Genetic and Immunological Mechanisms Regulating Neuroinflammation

Multiple Sclerosis (MS) is the most common neurological disorder in young adults and imposes both health and socioeconomic urdens on society. The cause and aetiology of MS are incompletely understood and current treatments are inadequate. Pathologically, prolonged chronic inflammation and widespread demyelination in the central nervous system leads to atrophy and progressive worsening of disease. This thesis combined use of in vivo animal models, in vitro cellular assays and in silico computational methods to characterise pathogenic mechanisms and translate findings from models to human disease. The animal model of MS, experimental autoimmune encephalomyelitis (EAE), was evaluated in light of novel findings in MS aetiology and further analyzed to explore differences in strain susceptibility. Susceptible rats had increased interleukin 7 receptor (Il7r) and Il2ra expression as well as altered isoform signatures in naïve lymphoid tissue, setting the stage for T cell differentiation towards pathogenic T helper 1 (TH1) and TH17 subtypes. Moreover, increased Il18r1 expression described in susceptible rats was explored in MS. Dysregulation of this receptor can mediate disease initiation through T cell differentiation as well as T cell and macrophage activation. IL18R1 levels were increased in peripheral immune and central nervous tissues in MS. Inflammatory molecules that are dysregulated in EAE likely represent true pathogenic mechanisms in humans. Multiple approaches were used to define tumour necrosis factor (TNF) regulation of disease severity. A region on chromosome 4 in the rat regulated TNF production in macrophages following innate inflammatory stimulation. Additional inflammatory molecules were also genetically regulated, modifying the cellular phenotype and severity of multiple diseases. This specific inflammatory control provides insight into disease pathogenesis and future treatment options. The approach of combining genetic and immunological approaches in both models and human samples will continue to improve disease understanding and provide novel therapeutics through identification of key regulators and general immune and non-immune pathways

Screening for type 2 diabetes : literature review and economic modelling

Objectives: To reconsider the aims of screening for undiagnosed diabetes, and whether screening should be for other abnormalities of glucose metabolism such as impaired glucose tolerance (IGT), or the ‘metabolic syndrome’. Also to update the previous review for the National Screening Committee (NSC) on screening for diabetes, including reviewing choice of screening test; to consider what measures would be taken if IGT and impaired fasting glucose (IFG) were identified by screening, and in particular to examine evidence on treatment to prevent progression to diabetes in these groups; to examine the cost-effectiveness of screening; and to consider groups at higher risk at which screening might be targeted. Data sources: Electronic databases were searched up to the end of June 2005. Review methods: Literature searches and review concentrated on evidence published since the last review of screening, both reviews and primary studies. The review of economic studies included only those models that covered screening. The new modelling extended an existing diabetes treatment model by developing a screening module. The NSC has a set of criteria, which it applies to new screening proposals. These criteria cover the condition, the screening test or tests, treatment and the screening programme. Screening for diabetes was considered using these criteria. Results: Detection of lesser degrees of glucose intolerance such as IGT is worthwhile, partly because the risk of cardiovascular disease (CVD) can be reduced by treatment aimed at reducing cholesterol level and blood pressure, and partly because some diabetes can be prevented. Several trials have shown that both lifestyle measures and pharmacological treatment can reduce the proportion of people with IGT who would otherwise develop diabetes. Screening could be two-stage, starting with the selection of people at higher risk. The second-stage choice of test for blood glucose remains a problem, as in the last review for NSC. The best test is the oral glucose tolerance test (OGTT), but it is the most expensive, is inconvenient and has weak reproducibility. Fasting plasma glucose would miss people with IGT. Glycatedhaemoglobin does not require fasting, and may be the best compromise. It may be that more people would be tested and diagnosed if the more convenient test was used, rather than the OGTT. Five economic studies assessed the costs and short-term outcomes of using different screening tests. None examined the long-term impact of different proportions of false negatives. All considered the costs that would be incurred and the numbers identified by different tests, or different cut-offs. Results differed depending on different assumptions. They did not give a clear guide as to which test would be the best in any UK screening programme, but all recognised that the choice of cut-off would be a compromise between sensitivity and specificity; there is no perfect test. The modelling exercise concluded that screening for diabetes appears to be cost-effective for the 40–70-year age band, more so for the older age bands, but even in the 40–49-year age group, the incremental cost-effectiveness ratio for screening versus no screening is only £10,216 per quality-adjusted life-year. Screening is more cost-effective for people in the hypertensive and obese subgroups and the costs of screening are offset in many groups by lower future treatment costs. The cost-effectiveness of screening is determined as much by, if not more than, assumptions about the degree of control of blood glucose and future treatment protocols than by assumptions relating to the screening programme. The very low cost now of statins is also an important factor. Although the prevalence of diabetes increases with age, the relative risk of CVD falls, reducing the benefits of screening. Screening for diabetes meets most of the NSC criteria, but probably fails on three: criterion 12, on optimisation of existing management of the condition; criterion 13, which requires that there should be evidence from high-quality randomised controlled trials (RCTs) showing that a screening programme would reduce mortality or morbidity; and criterion 18, that there should be adequate staffing and facilities for all aspects of the programme. It is uncertain whether criterion 19, that all other options, including prevention, should have been considered, is met. The issue here is whether all methods of improving lifestyles in order to reduce obesity and increase exercise have been sufficiently tried. The rise in overweight and obesity suggests that health promotion interventions have not so far been effective. Conclusions: The case for screening for undiagnosed diabetes is probably somewhat stronger than it was at the last review, because of the greater options for reduction of CVD, principally through the use of statins, and because of the rising prevalence of obesity and hence type 2 diabetes. However, there is also a good case for screening for IGT, with the aim of preventing some future diabetes and reducing CVD. Further research is needed into the duration of undiagnosed diabetes, and whether the rise in blood glucose levels is linear throughout or whether there may be a slower initial phase followed by an acceleration around the time of clinical diagnosis. This has implications for the interval after which screening would be repeated. Further research is also needed into the natural history of IGT, and in particular what determines progression to diabetes. An RCT of the type required by NSC criterion 13 is under way but will not report for about 7 years

