a larger allele than "2" and so on. Allele distributions are shown in Table Alleles for the SLC11A1 promoter polymorphism alleles identified in the study population revealed the following sequences:DNA sequence analysis of the three 5AC(GT)5AC(GT)11GGCAGA(G)6Allele 1: T(GT)5AC(GT)5AC(GT)10GGCAGA(G)6Allele 2: T(GT)5AC(GT)5AC(GT)9GGCAGA(G)6Allele 3: T(GT)These sequences corresponded to alleles 1, 2, and 3 as described by Searle and Blackwell . Allelesversus control comparisons of the studied markers are shown in Table n promoter polymorphism did not reveal any evidence of association. Additionally, we studied STR markers in genetic proximity to SLC11A1. The STR D2S1323 exhibited evidence of association with the test of the global null hypothesis of homogeneity of all groups exhibiting statistical significance for allele frequencies (q = 0.050, p = 0.008). To limit multiple testing, individual pair-wise disease versus control comparisons were only performed for D2S1323 alleles. These analyses showed over-representation of allele 1 in the UC group . The STR D2S434 did not reveal any evidence of association. Further analyses of genotype and allele data for all markers did not show a correlation with either age of onset or disease behavior.Results from global disease Family-based tests were performed for each of the three markers. Neither the likelihood ratio test nor the PDT revealed significant evidence of association for any of these markers.SLC11A1 promoter polymorphism alleles and any IBD sub-phenotypic group in this population, which unexpectedly refutes the significant evidence of others [SLC11A1 promoter polymorphisms to be involved in susceptibility to autoimmune disorders and disorders that are characterized by a high degree of immunological dysregulation, including IBD [SLC11A1 expression driven by alleles 1, 2, and 4; however, allele 3-driven SLC11A1 expression was enhanced. It has long been recognized that altered expression of SLC11A1 in response to LPS is a key event in the activation of macrophages upon contact with a variety of pathogens [No association was observed between any f others . This reding IBD . Searle ding IBD  investigathogens .SLC11A1 as an IBD candidate gene.Based on those observations, we hypothesized that over-representation of promoter allele 3 in CD patients could lead to hyperactivation of bowel wall macrophages that are chronically exposed to high levels of LPS. This could subsequently cause the autoimmune-like phenotype characteristic of IBD. This hypothesis must be rejected in this cohort due to the absence of association with promoter allele 3 in any IBD subgroup. Analysis of intragenic single nucleotide polymorphisms are required to conclusively exclude SLC11A1. This association was not, however, confirmed upon family-based statistical analysis. The current finding was particularly surprising to us, given that our previous study showed this STR to be associated with CD rather than UC [vice versa) may have been a confounding variable that may have in part led to what we now consider the false-positive association observed in the CD population. It should also be noted that the current study contains over two-fold the number of subjects as compared with the previous work, and thus statistical power has been significantly increased. Furthermore, our current statistical methodology is now far superior, and we argue that our stringent means of correcting for the effects of multiple testing effectively minimizes the likelihood of detecting a false-positive association. This is a vast improvement over our earlier study in which compounding of a type 1 error was not taken into account. Finally, genotyping in the earlier study was performed on specimens with a rather heterogeneous nature and consisted of genetic data derived from DNA extracted from both surgical specimens (i.e., somatic DNA) and leukocytes (i.e., germline DNA). In our current study, genetic material has only been isolated from leukocytes, thus reducing the possibility of detecting variations within somatic DNA.D2S1323 was associated with UC in this population. Indeed, this was the only evidence for association in any of the studied STR markers in proximity to  than UC . The sigSLC11A1 (GT)n promoter polymorphism in IBD sub-phenotypic groups and accept the null hypothesis as confirmed by family-based testing. We did, however, identify an association between an allele of the STR D2S1323 and UC upon case-control analysis. This association was not confirmed on family-based and haplotype testing, and its actual significance in IBD remains to be seen.In summary, we have characterized the The author(s) declare that they have no competing interests.NPSC performed genotyping, PDT analyses, and drafted the manuscript; MRE performed genotyping and was responsible for coordination of laboratory work; authors DWC and RKL performed genotyping; GAC performed statistical analyses; REP performed re-review of colonic IBD histology; SG collected all patient samples, participated in study design, coordination, and manuscript preparation and obtained funding for this work. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Endothelial nitric oxide synthase (eNOS) has previously been detected in the glandular part of the human gastric mucosa. Furthermore, nitric oxide (NO) has been shown to influence gastric secretion in various animal models. The present study was conducted to investigate the influence of exogenously and endogenously derived NO on histamine- and cAMP-stimulated gastric acid secretion in isolated human oxyntic glands.14C]aminopyrine.Oxyntic glands were isolated from human gastric biopsies and were subsequently pre-treated with NO donors and nitric oxide synthase inhibitors and then exposed to histamine or dibutyryl-cAMP (db-cAMP). The secretory response of the glands was determined as accumulation of [G-nitro-L-arginine methyl ester (L-NAME) or NG-nitro-L-arginine (L-NNA) enhanced the secretory response.The histamine- or db-cAMP-induced acid secretion was attenuated by L-arginine, a known source of endogenous NO, and also by the NO-donors sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP). Pre-treatment with