Articulating the value of health research.

Previous contributions to Quarterly Journal of Medicine have drawn attention to the work of FEAM, the Federation of European Academies of Medicine, in collaboration with others, in exploring and explaining the issues that will ensure an appropriate European Union (EU) policy framework for health research and innovation. In this article, we present a proposal for an archive of important research conducted in the EU that will act as a resource for illustrating and guiding the development of the necessary regulatory framework.

Short-term gain for long-term pain? Which patients should be treated and should we ration?

OBJECTIVES: Treatments of common conditions which do not affect mortality often become sidelined in the drive to improve efficiency and reduce costs. The rationing of patients is a divisive but crucial component to universal health care. How should this be accomplished? METHODS AND RESULTS: In this article we examine the outcomes of various rationing methods in varicose veins. CONCLUSIONS: No method is perfect and treatment of symptoms and complications should remain the target for all physicians.

Venous thromboembolism in the paediatric patient.

Venous thromboembolism is a concern in the paediatric population and its incidence seems to be increasing. Symptoms and signs may be subtle so a high degree of suspicion is needed. Paediatric patients represent a unique challenge in the provision of anticoagulation due to their unique physiology.

Treatment options, clinical outcome (quality of life) and cost benefit (quality-adjusted life year) in varicose vein treatment.

Varicose veins are an extremely common condition causing morbidity; however, with current financial pressures, treatment of such benign diseases is controversial. Many procedures allow the treatment of varicose veins with minimal cost and extensive literature supporting differing approaches. Here we explore the underlying evidence base for treatment options, the effect on clinical outcome and the cost-benefit economics associated with varicose vein treatment. The method of defining clinical outcome with quality-of-life assessment tools is also investigated to explain concepts of treatment success beyond abolition of reflux.

Parallel artefact rejection for epileptiform activity detection in routine EEG.

The EEG signal is very often contaminated by electrical activity external to the brain. These artefacts make the accurate detection of epileptiform activity more difficult. A scheme developed to improve the detection of these artefacts (and hence epileptiform event detection) is introduced. A structure of parallel Support Vector Machine classifiers is assembled, one classifier tuned to perform the identification of epileptiform activity, the remainder trained for the detection of ocular and movement-related artefacts. This strategy enables an absolute reduction in false detection rate of 21.6% with the constraint of ensuring all epileptic events are recognized. Such a scheme is desirable given that sections of data which are heavily contaminated with artefact need not be excluded from analysis.

Pilot translational study of dietary vitamin C supplementation in Barrett's esophagus.

The transcription factor Nuclear factor kappa B (NF-kappaB) is central to the regulation of genes encoding for mediators of inflammation and carcinogenesis. In the esophagus, NF-kappaB is progressively activated from inflammation to Barrett's metaplasia and adenocarcinoma. Vitamin C, an antioxidant, can inhibit NF-kappaB in in vitro models, and the aim of this study was to prospectively assess the effect of supplemental vitamin C on NF-kappaB and associated cytokines in patients with Barrett's esophagus. Twenty-five patients with long-segment Barrett's and specialized intestinal metaplasia received dietary vitamin C (1000 mg/day) orally for four weeks, and had pre- and post-vitamin C endoscopic biopsies. NF-kappaB activity (activated p50 and p65 subunits) of nuclear extracts was assessed using the Active Motif NF-kappaB assay, and cytokines and growth factors were measured using the Evidence Investigator biochip array. NF-kappaB and related pro-inflammatory cytokines and growth factors (IL-8, VEGF, IL-10) were activated in all Barrett's tissue pre-treatment. Down-regulation in activated NF-kappaB and cytokines was observed in 8/25 (35%) patients. Dietary vitamin C supplementation may down-regulate pro-inflammatory markers in a subset of Barrett's patients. Further studies with larger numbers of endpoints will be needed to further evaluate this effect.

High sensitivity cytokine detection in acute coronary syndrome reveals up-regulation of interferon gamma and interleukin-10 post myocardial infarction.