Non-pharmacological interventions to reduce the risk of diabetes in people with impaired glucose regulation : a systematic review and economic evaluation

Background The prevalence of type 2 diabetes mellitus (T2DM) is increasing in the UK and worldwide. Before the onset of T2DM, there are two conditions characterised by blood glucose levels that are above normal but below the threshold for diabetes. If screening for T2DM in introduced, many people with impaired glucose tolerance (IGT) will be found and it is necessary to consider how they should be treated. The number would depend on what screening test was used and what cut-offs were chosen. Objective To review the clinical effectiveness and cost-effectiveness of non-pharmacological interventions, including diet and physical activity, for the prevention of T2DM in people with intermediate hyperglycaemia. Data sources Electronic databases, MEDLINE (1996–2011), EMBASE (1980–2011) and all sections of The Cochrane Library, were searched for systematic reviews, randomised controlled trials (RCTs) and other relevant literature on the effectiveness of diet and/or physical activity in preventing, or delaying, progression to T2DM.The databases were also searched for studies on the cost-effectiveness of interventions. Review methods The review of clinical effectiveness was based mainly on RCTs, which were critically appraised. Subjects were people with intermediate hyperglycaemia, mainly with IGT. Interventions could be diet alone, physical activity alone, or the combination. For cost-effectiveness analysis, we updated the Sheffield economic model of T2DM. Modelling based on RCTs may not reflect what happens in routine care so we created a ‘real-life’ modelling scenario wherein people would try lifestyle change but switch to metformin after 1 year if they failed. Results Nine RCTs compared lifestyle interventions (predominantly dietary and physical activity advice, with regular reinforcement and frequent follow-up) with standard care. The primary outcome was progression to diabetes. In most trials, progression was reduced, by over half in some trials. The best effects were seen in participants who adhered best to the lifestyle changes; a scenario of a trial of lifestyle change but a switch to metformin after 1 year in those who did not adhere sufficiently appeared to be the most cost-effective option. Limitations Participants in the RCTs were volunteers and their results may have been better than in general populations. Even among the volunteers, many did not adhere. Some studies were not long enough to show whether the interventions reduced cardiovascular mortality as well as diabetes. The main problem is that we know what people should do to reduce progression, but not how to persuade most to do it. Conclusion In people with IGT, dietary change to ensure weight loss, coupled with physical activity, is clinically effective and cost-effective in reducing progression to diabetes

Increasing boys' and girls' intention to avoid teenage pregnancy: a cluster randomised control feasibility trial of an interactive video drama based intervention in post-primary schools in Northern Ireland