either of the NOS inhibitors NOur results show that NO inhibits gastric acid secretion in isolated human gastric glands, and that there is endogenous formation of NO within the glandular epithelium in the vicinity of the parietal cells. Nitric oxide (NO) is produced from L-arginine in a reaction catalyzed by the enzyme nitric oxide synthase (NOS),2. NO isAnimal experiments have provided conflicting information about the interaction between NO and gastric acid secretion. For instance, studies in vitro have shown that NO stimulates secretion of gastric acid in the mouse,18 and bIn an earlier study, we found morphological support that endogenous NO plays a role in regulation of parietal cell function . Also, tTwenty-four healthy men ranging in age from 22 to 31 years were recruited as paid volunteers. The selection criteria stipulated that the subjects had to be free from disease and should not have taken any medications or imbibed alcohol for at least one week prior to examination. The men fasted for at least six hours before examination.®, AstraZeneca, Södertälje, Sweden), after which routine gastroscopy was performed using an Olympus GIF-100 endoscope. Pinch biopsy forceps (Olympus FB 24K-1) were used to take tissue specimens from the greater curvature, immediately distal to the fundus. In all subjects, the gastric mucosa appeared to be normal, both macroscopically and histologically. All subjects tested negative for Helicobacter pylori infection in the urease breath test .Pharyngeal anaesthesia was induced with lidocaine spray , and all subjects gave informed consent.The current experiments were based on a technique that was first described in 1976 for use in rabbits in vitro  and is n2) collagenase enzyme solution -tosyl-L-lysine chloromethyl ketone [TLCK], 10 μM indomethacin, 10 mM glucose, 2 mg/ml human serum albumin [HSA; Sigma], and 1 mg/ml collagenase type IA [Sigma]). The mixture was placed in a 37°C water bath and was gently stirred for 120 minutes, after which most of the treated specimen had disintegrated, leaving mainly isolated gastric glands. The mixture was subsequently filtered through a 200 μm mesh. The isolated glands were washed and re-suspended in pre-warmed (37°C) respiratory medium (132.4 mM NaCl, 1.0 mM NaH2PO4, 1.2 mM MgSO4, 5.4 mM KCl, 5.0 mM Na2HPO4, 1.0 mM CaCl2, 10 μM indomethacin, 10 mM glucose, and 2 mg/ml HSA). The glands were then transferred to vials containing fresh respiratory medium to which we added one of the following: the NOS inhibitor NG-nitro-L-arginine methyl ester  or equivalent amounts of its biologically inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME); the NOS inhibitor NG-nitro-L-arginine ; either of the two NO donors sodium nitroprusside  and S-nitroso-N-acetyl-penicillamine ; the substrate for endogenous NO production, L-arginine (0.1 mmol/L) [The human oxyntic mucosal biopsies used in our study were washed and stored no longer than 15 minutes in ice-cold oxygenated phosphate-buffered saline (PBS). The tissue specimens were cut into smaller pieces with a pair of scissors and transferred to oxygenated (100% O14C-labeled aminopyrine (AP) in the glands themselves and in the supernatant after centrifugation and then calculate the ratio between those two values  [14C]labeled aminopyrine was added to the vials, which were then further incubated at 37°C for 90 minutes. Thereafter, the gland suspension was transferred to previously dried and weighed tubes, which were centrifuged at 4,000 rpm for two minutes. The supernatant was removed and transferred to scintillation vials. The pellets (glands) were dried at 100°C, and the dry weight was determined, and the glands were subsequently re-suspended in 0.5 mol/L NaOH at 60°C and transferred to scintillation vials. The radioactivity of the glands and the supernatant was determined in a liquid scintillation counter (1214 Rackbeta, LKB), and the AP ratio was calculated using the following formula[A well-established method used to indirectly measure acid secretion by isolated gastric glands is to determine accumulation of P ratio) . In shorg formula:where IGW is intraglandular water volume (= 2 × the dry weight of glands in mg).Background accumulation of AP is included in the values representing the secretory response. The AP ratios for background and histamine-and db-cAMP-stimulated conditions differed between the individuals. Therefore, for each subject, we determined the AP ratio for stimulated glands and considered that value to be 100% and used it as an individual reference value. All values are based on single analyses.Data were analysed by one-sample sign tests comparing median values using MINITAB™ Statistical Software. P values less than 0.05 were considered significant.® Avidin (Vector Laboratories) for 1 h. Following that treatment, the slides were washed and coverslipped using Vectashield® mounting medium. A Nikon Eclipse® E800 fluorescence microscope with a VFM EPI-fluorescence attachment was used to examine and evaluate the slides. A band-pass filter with a wavelength range of 520–560 nm and a long-pass filter with cut-on wavelength at 590 nm (for emitted light) were employed to visualize the Texas Red® molecules.Isolated gastric glands from five test subjects were placed on charged Super Frost*/Plus glass slides (Menzel-Gläser, Germany) and then washed with PBS and permeabilized with 100% ethanol at -70°C for 5 min. Thereafter, the slides were incubated at room temperature overnight with rabbit anti-NOS3 antibody  and then washed thoroughly in PBS and incubated for 1 h with biotinylated goat anti-rabbit secondary antibody. Slides where primary antibody had been left out served as negative controls. The slides were subsequently washed again, and biotinylated antibody was detected by exposure to 20 μg/ml Texas RedFor morphological evaluation, glands were fixed on glass slides and stained with Harris hematoxylin for five minutes and 0.5% eosin Y for two minutes. Each step was followed by a rinse in tap water.We studied the effects of NO on acid secretion induced by various stimulants in gastric glands isolated from stomach biopsies from human. Morphological examination of the hematoxylin-eosin-stained