Inflammation is an important element in the development and destabilization of atherosclerotic plaque. Using a high sensitivity multiplex assay, previously untested in the context of atherosclerotic disease, we determined serum concentrations of GM-CSF, IFNgamma, IL-1beta, IL-2, IL-10, IL-12p70, TNF alpha, IL-6, and IL-8 in 48 Myocardial Infarction (MI) patients, 14 Unstable Angina (UA) patients and 12 healthy controls. IFNgamma levels were significantly higher in MI compared to UA (p=0.0091) and Control groups (p=0.0014). IL-10 also showed higher expression levels between MI, UA groups and Controls (p=0.0299).This up-regulation may reflect the extent of plaque instability and/or rupture in MI patients.Our observations provide evidence that IFNgamma and IL-10 merit further investigation in atherosclerotic disease states as potential markers of disease and therapeutic targets.

Coeliac disease-associated risk variants in TNFAIP3 and REL implicate altered NF-kappaB signalling.

OBJECTIVE: Our previous coeliac disease genome-wide association study (GWAS) implicated risk variants in the human leucocyte antigen (HLA) region and eight novel risk regions. To identify more coeliac disease loci, we selected 458 single nucleotide polymorphisms (SNPs) that showed more modest association in the GWAS for genotyping and analysis in four independent cohorts. DESIGN: 458 SNPs were assayed in 1682 cases and 3258 controls from three populations (UK, Irish and Dutch). We combined the results with the original GWAS cohort (767 UK cases and 1422 controls); six SNPs showed association with p<1 x 10(-04) and were then genotyped in an independent Italian coeliac cohort (538 cases and 593 controls). RESULTS: We identified two novel coeliac disease risk regions: 6q23.3 (OLIG3-TNFAIP3) and 2p16.1 (REL), both of which reached genome-wide significance in the combined analysis of all 2987 cases and 5273 controls (rs2327832 p = 1.3 x 10(-08), and rs842647 p = 5.2 x 10(-07)). We investigated the expression of these genes in the RNA isolated from biopsies and from whole blood RNA. We did not observe any changes in gene expression, nor in the correlation of genotype with gene expression. CONCLUSIONS: Both TNFAIP3 (A20, at the protein level) and REL are key mediators in the nuclear factor kappa B (NF-kappaB) inflammatory signalling pathway. For the first time, a role for primary heritable variation in this important biological pathway predisposing to coeliac disease has been identified. Currently, the HLA risk factors and the 10 established non-HLA risk factors explain approximately 40% of the heritability of coeliac disease.

Comparison of three cell fixation methods for high content analysis assays utilizing quantum dots.

Semiconductor nanoparticles or quantum dots are being increasingly utilized as fluorescent probes in cell biology both in live and fixed cell assays. Quantum dots possess an immense potential for use in multiplexing assays that can be run on high content screening analysers. Depending on the nature of the biological target under investigation, experiments are frequently required on cells retaining an intact cell membrane or also on those that have been fixed and permeabilized to expose intracellular antigens. Fixation of cell lines before or after the addition of quantum dots may affect their localization, emission properties and stability. Using a high content analysis platform we perform a quantitative comparative analysis of three common fixation techniques in two different cell lines exposed to carboxylic acid stabilized CdTe quantum dots. Our study demonstrates that in prefixed and permeabilized cells, quantum dots are readily internalized regardless of cell type, and their intracellular location is primarily determined by the properties of the quantum dots themselves. However, if the fixation procedures are preformed on live cells previously incubated with quantum dots, other important factors have to be considered. The choice of the fixative significantly influences the fluorescent characteristics of the quantum dots. Fixatives, regardless of their chemical nature, negatively affected quantum dots fluorescence intensity. Comparative analysis of gluteraldehyde, methanol and paraformaldehyde demonstrated that 2% paraformaldehyde was the fixative of choice. The presence of protein in the media did not significantly alter the quantum dot fluorescence. This study indicates that multiplexing assays utilizing quantum dots, despite being a cutting edge tool for high content cell imaging, still require careful consideration of the basic steps in biological sample processing.

A calcified lung tumour and microcytic anaemia in a young woman: partial expression of the Carney triad.