Background: Adolescent men have a vital yet neglected role in reducing unintended teenage pregnancy (UTP). There is a need for gender-sensitive educational interventions. Objectives: To determine the value and feasibility of conducting an effectiveness trial of the If I Were Jack Relationship and Sexuality Education (RSE) intervention in a convenience quota sample of post-primary schools in Northern Ireland. Secondary objectives were to assess acceptability to schools, pupils (male/female, aged 14–15 years) and parents/guardians; to identify optimal delivery structures and systems; to establish participation rates and reach, including equality of engagement of different socioeconomic and religious types; to assess trial recruitment and retention rates; to assess variation in normal RSE practice; to refine survey instruments; to assess differences in outcomes for male and female pupils; to identify potential effect sizes that might be detected in an effectiveness trial and estimate appropriate sample size for that trial; and to identify costs of delivery and pilot methods for assessing cost-effectiveness. Design: Cluster randomised Phase II feasibility trial with an embedded process and economic evaluation. Intervention: A teacher-delivered classroom-based RSE resource – an interactive video drama (IVD) with classroom materials, teacher training and an information session for parents – to immerse young people in a hypothetical scenario of Jack, a teenager whose girlfriend is unintentionally pregnant. It addresses gender inequalities in RSE by focusing on young men and is designed to increase intentions to avoid UTP by encouraging young people to delay sexual intercourse and to use contraception consistently in sexual relationships. Main outcome measures: Abstinence from sexual intercourse (delaying initiation of sex or returning to abstinence) or avoidance of unprotected sexual intercourse (consistent correct use of contraception). Secondary outcomes included Knowledge, Attitudes, Skills and Intentions. Results: The intervention proved acceptable to schools, pupils and parents, as evidenced through positive process evaluation. One minor refinement to the parental component was required, namely the replacement of the teacher-led face-to-face information session for parents by online videos designed to deliver the intervention to parents/guardians into their home. School recruitment was successful (target 25%, achieved 38%). No school dropped out. Pupil retention was successful (target 85%, achieved 93%). The between-group difference in incidence of unprotected sex of 1.3% (95% confidence interval 0.55% to 2.2%) by 9 months demonstrated an effect size consistent with those reported to have had meaningful impact on UTP rates (resulting in an achievable sample size of 66 schools at Phase III). Survey instruments showed high acceptability and reliability of measures (Cronbach’s alpha: 0.5–0.7). Economic evaluation at Phase III is feasible because it was possible to (1) identify costs of delivering If I Were Jack (mean cost per pupil, including training of teachers, was calculated as £13.66); and (2) develop a framework for assessing cost-effectiveness. Conclusion: Trial methods were appropriate, and recruitment and retention of schools and pupils was satisfactory, successfully demonstrating all criteria for progression to a main trial. The perceived value of culture- and gender-sensitive public health interventions has been highlighted. Future work: Progression to a Phase III effectiveness trial. Trial registration: Current Controlled Trials ISRCTN99459996. Funding: This project was funded by the NIHR Public Health Research programme and will be published in full in Public Health Research; Vol. 5, No. 1. See the NIHR Journals Library website for further project information

A potential new, stable state of the E-cadherin strand-swapped dimer in solution

E-cadherin is a transmembrane glycoprotein that facilitates inter-cellular adhesion in the epithelium. The ectodomain of the native structure is comprised of five repeated immunoglobulin-like domains. All E-cadherin crystal structures show the protein in one of three alternative conformations: a monomer, a strand-swapped trans homodimer and the so-called X-dimer, which is proposed to be a kinetic intermediate to forming the strand-swapped trans homodimer. However, previous studies have indicated that even once the trans strand-swapped dimer is formed, the complex is highly dynamic and the E-cadherin monomers may reorient relative to each other. Here, molecular dynamics simulations have been used to investigate the stability and conformational flexibility of the human E-cadherin trans strand-swapped dimer. In four independent, 100 ns simulations, the dimer moved away from the starting structure and converged to a previously unreported structure, which we call the Y-dimer. The Y-dimer was present for over 90% of the combined simulation time, suggesting that it represents a stable conformation of the E-cadherin dimer in solution. The Y-dimer conformation is stabilised by interactions present in both the trans strand-swapped dimer and X-dimer crystal structures, as well as additional interactions not found in any E-cadherin dimer crystal structures. The Y-dimer represents a previously unreported, stable conformation of the human E-cadherin trans strand-swapped dimer and suggests that the available crystal structures do not fully capture the conformations that the human E-cadherin trans homodimer adopts in solution