slides revealed that the isolation procedure had successfully yielded whole-gland preparations and that parietal cells were present in the gastric glands. Immunohistochemical analysis showed that the isolated glands contained eNOS-immunoreactive cells Fig. , which aBackground AP accumulation was observed in the isolated glands, with a median AP ratio of 8.6 . After administration of 50 μmol/L of histamine or 1 mmol/L of db-cAMP, the median AP ratios were 24.7  and 38.2  respectively. The response to both histamine and db-cAMP exceeded the background by a factor of about 2–4 in all preparations  reduced the histamine response to a median of 48% (n = 7) of the response seen in non-pre-treated glands (100%). In four out of five gland preparations, the substrate for endogenous NO formation, L-arginine (0.1 mmol/L), decreased the AP ratio. This effect however was not statistically significant Fig. . When isExposing the isolated glands to db-cAMP increased the secretion of gastric acid compared to the background level. Compared to untreated glands, those that were pre-treated with SNP (1 mmol/L) or SNAP (0.1 mmol/L) accumulated less AP after stimulation with db-cAMP; 63%(n = 9) and 81%(n = 8) respectively Fig. . MoreoveThe method of using isolated gastric glands in vitro is well suited for studying the interaction between gastric acid secretion and various endocrine signals. This technique has been thoroughly evaluated, chiefly in experiments on animals, and it has been reported to offer good reproducibility . FurtherIn a previous study conducted by our research group , examinaThe exact mechanisms behind this paracrine regulation of gastric acid secretion is yet to be elucidated. There are several different pathways within the parietal cell that might be affected by NO. It can induce ADP ribosylation of G-actin , therebySince it is known that guanylate cyclase is a general target of NO in many cell systems, some investigators have suggested that NO exerts its effects via cyclic guanosine 3',5'-monophosphate (cGMP) in both rat and rabbit parietal cells ,15. An oFurther investigations are needed to clarify the role of NO in parietal cell function.The findings of the present study suggest that NO produced endogenously in the human oxyntic mucosa can reduce the stimulatory effects of histamine or db-cAMP on gastric acid secretion. We obtained uniform results for gastric glands isolated from different healthy human subjects, which implies that NO released from specific cells within the secretory mucosa plays an important physiological role in the regulation of gastric acid secretion.None declared.AB participated in the design and coordination of the study, performed the secretory studies, carried out the immunohistochemical procedures, and drafted the manuscript. SR was the surgeon in charge and carried out all gastroscopic procedures. ACE and SES were involved in the design of the study and in drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The study investigated the extent to which approaches to work, workplace climate, stress, burnout and satisfaction with medicine as a career in doctors aged about thirty are predicted by measures of learning style and personality measured five to twelve years earlier when the doctors were applicants to medical school or were medical students.Medical Register. The current questionnaire included measures of Approaches to Work, Workplace Climate, stress , burnout (Maslach Burnout Inventory), and satisfaction with medicine as a career and personality (Big Five). Previous questionnaires had included measures of learning style (Study Process Questionnaire) and personality.Prospective study of a large cohort of doctors. The participants were first studied when they applied to any of five UK medical schools in 1990. Postal questionnaires were sent to all doctors with a traceable address on the current or a previous Doctors' approaches to work were predicted by study habits and learning styles, both at application to medical school and in the final year. How doctors perceive their workplace climate and workload is predicted both by approaches to work and by measures of stress, burnout and satisfaction with medicine. These characteristics are partially predicted by trait measures of personality taken five years earlier. Stress, burnout and satisfaction also correlate with trait measures of personality taken five years earlier.Differences in approach to work and perceived workplace climate seem mainly to reflect stable, long-term individual differences in doctors themselves, reflected in measures of personality and learning style. Sir William Osler (1849–1919), one of the most distinguished physicians of the nineteenth and early twentieth century, recognised that only some doctors are happy in their professional lives:"To each one of you the practice of medicine will be very much as you make it – to one a worry, a care, a perpetual annoyance; to another, a daily joy and a life of as much happiness and usefulness as can well fall to the lot of man."The modern medical workplace is a complex environment, and doctors respond differently to it, some finding it stimulating and exciting, whereas others become stressed and burned out from the heavy workload. The medical workplace also provides an environment where new skills are continually being learned, both as a result of medical knowledge evolving and because a doctor's work changes, in part due to career development and progression through different jobs.et al [Surface-Rational, Surface-Disorganised, and Deep  or burnout, which has three separate components of emotional exhaustion, depersonalisation and reduced personal accomplishment , which assesses the motivations and approaches used by students in higher education . Included in the present questionnaire (described in the results as '2002') were the 12-item General Health Questionnaire (GHQ) ,14; a thtion see  [2]. Theth March 2003, a few questionnaires were returned up until the end of August 2003.Questionnaires, along with a postage-paid return envelope, were posted at the beginning of December 2002. Two reminders were sent to non-respondents. Although the official closing date