The Carney triad is the non-familial addociation of gastric stromal tumours (GISTs), pulmonar chondromas and extra-adrenal paragangliomas. Fewer than 100 cases of the disorder have been reported since its description in 1977. The condition has a predeliction for young women. Most patients exhibit only two of the three components. The tumours tend to be multifocal in the affected organ or system. Herein, we describe the case of a 27-year-old woman with multiple gastric GISTs and a pulmonary chondroma, partial expression of the Carney triad. It is important to be aware of the Carney triad when one of its constituent tumours is found, particularly if the patient is a young woman, so that a search can be made for and surveillance instituted for the other components. Treatment for the gastric tumours (sarcomas) and the paragangliomas (potentially malignant) is surgical. The lung chondromas are benign neoplasms and ordinarily not symptomatic. If a diagnosis of the tumour can be established by biopsy, surgical resection may not be necessary.

Characterization of tumour necrosis factor-alpha genetic variants and mRNA expression in patients with severe sepsis.

Tumour necrosis factor-alpha (TNFalpha) has been implicated in the pathogenicity of severe sepsis by both genetic association studies and animal models. Conflicting functional data have emerged in relation to genetic variants and TNFalpha protein production. Therefore, we assessed the functionality of TNFalpha genetic variants in terms of mRNA production and their potential influence on outcome in the setting of severe sepsis. Sixty-two Irish Caucasian patients presenting with severe sepsis were recruited and TNFalpha mRNA and protein levels were quantified. Patient DNA was analysed for the presence of common promoter polymorphisms and haplotypes were inferred. An A allele at position -863 was associated with more TNFalpha mRNA on day 1 compared to C homozygotes (P = 0.037). There was a trend for G homozygotes at position -308 to produce more TNFalpha mRNA on day 1 than those carrying an A allele (P = 0.059). The presence of an A allele at -863 was associated with greater levels of TNFalpha mRNA in comparison with patients carrying the A allele at -308 on day 1 (P = 0.02). Patients homozygous for the A allele at position -308 had a higher mortality than those carrying the G allele (P = 0.01). Our data are consistent with recent reports suggesting that a deficient proinflammatory response may be harmful in human sepsis. This deficient inflammatory response may be mediated in part by polymorphisms in the TNFalpha promoter.

The impact of inherited prothrombotic risk factors on individuals chronically infected with hepatitis C virus from a single source.

Intrahepatic thrombotic events have been postulated to play a key role in the pathogenesis of hepatic fibrosis. Genetic and acquired thrombotic risk factors may therefore contribute to the varying rates of fibrosis progression observed in patients with chronic hepatitis C virus (HCV) infection. The aim of this study was to assess the impact of inherited mutations in factor V and factor II (prothrombin) on hepatic fibrosis progression rates in individuals infected with HCV. Two hundred and ten Irish women infected with HCV genotype 1b, contracted from a single source (HCV-contaminated anti-D immunoglobulin) were genotyped for the factor V Leiden G1691A and prothrombin G20210A polymorphisms, and compared with Irish Caucasoid controls. Index and subsequent liver biopsies were scored (Ishak scoring system) by a single pathologist. Statistical analysis was performed using SPSS. Factor V Leiden and prothrombin G20210A heterozygosity were determined in 3.7% and 1.85%, respectively, of the study population. There was no association between these polymorphisms and fibrotic score on the index biopsy, or degree of change in fibrotic score on subsequent biopsies. The mean fibrotic score for factor V wild type was 1.06 vs 0.71 for the heterozygotes (P = 0.89). The mean change in fibrotic scores between subsequent biopsies was 0.72 for factor V wild type vs 0.50 for heterozygotes (P = 0.68). Similarly, there was no significant difference in fibrotic score for those with the prothrombin G20210A polymorphism (P = 0.936). Alanine aminotransferase levels for factor V wild type were significantly lower than those for the heterozygotes, 45.9 vs 57 (P = 0.032). Factor V Leiden and prothrombin G20210A heterozygosity rates were infrequently detected in this HCV cohort and were similar to rates seen in a Caucasian Irish control population. In this cohort, neither factor V Leiden nor prothrombin G20210A polymorphisms had a significant impact on fibrotic scores or degree of change between subsequent biopsies. These data do not support a key role for thrombotic risk factors in fibrogenesis in HCV-infected patients.