Osteoprotegerin antibodies in the pathogenesis of osteoporosis

Osteoporosis is a common complication of many autoimmune diseases that is typically attributed to disease specific factors rather than a direct autoimmune process. This thesis arises from the investigation of a patient with severe high bone turnover osteoporosis who was identified as having autoimmune disease but whose osteoporosis deteriorated despite appropriate treatment. This presentation led to the hypothesis that neutralising autoantibodies to the bone protective cytokine osteoprotegerin (OPG) may have developed. Serum from the index patient, but not healthy controls, was able to immunoprecipitate recombinant OPG protein, demonstrating that OPG had become the target of an autoimmune response. Purified immunoglobulins from the index case were able to inhibit the function of OPG in vitro, by suppressing OPG-mediated inhibition of a luciferase reporter cell line. This represents the first description of disease associated with neutralising antibodies to OPG. Whilst the immunoprecipitation assay did identify OPG antibodies in further patients these results were difficult to quantify. A more robust enzyme linked immunosorbent assay for OPG antibodies was developed using OPG as a capture antigen, which allowed the screening of patient cohorts. Presence of OPG antibodies was defined as a titre greater than the mean plus three standard deviations of 101 healthy volunteers. A low prevalence of 14/864 (1.6%) was seen in a general population cohort and no association with bone density or turnover was seen. An association with higher vascular calcification score in this cohort requires replication. A prevalence of 37/315 (11.7%) was seen in an osteoporosis cohort though no association was seen with bone density or response to treatment. In a coeliac cohort OPG antibodies were identified in 14/282 (5.0%) patients and presence of antibody was independently associated with reduced spine bone density. Functional inhibition of OPG was shown in vitro in 3/14 (21.4%) of the positive cases. Case finding of osteoporosis in the coeliac cohort was not improved by identification of OPG antibodies. These results are consistent with OPG antibodies being pathological in a small number of patients with osteoporosis but a clinical utility of measuring OPG antibodies has not been established

Natural Polymorphisms in Tap2 Influence Negative Selection and CD4 : CD8 Lineage Commitment in the Rat

Contains fulltext : 136368.pdf (publisher's version ) (Open Access)Genetic variation in the major histocompatibility complex (MHC) affects CD4ratioCD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4ratioCD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4ratioCD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of approximately 0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells

Genetic predisposition to in situ and invasive lobular carcinoma of the breast.

Invasive lobular breast cancer (ILC) accounts for 10-15% of all invasive breast carcinomas. It is generally ER positive (ER+) and often associated with lobular carcinoma in situ (LCIS). Genome-wide association studies have identified more than 70 common polymorphisms that predispose to breast cancer, but these studies included predominantly ductal (IDC) carcinomas. To identify novel common polymorphisms that predispose to ILC and LCIS, we pooled data from 6,023 cases (5,622 ILC, 401 pure LCIS) and 34,271 controls from 36 studies genotyped using the iCOGS chip. Six novel SNPs most strongly associated with ILC/LCIS in the pooled analysis were genotyped in a further 516 lobular cases (482 ILC, 36 LCIS) and 1,467 controls. These analyses identified a lobular-specific SNP at 7q34 (rs11977670, OR (95%CI) for ILC = 1.13 (1.09-1.18), P = 6.0 × 10(-10); P-het for ILC vs IDC ER+ tumors = 1.8 × 10(-4)). Of the 75 known breast cancer polymorphisms that were genotyped, 56 were associated with ILC and 15 with LCIS at P<0.05. Two SNPs showed significantly stronger associations for ILC than LCIS (rs2981579/10q26/FGFR2, P-het = 0.04 and rs889312/5q11/MAP3K1, P-het = 0.03); and two showed stronger associations for LCIS than ILC (rs6678914/1q32/LGR6, P-het = 0.001 and rs1752911/6q14, P-het = 0.04). In addition, seven of the 75 known loci showed significant differences between ER+ tumors with IDC and ILC histology, three of these showing stronger associations for ILC (rs11249433/1p11, rs2981579/10q26/FGFR2 and rs10995190/10q21/ZNF365) and four associated only with IDC (5p12/rs10941679; rs2588809/14q24/RAD51L1, rs6472903/8q21 and rs1550623/2q31/CDCA7). In conclusion, we have identified one novel lobular breast cancer specific predisposition polymorphism at 7q34, and shown for the first time that common breast cancer polymorphisms predispose to LCIS. We have shown that many of the ER+ breast cancer predisposition loci also predispose to ILC, although there is some heterogeneity between ER+ lobular and ER+ IDC tumors. These data provide evidence for overlapping, but distinct etiological pathways within ER+ breast cancer between morphological subtypes

Carboplatin in BRCA1/2-mutated and triple-negative breast cancer BRCAness subgroups: the TNT Trial

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative ‘BRCAness’ subgroups—tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes—may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker–treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected population (376 subjects; 188 carboplatin, 188 docetaxel), carboplatin was not more active than docetaxel (ORR, 31.4% versus 34.0%, respectively; P = 0.66). In contrast, in subjects with gBRCA-BC, carboplatin had double the ORR of docetaxel (68% versus 33%, respectively; biomarker, treatment interaction P = 0.01). Such benefit was not observed for subjects with BRCA1 methylation, BRCA1 mRNA-low tumors or a high score in a Myriad HRD assay. Significant interaction between treatment and the basal-like subtype was driven by high docetaxel response in the nonbasal subgroup. We conclude that patients with advanced TNBC benefit from characterization of BRCA1/2 mutations, but not BRCA1 methylation or Myriad HRD analyses, to inform choices on platinum-based chemotherapy. Additionally, gene expression analysis of basal-like cancers may also influence treatment selection