was 25Statistical analysis used SPSS version 10.5, and structural equation modelling used LISREL 8.52.Supplementary Information see The tracing exercise looked for 2,912 individuals thought to have completed basic medical sciences and entered a clinical course. Eighty-nine had never been on the UK Medical Register, and either had failed finals, had never registered, or had emigrated. Of 2,823 individuals who were traced, 2,754 doctors were on the 2002 Register, 7 returned to the Register during 2002, and 64 were on an earlier Register. Of 2,823 questionnaires sent, 176 were returned by the Post Office as undeliverable, 10 doctors were travelling and hence uncontactable, and2 had died. Of the remaining 2,635 doctors, 1,668 returned questionnaires, giving a response rate of 63.3%. There was no evidence of response bias (see st December 2002 was 30.4 years (SD 1.86, range 28.3 – 49.2). There was substantial variation in the scores on the aAWQ and the aWCQ, and the factor structures of the aAWQ and aWCQ were similar to those reported elsewhere [Supplementary Information see The mean age of respondents on 1lsewhere  (see Supet al study, the highest correlations were for a surface-disorganised approach correlating with high workload, and a deep approach correlating with a supportive-receptive environment and with choice-independence (table Approaches to work and learning were correlated with climate in the workplace, and as in the Delva ce table .Table Table Table Supplementary Information see Tables A surface-disorganised approach to work is predicted by surface learning in medical school and by higher neuroticism scores and lower conscientiousness (see tables A workplace climate dominated by a high workload is predicted by higher stress and emotional exhaustion measures five years earlier, and by lower openness to experience (see tables Supplementary Information see Although in the previous analyses, stress and burnout have been used as predictors of approaches to work and workplace climate, they are also important outcome measures in their own right. Table Supplementary Information (see The complex relationships described by the various correlations are best analysed and described by means of path analysis or causal modelling , which ation see . AlthougSupplementary Information (see Although the path diagram in figure tion see .Stress in our model is caused by personality differences, being greatest in those having high neuroticism scores, low extraversion scores, and low conscientiousness scores. It is unrelated to learning style.Learning styles at medical school relate to different personality measures, in particular showing no relationship to neuroticism. Deep learning is highest in extraverts who are open to experience, whereas strategic learning is highest in highly conscientious individuals with low openness to experience. Surface learning style is higher in introverts who are low in openness to experience. These findings are similar to those of others .Approaches to work are mainly but not entirely driven by learning styles. A deep approach to work occurs in extraverts who are open to experience and have a deep learning style. The surface-rational and surface-disorganised approaches to work are both greater in those with a surface learning style. However, a surface-disorganised approach occurs in individuals with higher neuroticism scores, in those with lower conscientiousness scores, and in those who have been stressed, whereas the surface-rational approach to work occurs in strategic learners and in those who are low in openness to experience.Workplace climate has a range of influences. High perceived workload occurs in those with a surface-disorganised approach to work, who have been stressed and are more neurotic. In contrast, choice-independence and a supportive-receptive environment both occur in individuals who have not previously been stressed, the choice-independence approach occurring in those with a deep approach to work, whereas the supportive-receptive approach occurs in those who have higher scores on the personality trait of agreeableness.Many doctors at the age of 30 are unhappy in their jobs, and a fifth of our sample reached the conventional GHQ criterion of psychiatric 'caseness'. In contrast, many doctors reported high levels of personal accomplishment, choice and independence in their work environment, satisfaction with medicine as a career, and intellectual and emotional satisfaction from their work. That is not new; Sir William Osler in 1905 contrasted doctors "whose stability of character and devotion to duty make one proud of our profession" with those who find it difficult to keep "the flame alive, smothered as it is apt to be by the dust and ashes of the daily routine" [BMJ Careers devoted to "Doctors' Wellbeing", that excessive workload and absence of support are directly caused by poor working conditions: "the way in which the NHS is run generates stress for members of the workforce every day" [with stress and burnout reported five or six years earlier, when the doctors were PRHOs and carrying out entirely different jobs. High perceived workload and poor support are therefore determined as much by doctors themselves as by specific working conditions. That view was expressed in another article in the special edition of BMJ Careers: "A critical element contributing to the stress that many conscientious doctors experience is internal..." [In 2001, Richard Smith asked "Why are doctors so unhappy?" and concluded that "The most obvious cause of doctors' unhappiness is that they feel overworked and undersupported" . Certainery day" . Howeverery day" . It is prnal..." . A similrnal..." .et al [surface-disorganised approach to work, which in turn is correlated with being a surface learner at application to medical school, a dozen years previously. Surface-disorganised doctors are also high on the personality trait of neuroticism and low on the trait of conscientiousness; and again those correlations are with measures taken six years earlier when the doctors were PRHOs. Doctors reporting a work climate low in support were lower on the personality scale of agreeableness in the measures