Levels of interpopulation differentiation among different functional classes of immunologically important genes.

It has been postulated that gene function may influence the degree to which allele frequencies differ among populations. In order to evaluate this effect, genotypic data from resequencing studies of genes classified as cytokines, cytokine receptors, cell adhesion molecules, Toll-like receptors and coagulation proteins were analysed for genetic differentiation (FST) between population samples of European and African descent. FST values did not differ statistically among functional groups when all polymorphic sites were included in the analyses. However, analysis based on nonsynonymous SNPs alone suggested weak heterogeneity among functional classes (P=0.0424). Particularly high levels of differentiation were shown by individual nonsynonymous SNPs at some genes, most notably ICAM1 and some Toll-like receptors. These genes interact directly with pathogens, and may therefore have been subject to geographically localised natural selection. Such loci warrant particular attention in studies of genetic disease risk and local adaptation to environmental conditions.

Haplotypes in the CTLA4 region are associated with coeliac disease in the Irish population.

Chromosomal region 2q33 encodes the immune regulatory genes, CTLA4, ICOS and CD28, which are involved in regulation of T-cell activity and has been studied as a candidate gene locus in autoimmune diseases, including coeliac disease (CD). We have investigated whether an association exists between this region and CD in the Irish population using a comprehensive analysis for genetic variation. Using a haplotype-tagging approach, this gene cluster was investigated for disease association in a case-control study comprising 394 CD patients and 421 ethnically matched healthy controls. Several SNPs, including CTLA4_CT60, showed association with disease; however, after correction for multiple-testing, CTLA4-658C/T was the only polymorphism found to show significant association with disease when allele, genotype, or carrier status frequency were analysed (carrier status (Allele C), P = 0.0016). Haplotype analysis revealed a haplotype incorporating the CD28/CTLA4 and two 5' ICOS polymorphisms to be significantly associated with disease (patients 24.1%; controls 31.5%; P = 0.035), as was a shorter haplotype composed of the CTLA4 markers only (30.9 vs 34.9%; P = 0.042). The extended haplotype incorporating CD28/CTLA4 and 5' ICOS is more strongly associated with disease than haplotypes of individual genes. This suggests a causal variant associated with this haplotype may be associated with disease in this population.

Vitamin C enhances chemosensitization of esophageal cancer cells in vitro.

Chemotherapy is increasingly utilised in multimodal protocols to try and improve outcomes. Cisplatin and 5-fluorouracil (5-Fu) are the mainstay of chemotherapeutic regimens, and an understanding of sensitivity and resistance of esophageal cancer to these agents is of considerable clinical importance. Antioxidants may modulate the response to chemotherapy, and in this study we examined the effect of vitamin C on 5-Fu and cisplatin cytotoxicity and related pathways in the esophageal cancer cell lines OE33 and SKGT-4. The antiproliferative effect of antitumor agents was measured by the MTT assay, and the transcription factors NF-kappaB and AP-1 pathways were assessed by electrophoretic mobility gel shift assay. 5-Fu and cisplatin demonstrated marked morphological changes and decreased cell proliferation. A combination of vitamin C with 5-Fu or cisplatin exerted a significantly enhanced cytotoxic effect compared to both drugs individually. Treatment of esophageal cancer cells with 5-Fu and cisplatin induced NF-kappaB and AP-1 activation. Pretreatment with vitamin C inhibited 5-Fu or cisplatin induced NF-kappaB nuclear translocation and DNA binding activity, but vitamin C had no effect on IkappaB-alpha protein levels. Vitamin C also inhibited 5-Fu- and cisplatin-induced AP-1 activation. Our data demonstrate that vitamin C enhances the antitumor activity of 5-Fu and cisplatin, in part by inhibiting translocation of NF-kappaB and AP-1, and sensitizes cancer cells to drug-induced cell death. The data suggest that vitamin C supplementation may improve the efficacy of chemotherapy for esophageal cancer.