collected when they were PRHOs.If differences in reported workload are partly explained by differences among doctors, what in turn explains those differences? Doctors reporting a high workload also have what Delva et al  describeSome doctors may be stressed and burned out, but what predicts those others who are happy in their work? Doctors reporting high satisfaction with medicine as a career have a deep approach to work, and that approach is more common in those who also had a deep learning style when they applied to medical school. Satisfaction with medicine also relates directly to the personality traits of greater extraversion and lower neuroticism, and the deep approach to work correlates with greater extraversion and more openness to experience. Doctors who describe their colleagues as receptive and supportive score more highly on the personality trait of agreeableness; and as in many other correlations reported here, that correlation is stable across time – those who are more agreeable at the age of 24 have a more receptive and supportive work environment when aged 30.An overview of our findings is that approaches to work are predicted by earlier measures of study habits and learning styles, whereas perceived work climate, and its pathologies such as stress and burnout, are predicted mainly by personality. Although unfortunately our study did not measure personality during selection, the high stability of the Big Five measures across the life-span -29 (and If, as William Wordsworth said, "the child is father to the man", then the seeds of subsequent job satisfaction and dissatisfaction in doctors may be visible in the personality, motivations and learning styles of medical school applicants. This argument may provide some justification for using such measures in selection, particularly given the general association of job performance and satisfaction with personality  and motiHowever, just as genes are not destiny, so neither personality nor learning style is destiny. Nurture interacts with nature , the envFormal education, particularly effective formal education , can alscorrelates of approaches to work, workplace climate, stress, burnout and satisfaction with a medical career, but are causes, events later in time being predicted by events earlier in time [Longitudinal data suggest that personality and learning style are not merely  in time . DoctorsThese results imply that differences in approach to work and workplace climate in our study result from differences among doctors themselves, as much as they do from differences in working conditions.None declared.The cohort study was designed by ICMcM. The present follow-up was designed by ICMcM and EP, who also prepared the questionnaire. AK was responsible for day-to-day running of the study, and for data entry and cleaning. ICMcM was primarily responsible for data analysis and for writing the first draft of the paper. ICMcM, EP and AK were all involved in preparing the final draft of the paper.The pre-publication history for this paper can be accessed here:Click here for file


The European Union recently awarded 12 million Euros to the ZF-MODELS research consortium to study zebrafish models for human development and disease Danio rerio) in 1822, would be astounded to see the scientific attention now afforded to this two-inch-long native of Indian rivers. A fish with no economic worth was how he described this little creature. Yet recently, the European Union awarded 12 million Euros to the ZF-MODELS research consortium to study zebrafish models for human development and disease. When and why did zebrafish swim from home aquaria into research labs, and what can we learn about our biology from this surprising source?Francis Hamilton, the Briton who first described zebrafish , ‘who laid the groundwork for the use of zebrafish as a developmental model’.Xenopus neural development but soon became attracted to zebrafish as a model organism. By the early 1980s, she explains, Streisinger had worked out many of the genetic tricks needed to tackle zebrafish development. What's more, the fish had ‘wonderful embryology’. The embryo, which develops outside its mother, is transparent. ‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists.Eisen, who now heads her own research group, went to Oregon in 1983 to work on The properties of zebrafish that attracted Eisen soon attracted people interested in other aspects of vertebrate development to the stripy tiddler . As EiseDevelopment was dedicated to the mutants that had been isolated and characterised. These screens, says Ralf Dahm , project manager of the ZF-MODELS consortium, ‘were the first major zebrafish projects, and they showed that zebrafish was a model organism to be reckoned with’.The speedy expansion was driven in great part by two genetic screens initiated in 1992–1993 by Christiane Nüsslein-Volhard in Tübingen, Germany, and Wolfgang Driever and Mark Fishman in Boston, Massachusetts. The aim of both screens was to identify genes with unique and essential functions in zebrafish development, and in 1996 an issue of the journal ‘That was a fantastic time’, says Derek Stemple, then a postdoc with Driever but now a group leader at the Wellcome Trust Sanger Institute (Cambridge, United Kingdom) and a ZF-MODELS participant. ‘From Wolfgang's lab, I was able to take the mutations that affected notochord development, and have been studying them ever since’. The notochord is an embryonic structure that forms the primitive axial skeleton of the developing embryo, and because mutations affecting notochord development result in shortened embryos, seven of the affected genes have been named after the dwarves in Snow White—zebrafish, like some other developmental models, have many imaginatively named mutants. Stemple now knows the identity of six of these mutated genes, all of which lead to disruption of basement membrane around the notochord.Many mutants from those first two screens are still used by developmental biologists, but another set of mutants has recently been isolated by Nancy Hopkins, Amgen Professor of Biology at the Massachusetts Institute of Technology (Cambridge, Massachusetts, United States). About ten years ago, Hopkins started to develop insertional mutagenesis in zebrafish . In thisThe Tübingen researchers did another chemical mutagenesis screen between 2000 and 2001, and are now starting a third screen of 6,000 genomes as part of the ZF-MODELS project. ‘Each of our screens has built on the previous one by including more specific assays’, explains Dahm. Mutagenesis for the third screen is underway, but the assays, which include looking for defects that specifically affect adults, are still at the pilot stage; this autumn, the project's executive committee, which is headed by Nüsslein-Volhard, will decide which assays to use in the full-scale screen. ‘Just over half the 17 partners in the consortium will come to Tübingen to do screens’, predicts Dahm. ‘By bringing in expertise in different systems in this way we should greatly increase the efficiency of the screen’.The ZF-MODELS consortium, which is funded under the European Union's Sixth Framework Programme, aims to establish zebrafish models for human diseases, discover genes that will lead to the identification of new drug targets, and gain fundamental insights into human development. ‘We will mainly focus on using advanced technologies that have recently become available’, says scientific coordinator Robert Geisler . For example, Geisler's lab will use DNA chip technology to investigate gene expression patterns in zebrafish mutants and so provide increased knowledge of the regulatory pathways that act in zebrafish development.Consortium members will also use ‘reverse genetics’ to investigate these pathways. In reverse genetics, researchers start with a gene of interest and investigate the phenotypic effect of altering its activity; by contrast, in ‘forward genetics’ the starting point is to look for a particular phenotype and then hunt out the altered gene that is causing it. Two reverse genetics approaches will be used by the consortium. Gene expression will be transiently knocked down with morpholinos, short segments of the gene that block its function. In addition, a recently developed technique known as TILLING  will be used to knock out gene activity permanently.The first step in TILLING is to mutagenise male zebrafish and mate them with untreated female fish, explains Stemple, whose group is one of three ZF-MODELS partners who will use this approach. Offspring are raised to adulthood, and the DNA of each individual is then genotyped for the exon of interest. The consortium already has a collection of 6,000 such individuals, and once a fish carrying a mutation in the gene of interest has been identified, it will be outcrossed to produce offspring, half of which will carry the desired mutation on one of their chromosomes. ‘It is then a matter of identifying these heterozygote fish and incrossing them to get homozygous fish in which you can see the phenotype that correlates with that mutation’, says Stemple.As well as helping to produce knock-outs for other researchers, Stemple is also using the TILLING technique to develop zebrafish models for muscular dystrophy. Among the genes that are important in notochord development are those that encode laminins. This led Stemple into studying muscular dystrophy because laminins are involved in the human disease. ‘When we used morpholinos to disrupt [the production of] dystroglycan, a laminin receptor, we got a good model for muscular dystrophy’, he explains. Now, he plans to use TILLING to disrupt up to 30 other genes known to be involved in human muscular dystrophy. ‘In particular, we will look for hypomorphic mutants, fish that are viable but on the edge of falling apart’. These mutants can be used to identify small molecules that push the fish into muscular dystrophy. Finding molecules that can cause a disease in this way ‘might give us a handle on something to fix the disease’, says Stemple.In another strand of the ZF-MODELS project, zebrafish expressing green fluorescent protein (GFP) in specific cells or tissues will be generated and characterised . In suchWilson's own interest is in neuroanatomy. Together with Jon Clarke, another developmental neurobiology group leader at University College London, he plans to analyse GFP lines in which small groups of neurons or particular parts of neurons are labelled, and in this way start to build a detailed reconstruction of early brain neuroanatomy. This, in combination with other work on zebrafish carrying mutations affecting neural development, will give the team ‘a better picture of how a vertebrate brain is built’.A final, important aspect of the ZF-MODELS project, adds Dahm, is database construction. ‘We will be developing a set of databases that will integrate all of the project data’, he explains. ‘In addition, we hope to integrate our data with that of ZFIN in the United States to make one central zebrafish resource’.The researchers of the ZF-MODELS consortium are understandably excited about participating in what will, says Geisler, bring an already strong European zebrafish community closer together. But zebrafish researchers in the United States are also excited by the ZF-MODELS project. ‘We need big lab models like ZF-MODELS in developmental biology’, says Hopkins, noting that the days of small groups working in isolation are long gone. This consortium, adds Howard Hughes Medical Institute Investigator Leonard Zon , ‘will not only have an effect on European zebrafish science but also on how it is done in the United States’.But how much can zebrafish tell us about human development and disease? A lot, say zebrafish researchers. ‘Fish really are just little people with fins’, says Hopkins. ‘Of course, there are developmental differences between people and fish, and no one pretends that we can answer every question about human development in zebrafish’. Nevertheless, zebrafish studies can provide valuable clues to the genes involved in human diseases and to potential targets for therapeutic interventions. Hopkins provides the following illustration: ‘We have been doing “shelf screens”, in which we go back to our collection of mutants to find all those that affect the development of a single organ. When Zhaoxia Sun, a postdoc in my lab who now has an independent position at Yale Medical School, screened three-day-old fish for cystic kidney disease, she found 12 different genes. Two were known to cause human cystic kidney disease, so we knew we were in the human disease pathway somewhere, but we had no idea what the other genes were’. Hopkins and Sun have since identified the remaining genes, and these point to a single pathway being involved in the human disease.Developmental geneticist Didier Stainier  is also using zebrafish to study organ development, in particular, heart development. The zebrafish heart is like the early human heart—a tube with an atrium, ventricle, and valves. ‘Everything we have found in the fish is relevant to the human heart’, says Stainier. ‘Obviously, there are additional processes involved in humans, but the basic outline of heart development in fish and people is largely similar’. Stainier has a collection of zebrafish in which valve formation is faulty. ‘Some of the genes we have found will be involved in human congenital valve defects’, he predicts. Knowing the identity of these genes will be useful diagnostically, but, in addition, zebrafish studies can reveal exactly what has gone wrong at a cellular level. The ability to follow individual cells as organs develop is key to this, says Stainier, who reported in March that fibronectin is required for heart development because, by regulating the polarisation of epithelial cells, fibronectin ensures the correct migration of myocardial cells. And in this issue of PLoS Biology, Stainier's lab have identified another zebrafish gene that is involved in heart development—cardiofunk, which encodes a special type of muscle protein.Many researchers are now recognising the value of zebrafish models of human disease. Over the past three to four years, says Zon, this area of research has become a growth industry. The interest in disease models has grown hand-in-hand with the development of morpholinos to knock out specific genes, and the advent of TILLING, says Zon, ‘has set off a whole new fury. There are now large numbers of investigators who will try to knock out their favourite gene and come up with a model’.Zon has worked on disease models for blood , blood vBigger and bigger seems to be the consensus. Chemical screens like Zon's for anticancer drugs can be set up for other human diseases such as muscular dystrophy. Work like Stainier's on organ development may have applications in tissue engineering. ‘If we can find out what drives differentiation in zebrafish’, he suggests, ‘we might be able to do the same for human cells’, making human tissue replacement therapy a practical possibility. And while many zebrafish researchers will continue to study development, others are now moving into the realms of physiology and behavioural studies.Geisler sums up zebrafish developmental research past, present, and future as follows: ‘No other [vertebrate] organism offers the same combination of transparent and accessible embryos, cost-effective mutagenesis screening, and, more recently, a sequenced genome, [DNA] chip, GFP, and knockout technology’. Add to that the potential of zebrafish embryos as a screening platform for small molecule libraries and the new technologies that allow forward and reverse genetics, and it is clear that zebrafish are not about to revert to being pretty pets swimming in small tanks in the corner of the living room.ZF-MODELShttp://www.zf-models.orgMore details of the work included in this European Union Integrated Project can be found at ZFINhttp://z.n.org/ZFIN, provides an extensive database for the zebrafish community including genetic, genomic, and developmental information; search engines for zebrafish researchers and laboratories; listings of meetings; and links to many other zebrafish sites, including sites with movies of zebrafish development.The ZFIN Web site, at Development  on the first two mutagenesis screens contains 37 research articles and can be freely accessed at http://dev.biologists.org/content/vol123/issue1/index.shtmlThe special issue of


Epidemiological studies have shown that individuals who regularly consume NSAIDs have lower rates of mortality associated with colorectal cancer. Because COX-2 inhibitors prevent tumor growth through some mechanisms, we assessed the effect of JTE-522, a selective COX-2 inhibitor, on pulmonary metastases of colon cancer in a rat model.6 RCN-9 (rat colon cancer cells) was injected into the tail vein of 24 anesthetized male F344/DuCrj rats. Oral JTE-522 (0, 3, 10, or 30 mg/kg/day) was administered from the day before RCN-9 injection until the end of the study. Twenty-four days later, the lungs were removed from sacrificed rats and weighed. Pulmonary metastatic tumors were microscopically evaluated in the largest cross sections. We also performed immunohistochemical staining for both COX-2 and VEGF.A suspension of 5 × 10JTE-522 dose-dependently decreased lung weight (p = 0.001) and the size of pulmonary metastatic tumors (p = 0.0002). However, the differences in the number of metastatic tumors among 4 groups were insignificant. Significant adverse effects of JTE-522 were undetectable. Immunohistochemical staining showed high levels of both COX-2 and VEGF in pulmonary metastatic tumors.JTE-522 dose-dependently decreased the size, but not the number of pulmonary metastases. COX-2 inhibitors might block metastatic tumor growth, but not actual metastasis. Selective COX-2 inhibitors might be useful as therapeutic agents that inhibit the growth of metastatic tumors, as well as the tumorigenesis of colorectal cancer. They fo in 1991 ,6. Many  in 1991 . Oshima out mice . In addiat model , and anoat model . We repoat model . Tomozawl cancer .It is well known that colorectal metastasis occurs primarily in the liver and secondarily in the lung . A laparThe selective COX-2 inhibitor, JTE-522 , was susTwenty-four 4-week-old male F344/DuCrj rats, weighing 50 to 70 g purchased from SLC (Shizuoka, Japan) were acclimatized for one week before use in this study. The rats were maintained in an environment with a controlled temperature (22 to 24°C), humidity (40 to 50%), and lighting (12 hours light-dark cycle) with free access to tap water and standard rodent chow (CE-2, Nihon Clea, Tokyo, Japan).et al. after inbred F344/DuCrj rats were subcutaneously injected with neutralized 1, 2- dimethylhydrazine (DMH) [The RIKEN CELL BANK donated the rat colon cancer cell line, RCN-9, which was established by Inoue ne (DMH) . The tum6 RCN-9 cells in 0.4 ml of PBS was injected into the tail vein.F344 male rats were anesthetized with ether and then a tumor suspension containing 5 × 10Twenty-four rats were divided into four groups. Group 1 (control) was not given JTE-522. The other three groups all orally received daily doses of 3, 10 and 30 mg/kg JTE-522 (groups 2, 3 and 4, respectively) by gavage from the day before RCN-9 injection until the end of the study. The rats were sacrificed 24 days after injection and the lungs were removed and weighed. After 3-day formalin fixation, lungs were embedded in paraffin. Paraffin-embedded sections were cut into 3-μm serial sections, which stained with hematoxylin and eosin to evaluate the pharmacological effects of JTE-522 on pulmonary metastases. The number and the largest diameter of metastatic tumors were examined microscopically in the largest sagittal cross section. The largest metastatic tumor was selected after measuring the diameter of 10 tumors in each slide.2O2 in methanol for 20 minutes. After a short rinse in phosphate-buffered saline (PBS), non-specific binding was blocked by incubating the sections in 10% normal goat serum for 10 minutes at room temperature. Thereafter, the slides were incubated with primary antibodies directed against COX-2  for 2 hours or vascular endothelial growth factor (VEGF)  for 1 hour at room temperature. The slides were washed in PBS (3 × 5 minutes) and incubated with secondary antibody (N-Histofine® Simple Stain MAX PO, Nichirei, Tokyo, Japan) for 30 minutes at room temperature. After washing in PBS, color was developed using 3, 3'-diaminobenzidine tetrahydrochloride (Sigma) in 50 mmol Tris-HCl (pH 7.5) for 5 minutes and counterstaining with 1% Mayer's hematoxylin. Sections known to show intense immunostaining for COX-2 and VEGF were included in each experiment, having been incubated with either primary antibody or PBS as positive and negative controls, respectively. The positive controls were always obviously stained and negative controls were never stained.Slides were immunohistochemically examined using the Universal Immuno-enzyme Polymer (UIP). Sections of 3 μm thick were cut from formalin-fixed and paraffin-embedded blocks of rat tissues. The slides were deparaffinized in xylene, rehydrated through a graded series of ethanols and washed with distilled deionized water. Antigen was retrieved by incubating the sections in 10 mmol/L citrate buffer (pH 6.0) in an autoclave (120°C) for 5 minutes. Thereafter, slides were cooled to room temperature and then washed with distilled deionized water. To quench endogenous peroxidase activity, slides were immersed in 3% Het al [The specimens immunostained for COX-2 and VEGF were independently scored by two blinded investigators. Both the distribution (the percentage of positive cells) and the intensity of staining were assessed in a semiquantitative fashion. Sections were scored, according to the method of Yukawa et al . Both thThe effects of JTE-522 on pulmonary metastasis were analyzed by the Kruskal-Wallis test and are expressed as means ± standard deviation. A correlation between COX-2 and VEGF expression was analyzed by Spearman's rank test. In all instances, statistical significance was established at p < 0.05.Each group consisted of 6 rats. JTE-522 significantly and dose-dependently decreased the weight of the lungs  supplied from the date of implantation [et al. evaluated the pharmacological effects of celecoxib on lung metastasis by counting the metastatic tumors using a stereomicroscope and by histochemical analyses of consecutive lung sections. Tomozawa et al. investigated the effect of JTE-522 on lung metastasis of colon cancer in mice [JTE-522 dose-dependently reduced the weight of the lung with metastases and the size of metastatic tumors. These observations are consistent with those of other studies of selective COX-2 inhibitors. In the present study, however, the number of metastatic lesions determined microscopically was not reduced by JTE-522, which conflicts with the earlier findings. Masferrer antation . Celecox in mice . Their sxib 160–30 ppm supet al. identified a significant correlation between COX-2 and VEGF, the latter of which might be one of the most important mediators of the COX-2 angiogenic pathway [et al. reported that hypoxic animals have significantly higher VEGF concentrations compared with normoxic controls [Here, we immunohistochemically stained COX-2 and VEGF because some reports indicate that prostaglandins regulate VEGF expression ,22 and t pathway . Immunoh pathway . Our res pathway . VEGF wacontrols . On the We reported that COX-2 is related to human colorectal tumorigenesis. COX-2 mRNA expression is obviously increased in sporadic adenomas compared with normal colorectal mucosa and is significantly greater in adenomas with larger diameters . Additioet al. demonstrated that rofecoxib decreased the growth and metastatic potential of colorectal cancer in mice through multiple mechanisms [et al. reported that refecoxib negatively regulated angiogenesis in human colorectal cancer liver metastases [Recent reports have shown the inhibitory effect of rofecoxib on liver metastases. Yao chanisms . Fenwicktastases . It is cIn conclusion, JTE-522, a selective COX-2 inhibitor, dose-dependently inhibited the growth of pulmonary metastasis from colorectal cancer in a rat model. Selective COX-2 inhibitors may constitute useful therapeutic agents that can interfere with the growth of hematogenous metastasis, as well as the tumorigenesis of colorectal cancer.The author(s) declare that they have no competing interests.HK carried out the animal experiment and drafted the manuscript. HK and TH performed immunostaining and its evaluation. HU performed the statistical analysis. ME and KS participated in the design of the study and its coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: