Dissertationen zum Thema „Rheumatoid arthritis“

The literature is replete with reports of osteoporosis in rheumatoid arthritis, but the mechanism of bone loss remains obscure. This is probably due to the overlap with bone loss of aging and the menopause, whose exact mechanisms are also poorly understood. Against this background, a study was designed to evaluate generalised bone loss in young, premenopausal (if female), patients with rheumatoid arthritis. The protocol was designed to record demographic data, as well as information pertaining to the disease. Cortical bone mass was measured at the metacarpals and left femur, using an automated, computer-controlled technique. Trabecular bone was evaluated at the left femur (Singh index) as well as at the 3rd lumbar vertebra (Saville index). Bone kinetics were studied by the measurement of urinary excretion of calcium, phosphate and hydroxy-praline (resorption) and serum alkaline phosphatase (formation). Disease activity was measured clinically and with laboratory indices. Physical activity was indirectly measured by quantitating the disability, using the Keitel function test as well as a modified health assessment questionnaire (HAQ). The radiograph of the right wrist was scored by the Larsen index. The carpometacarpal ratio was also calculated from the radiograph. Numerous statistical techniques were applied in the analysis of the data. Healthy volunteers were used as controls. Patients with SLE were also studied, in order to compare the 2 inflammatory diseases. Patients with RA had generalised cortical bone loss (metacarpal and femur) (p < 0.001). Trabecular bone measurements were not significantly different from normals, using the crude radiographic techniques. Duration of disease was the most important clinical determinant of this bone loss. The relative contributions of disease activity and lack of physical activity to the loss of bone could not be adequately separated using conventional statistical techniques. Corticosteroid therapy did not promote metacarpal bone loss in these subjects, but may have contributed to thinning of the femoral cortex. Nonsteroidal anti-inflammatory drugs and disease modifying agents did not seem to influence the extent of the bone loss. Nutritional status and skinfold thickness did not correlate with bone mass. Dietary factors played no role in the genesis of bone loss, but may have had some effect on disease activity. Metacarpal measurements showed a sensitivity of 80% and specificity of 85% in discriminating between osteopaenic and normopaenic groups with RA. Osteopaenia could not be adequately predicted in the absence of metacarpal measurements. Metacarpal bone loss in RA was due to endosteal resorption, while in SLE it was due to periosteal resorption. The semi-automatic technique for measurement of metacarpal bone mass showed good reproducibility among 5 observers and at 2 different centres. The pathogenesis of bone loss in RA was multifactorial, the largest contribution probably coming from a humoral factor in the circulation, closely related to disease activity. Ionised calcium was elevated in 55% of RA patients, but only 5% of SLE patients. Serum PTH levels were normal in 99% of the RA subjects. Elevations in alkaline phosphatase. (25%) probably reflected disease activity rather than increased bone formation. Factor analysis of 27 variables showed that disease activity was central to the development of OP in RA. CS therapy tended to be used in the presence of active disease. Disability was not an important determinant of bone loss in RA, but may be a useful measure of activity of the disease. This study did not evaluate the relationships with sex hormonal status or vitamin D metabolism. Future research should aim at cohort analysis at 2 different periods, in order to improve our understanding of the pathogenesis of bone loss in RA.

Introduction: Rheumatoid arthritis (RA) is known to be associated with an increased risk of cardiovascular disease (CVD), resulting in excess mortality. Dyslipidaemia has been identified as a major CVD risk factor in the general population. Current evidence would suggest that lipid metabolism is altered in RA due to inflammation, and that use of anti-inflammatory therapy may reverse some of these changes. However, the impact of such lipid changes on CVD in RA remains unknown. Data regarding the effects of RA/drug therapy on lipid structure and function are sparse. Genetic factors are important in the pathogenesis of RA and play a central role in the regulation of lipid metabolism. To date, no studies have assessed the impact of genetic polymorphisms on lipids in RA.The aim of this thesis is to: 1) assess the prevalence of dyslipidaemia in RA and the CVD risk this confers 2) establish the effects of inflammation on lipid levels and lipid ratios 3) assess the impact of anti-inflammatory drug therapy (anti-TNF, rituximab and intravenous glucocorticoids) on lipid levels, structure and function 4) assess the prevalence and associations of particular genetic polymorphisms (RA susceptibility and lipid metabolism regulating genes) with lipids in RA.Methods: Data from 400 RA patients were used to address aims 1, 2 and 4 in cross-sectional studies. All patients had a clinical assessment and fasting blood taken. Blood was processed to provide data on the lipid profile, ESR, CRP and DNA was extracted for genotyping. Aim 2 and 4 also utilised a retrospective longitudinal cohort of 550 RA patients and the DNA from 400 healthy controls, respectively. Aim 3 was addressed using a longitudinal cohort including: patients due to commence anti-TNF (n=35), rituximab (n=10), intravenous glucocorticoids (n=12); 15 RA controls on stable therapy; and 40 healthy controls. Assessments and blood samples were taken at baseline, 2 weeks and 3 months. Results: Dyslipidaemia was highly prevalent (56.8%), but undertreated in many RA patients at risk of developing CVD. Systemic inflammation associated with many of the changes in lipid levels and structure. Lipid ratios were found to be less susceptible to fluctuations due to inflammation. The use of anti-inflammatory drug therapy produced changes in lipid structure and function through both generic suppression of inflammation and drug specific mechanisms (particularly in the case of glucocorticoids). The prevalence of cholesterol ester transfer protein (CETP) and Apolipoprotein C3 genetic polymorphisms differed between RA patients and local population controls. RA susceptibility genes (HLA-DRB1-SE and TRAF1C5) and several ’lipid metabolism genes’ (Apolipoprotein E, ATP-binding cassette transporter 1 (ABCA1) and CETP) were found to associate with lipid levels within the RA population. Conclusion: Dyslipidaemia is highly prevalent in RA and currently undertreated. Dyslipidaemia in RA is regulated by numerous factors including inflammation, drug therapy and genetic factors. Further longitudinal studies are required to assess whether these findings have an impact on hard CVD endpoints.

This study was designed to investigate the experience of having arthritic pain from the children's perspective. Previous research on how Canadian children perceive and manage arthritic pain and how it affects their daily lives is nonexistent. Therefore the purpose of this qualitative descriptive study was to explore and describe the arthritic pain experience of school age children with juvenile rheumatoid arthritis (JRA) and to understand the impact/influence of various factors on the construction of that experience. Ten children, aged 10 to 13 years, with either early (at 2 to 4 years) or late (at 7 to 11 years) onset arthritis participated in this study. Descriptive data were obtained during two open-ended in depth interviews with the children in their homes. Using content analysis, data were analyzed for themes and their elements. An analytical framework of themes and their elements was developed that reflected the children's descriptions of and explanations for arthritic pain in the context of their day to day in the context of their day to day living with arthritis, both in the past and currently. The children perceived pain to be synonymous with arthritis and the mediating factor in how they functioned. They described arthritic pain in relation to distinguishing factors: intensity, duration, and frequency. Intermittent arthritic pain was attributed to cessation of medications, arthritis "flare-ups," inactivity, and activity. A current concern for most children was pain attributed to activity because it meant limitations in activities with peers. The children identified strategies they used to manage pain and cope with pain's unpredictability. The findings of this study were discussed in relation to selected research studies that either supported or refuted the findings of this study. Implications for nursing practice and research were addressed.<br>Applied Science, Faculty of<br>Nursing, School of<br>Graduate

These findings suggest that systemic and vascular wall levels of matrix metalloproteinase-9, related to inflammation at the joint site, may play a prominent role in the development of vascular dysfunction in this experimental model. This thesis goes someway to elucidating the potential mechanisms of vascular dysfunction in rheumatoid arthritis.

Genetic as well as environmental factors are believed to be of importance in the etiology of rheumatoid arthritis (RA). There are a number of previous studies of genetic markers in RA, but so far no genetic linkage and only a few associations have been found. Of the associations only one (with the HLA antigen DR4) appears to be well documented. In most previous association studies the patients have not been divided according to sex and family history of RA. In this investigation the HLA antigens A, B and DR and five serum protein systems (Bf, C3, Pi, Hp and Tf) were studied in patients with erosive rheumatoid arthritis (RA), from northern Sweden. Special attention was paid to variations in the strength of associations accord­ing to sex and family history of polyarthritis. The following results were found:  The frequency of the HLA antigen B27 was significantly increased in the North-Swedish population (16.6%) and among patients with a family history of polyarthritis (42.6%). In agree­ment with previous investigations a significantly increased frequency of the DR4 antigen was found in the RA patients.  In the properdin factor B (Bf) system the S phenotype was found to be significantly in­creased in male patients and in patients with a family history of polyarthritis, more severe form of RA and high titres of rheumatoid factor.  No significant differences with respect to phenotype or gene frequencies were found in the C3 complement system. Thus, the association between RA and C3 found in previous investiga­tions was not confirmed.  A significant increase of rare alpha-1-antitrypsin (Pi) types (MS, MZ, MF and SZ) was found among RA patients. However, the increase concerned mainly Z heterozygotes and was more strongly pronounced among male patients.  In the haptoglobin system a significant increase of the Hp^ gene and the Hp2-2 type was found among patients with a family history of polyarthritis, more pronounced among males.  A significant increase of the transferrin gene and of the 2 type was found among male RA patients, more pronounced among patients with a family history of polyarthritis. In 6 out of 8 gene loci studied significant associations were found, which is in agreement with a multifactorial etiology of RA. The results were largely in agreement with the hypothesis that associations would be expected to be stronger in males and in patients with a family history of polyarthritis. A notable finding was the high frequency of first degree relatives (around 40%) with symmetric peripheral polyarthritis of which more than 70% had a diagnosis of RA verified by hospital records.<br><p>Diss. (sammanfattning) Umeå : Umeå universitet, 1985, härtill 6 uppsatser.</p><br>digitalisering@umu

Biologic therapies have revolutionized the treatment of rheumatoid arthritis, a common chronic inflammatory disease, mainly characterized by the chronic inflammation of the joints. The activity and progression of the disease are highly variable, both between subjects and between the successive assessments for the same subject. Standardized assessments of clinical variables have been developed to reflect the disease activity and evaluate new therapies. Pharmacokinetics-pharmacodynamic (PKPD) models and methods for analyzing the generated time-course data are needed to improve the interpretation of the clinical trials’ outcomes, and to describe the variability between subjects, including patients characteristics, disease factors and the use of concomitant treatments that may affect the response to treatment. In addition, good simulation properties are also desirable for predicting clinical responses for various populations or for different dosing schedules. The aim of this thesis was to develop methods and models for analyzing pharmacokinetic and pharmacokinetic-pharmacodynamic (PKPD) data from rheumatoid arthritis patients, illustrated by treatment with a new anti-TNFα biologic drug under clinical development, certolizumab pegol. Two models were developed that characterized the relationship between the exposure to the drug and the efficacy ACR variables that represent improvement of the disease; a logistic-type Markov model for 20% improvement (ACR20) and a continuous-type Markov model for simultaneous analysis of 20% (ACR20), 50% (ACR50) and 70% (ACR70) improvement. Both models accounted for the within-subjects correlation in the successive clinical assessments and were able to capture the observed ACR responses over time. Simulations from these models of the ACR20 response rate supported dosing regimens of 400 mg at weeks 0, 2 and 4 to achieve a rapid onset of response to the treatment, followed by 200 mg every 2 weeks, or alternative maintenance regimen of 400 mg every 4 weeks. The immunogenicity induced by the biologic drug was characterized by a time to event model describing the time to appearance of antibodies directed against the drug. The immunogenicity was predicted to appear mainly during the first 3 months following the start of the treatment and to be reduced at higher trough concentrations of CZP, as well as with concomitant administration of MTX. The full time-course of sequential events, such as dose-exposure-efficacy relations, is most accurately described by a simultaneous analysis of all data. However, due to the complexity and runtime limitations of such an analysis, alternatives are often used. In this thesis, a method, IPPSE, was developed and compared to the reference simultaneous method and to existing alternative methods. The IPPSE method was shown to provide accuracy and precision of estimates similar to the simultaneous method, but with easier implementation and shorter run times. In conclusion, two PKPD models and one immunogenicity model were developed for evaluation of the response of a biologic drug against rheumatoid arthritis that allowed accurate analysis and simulation of clinical trial data, as well as serving as examples for how a model-informed basis for decisions about biological drugs can be created.

Rheumatoid arthritis (RA) is a chronic inflammatory condition with poorly understood pathophysiology and increased cardiovascular risk. The mechanisms for increased cardiovascular risk are not fully known, however one novel mechanism explored in this thesis is autonomic nervous system (ANS) dysfunction. The thesis comprises of: a systematic literature review; two case-control studies (n=30 RA patients, n=34 controls; a longitudinal case-study (n=1 RA patient)); a cohort study (n=112 RA patients); and a randomised placebo controlled crossover study (n=10 healthy controls). The work presented in this thesis demonstrates that ANS dysfunction is prevalent in ~60 % of RA patients and characterised by heightened sympathetic outflow to the peripheral vasculature (determined by muscle sympathetic nerve activity using microneurography), depressed baroreflex control of heart rate (determined using the modified Oxford technique), depressed heart rate variability and heightened vascular responses to stressors (cold pressor test and mental stress). Inflammation was associated with ANS dysfunction, and may well contribute to the increased cardiovascular risk seen in RA. Further studies are required to: confirm these findings; determine whether therapeutic strategies to restore ANS function improve prognosis in RA; and further explore the precise mechanisms by which inflammatory cytokines may influence ANS function in health and disease.

Identification of the role of T cells and their interaction with other cell types remains a major challenge to our understanding of the pathogenesis of rheumatoid arthritis. In this study we have investigated the regulation of the response of T cells infiltrating the rheumatoid joint to IL-6. Furthermore we have investigated the level of T cell activation in the early stages of rheumatoid arthritis. Interleukin-6 is an important regulator of T cell differentiation and survival. It exerts its biological function by either directly binding to the complete IL-6 receptor consisting of CD126 CD130 or via transsignaling, when sIL6R-IL6 complexes bind to CD130. This study addresses the expression and regulation of these receptor components on the T cells infiltrating the rheumatoid joint. While compared to blood T cells, CD126 expression was found at low levels on synovial fluid and tissue T cells, expression of CD130 on synovial tissue T cells was comparable to that of blood T cells, with lower levels in synovial fluid T cells, both at protein and mRNA level. When exposed to sIL6R-IL6 complexes, tissue derived T cells responded with a higher level of STAT3 phosphorylation compared to cells incubated with IL-6, suggestive of transsignaling. High CD130 expression was demonstrable in T cells in the perivascular cuff area. Among a range of cytokines tested, IL-6 reduced CD126 and CD130 expression while IL-10, which is expressed at high levels in the perivascular infiltrate, induced expression of CD130. Taken together these data suggest that the inflammatory microenvironment maintains responsiveness to IL-6 transsignalling by cytokine driven CD130 expression on CD4 positive T cells. To address the question whether the role of T cells changes during the course of progression of RA, we analysed the expression of T cells activation markers on synovial fluid and peripheral blood T cells from patients at the very early stage of disease (within 3 months of disease onset) compared to patients with established or self resolving arthritis. Expression of CD69, CD71 and HLA-DR was upregulated on synovial fluid T cells compared to peripheral blood but there were no differences between the different groups of patients. Furthermore, we quantified the proportion of T cells expressing the invariant TCR Vα24Jα18 in synovial fluid and blood of the same groups of patients. We found a lower frequency of iNKT cells in the synovial fluid of very early arthritis patients compared to other patients. While this is a preliminary result, it suggests that there may be a role for these cells in the regulation of disease susceptibility.

The principle aim of this study was to test the hypothesis that heavy smoking is an aetiological factor in RA and generates a distinct subgroup of the disease definable in terms of clinical phenotype, particularly severity. A second aim was to investigate possible molecule mechanisms linking smoking with RA and what I believe to be candidate mechanisms involving the detoxifying glutathione S transferase Mu 1 (GST M1) gene and oxidative damage to alpha 1 proteinase (alpha1 PI). These studies involved a review of the literatures regarding the link between RA and both smoking and alpha1 PI deficiency. I investigated the relationship between heavy cigarette smoking and hospital based, more severely affected RA patients. Additionally, the age of onset and smoking history was compared in familial and sporadic RA cases. Regarding smoking and severity of RA, a cohort of RA patients were studied to determine if smoking was an independent risk factor for severe RA and whether this effect was influenced by the presence (GSTM1-1) or absence (GSTM1-0) of the GST M1 gene. Oxidative damage in RA to the alpha1 PI protein was studied in relation to rheumatoid disease activity, GST M1 and cigarette smoking. The oxidative damage to alpha1 PI was measured in terms of serum levels of Immunoglobulin A-1 PI was measured in terms of serum levels of Immunoglobulin A-alpha1 PI (IgA-alpha1 PI). In summary, I have shown that heavy smoking is strongly associated with hospital based RA. Secondly, that familial RA presents at an earlier age than sporadic RA in individuals smoking at disease onset only, and that sporadic RA patients are significantly more likely to smoke at disease onset that familial RA patients. I have confirmed previous findings that raised serum IgA-alpha1 PI levels are associated with erosive as opposed to non-erosive RA cases.

Thesis: S.B. in Art and Design, Massachusetts Institute of Technology, Department of Architecture, May, 2020<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (pages 34-36).<br>The purpose of this thesis is to investigate and create more responsive and adaptive assistive technology for patients with rheumatoid arthritis (RA), using computational design methods to embed individualized data within the design and materiality. Rheumatoid arthritis is a chronic, autoimmune disease that attacks the joints and causes progressive deformity and bone erosion directed mostly at joint linings and cartilage. Living with RA means sudden flare-ups of pain and inflammation that can last anywhere from hours to months and dramatically impact the ability to accomplish ordinary tasks. While there is no cure, the disease can be slowed down through intensive drugs and or mitigated with assistive wearable devices such as braces, splints, and compressive gloves. These wearables are used to minimize swelling in affected joints, lessen ulnar deviating forces, and reduce pain. However, many people are unwilling to wear these devices because they can be quite obtrusive and hinder patients' lifestyles. Most wearables are only available in set sizes, and when sized incorrectly can aggravate pain and symptom flare-up or have no healing benefits. This thesis asks whether and how computational design methods can be applied to alleviating unique pain points faced daily by people with chronic health issues such as RA and other physical joint or musculature needs. Given that each person suffering from rheumatoid arthritis manifests the debilitating effects of the disease in different ways, this leads to the question of how more effective and personalized assistive devices can be designed using computational design methods that do not put the onus on the user to perform corrective action, but rather automatically offer responsive support as needed.<br>by Jierui Fang.<br>S.B. in Art and Design<br>S.B.inArtandDesign Massachusetts Institute of Technology, Department of Architecture

As rheumatoid arthritis (RA) is a heterogeneous disease whose course and treatment response varies between patients, a stratified approach to its management is required. This thesis aimed to facilitate the risk prediction that underpins stratified medicine in RA. Its primary aim was to improve the knowledge of which clinical and genetic factors predict RA’s onset, disease course and treatment responses. Its secondary aim was to develop a prediction modelling framework that harnessed these factors to inform clinical care. There were five key findings. Firstly, it demonstrated a significant inverse association between alcohol consumption and RA development when the evidence across published studies was pooled using meta-analytical techniques. This suggests alcohol may protect against RA. Secondly, it demonstrated that only HLA RA susceptibility variants associated with radiological progression in a clinical trial cohort of early, active RA patients. This suggests the non-HLA genetic architectures of RA susceptibility and severity may, at least partially, differ. Thirdly, it provided evidence that anti-citrullinated protein antibodies (ACPA) can identify patients with early, active RA that are most likely to benefit from combination treatments. Fourthly, it demonstrated that estimating an asymptomatic individual’s risk of RA is possible, through developing and validating a risk prediction model that uses computer simulation to improve upon the discriminative abilities of existing RA prediction models. Finally, it highlighted the importance of considering RA’s heterogeneity when assessing its predictive factors; alcohol’s likely protective effect was predominantly seen in ACPA-positive disease and genetic and environmental factors had different impacts on the risk of developing younger and older onset RA. In conclusion this thesis has contributed to stratified medicine in RA by better characterising which predictive factors are relevant to its development, severity, treatment needs and responses and developing a risk prediction modelling framework that may be applicable to many aspects of stratified care.

The heritability of Rheumatoid Arthritis is approximately 60%. Although association with the HLA region is well recognised, these genes account for only 40% of the total genetic contribution. Linkage mapping and association studies need to proceed in parallel to identify which non-MHC genes are involved in the remaining 60% of the genetic contribution. The work described in this thesis represents a systematic approach to identifying these non-HLA effects, using genome-wide linkage mapping and candidate gene methods. To identify regions exhibiting genetic linkage to rheumatoid arthritis a systematic, whole genome linkage analysis was undertaken. Two hundred and fifty-one affected sibling pairs from 182 United Kingdom families were studied using 365 highly informative microsatellite markers. Highly significant linkage was identified around the HLA region on chromosome 6 (max LOD = 4.8 at 44.9cM, p=0.000001). Eighteen other sites of nominal linkage (p < 0.05) were identified on chromosomes 1, 2, 3, 4, 6, 7, 10, 12, 14, 16, 21 and the X chromosome by single point analysis (23 markers). Eight of the non-MHC regions (on chromosomes 1, 6, 7, 14, 16, 21, and X) also showed evidence of linkage by multi-point analysis. A parallel linkage study designed to replicate the 25 regions of nominal linkage (p < 0.05) reported following a genome-wide linkage study of 97 European affected sibling pair families was also undertaken. Fifty-nine microsatellites within the 25 regions of interest (including IDDM6 on chromosome 18 and IDDM9 on chromosome 3) were used to genotype 368 affected sibling pairs from 280 families. Markers on chromosomes 12, 15, and 21 (d12s95, CYP 19, d21s1252) showed evidence of nominal linkage with p values ≤ 0.05. Markers close to IDDM 6 on chromosome 18 showed p values of between 0.1 and 0.5, not lending additional support to a locus near IDDM6 in RA. Interleukin 10 (IL10), an immunoregulatory cytokine, is a potent up-regulator of B cell production and differentiation but has anti-inflammatory capabilities and can directly down-regulate TNF, IL1, IL8 and IFNy production. Data from twin and family studies suggest large inter-individual variations in secretion which are 75% heritable and as such IL10 is a plausible candidate gene for involvement in RA. It is highly polymorphic with point mutations in the promoter region and two microsatellite loci IL10.R and IL10.G, 1.1 and 4kb upstream of the transcription initiation site. Higher levels of IL10 secretion have been associated with allele 2 of IL10.R (IL10.R2) and IL10.R3 has been associated with decreased secretion. Additionally, a case-control study including two independent Caucasian populations and one African-American cohort found an over-representation of the IL10.R2 allele with a concomitant reduction of IL10.R3 in all three rheumatoid arthritis populations. Three studies investigating IL10 were undertaken. Two case-control studies including two cohorts of racially distinct RA patients (186 UK Caucasians with severe rheumatoid arthritis and 138 South Africans of Zulu or Sotho origin) were performed. An association with RA was not confirmed in either study but demonstrated significantly different frequencies of the IL10.R2 allele in the two study populations. The third study was a family-based association study and included 163 probands and their families. Single marker and haplotypic association analysis was performed by transmission disequilibrium testing (TDT analysis) using the software package TRANSMIT. The IL10.R1 allele was transmitted to affected individuals more frequently than expected (p < 0.05) and the IL10.R3 allele was transmitted less frequently than expected (p < 0.05). This effect was particularly pronounced with the IL10.R3/IL10.G10 haplotype (p < 0.005). The still stronger negative association identified with the IL10.R3/IL10.G10 haplotype suggested it was not IL10.R itself but another polymorphism on the particular haplotype that may be primarily involved with RA. The application of these methods to the examination of the genetic component of RA is discussed. Plans for future work include systematic genome-wide screening of positional candidates and evaluation of candidates from studies of other human and animal models of inflammatory disease.

This research investigated the role of coping strategies in reducing the pain experience of rheumatoid arthritis (RA) patients over a seven-day period. Sixty-three patients completed a twice-daily structured dairy consisting of an eight scale revision of the Ways of Coping (WOC), the depression subscale of the Affects Balance Scale (ABS), and a pain visual analogue scale (VAS). Multivariate analyses for repeated measures identified two coping strategies, Self-Care and Positive Reappraisal, as significantly effective in pain reduction. Self-Care includes behavioral attempts to manage the symptoms of RA. Positive Reappraisal involves cognitive efforts to redefine pain experience in positive terms. The other six coping strategies also demonstrated a trend to increased use on days when pain decreased. These data imply that intraindividual approaches in examining the coping/pain association have potential benefit for determining a causal relation between coping and pain.<br>Medicine, Faculty of<br>Cellular and Physiological Sciences, Department of<br>Graduate

This thesis describes the use of phage display for the isolation of autoantigens in rheumatoid arthritis. The potential of the technology is demonstrated by the isolation of an autoantigen, eukaryotic translation elongation factor 1a1 (eEF 1 (x 1)) from a fibroblast cDNA library using rounds of selective enrichment with IgG from RA patients. Subsequently in order to isolate joint-specific antigens a phage-displayed cDNA library from rheumatoid pannus was generated and screened with analogous procedures. From the clones isolated, putative candidate autoantigens were identified. The presence of anti- eEF 1a1 autoantibodies in approximately 20% of patients with RA was confirmed and extended in larger panels of sera, and the finding of anti-eEF la1 shown to be relatively specific for RA. In contrast autoantibodies to the activation-induced negative regulator of T cells, CTLA-4 were not found in contrast to a previous report. The relevance of these findings for the use of antibodies in the diagnosis and prediction of disease characteristics in RA are discussed.

This thesis is about what people can do with their hands in the early stages of rheumatoid arthritis (RA). The wrist and hand are affected early in RA. Wrist and hand joint swelling, pain and deformity are all likely to contribute to the functional performance of the hand. Static splinting alongside joint protection education and active exercise is one of the most common conservative intervention strategies used by therapists. Splinting attempts to reduce local inflammation and pain, correctly position joints, minimise the occurrence or progression of deformity and maintain hand function. This thesis reviews the impact that RA can have on the hands and upper limb, examines what factors may influence function and compares the outcome measures that are used to measure this. A clinical effectiveness randomised controlled trial, examining the effectiveness of static hand splinting in early RA is described. A total of one hundred and sixteen patients with early RA were recruited onto the main trial and randomly allocated to either a splint or non-splint group. Assessments were carried out at baseline, six and 12- months. There was insufficient evidence from the results of this 12 month randomised controlled trial to suggest that using static splinting alongside standardised occupational therapy intervention was any more effective than standardised occupational therapy intervention alone in the maintenance of hand functional ability. However, when male and female participants were analysed separately young male participants could potentially experience clinically significant deleterious effects when issued with static resting splints.

Bibliography: leaves 190-206.<br>This study investigated both the mediating role of psychological adjustment in determining pain experience, disease · status, and immune function in Rheumatoid Arthritis (RA), and the value of cognitive-behavioural intervention in improving the overall health status of such patients. Two related hypotheses were tested in a matched-random assigned two-groups design, with pre-, mid-, and post-intervention assessment. Fourteen (N = 14) female RA outpatients, selected along established inclusion criteria, were allocated to either treatment (n=8) or control (n=6) groups after being matched on date of disease onset and ratings, of coping efficacy. The treatment group received an eight week Stress Inoculation and Pain Management Training programme (sixteen 2-hour sessions) based on the conceptual approach of Meichenbaum (1985) and adopted from a program by O'Leary, Shoor, Lorig and Holman (1988). The program included educational material, instruction in palliative and cognitive pain management strategies and the application thereof in daily living, goal setting to improve activity function, and group discussion. The program was designed to nurture and develop existing coping skills, and to impart new strategies to cope with daily stress and pain. Pre-intervention correlational analyses tested the extent to which mood disturbance, self-perceptions of coping efficacy, health locus of control and stressful life experience were related to intensity and quality of pain, disease activity, functional status and lymphocyte proliferation rate. Intra- and inter-group analyses were conducted to determine treatment effects in terms of change scores .on the dependent measures, and case studies were conducted to evaluate individual response both to disease and cognitive-behavioural intervention.

Objectives. To (a) determine the immunological effects of a PRIMATIZED® anti-CD4 antibody alone or in combination with methotrexate in RA patients, (b) determine the immunological effects of a chimeric anti-CD25 antibody in RA patients who are partially refractive to methotrexate and (c) compare interleukin-15 levels in the serum of RA patients and healthy controls and determine if there is a correlation between this cytokine and serum TNF-α, CD 122 expression, and disease activity. Patients and methods. (a) Eight RA patients were selected, four received anti-CD4+ placebo and the other four received anti-CD4+ methotrexate for 4 weeks. The immunological effects were assessed on peripheral blood by flow cytometry and thymidine incorporation assays. (b) Six RA patients were given anti-CD25 antibody (0.02-60mg) along with methotrexate for 26 days. The immunological effects were assessed on peripheral blood by flow cytometry, thymidine assays, and ELISA. (c) Blood and disease activity from twenty-one RA patients were obtained and serum IL-15 and TNF-α levels were measured by ELISA. IL-15R β chain (CD 122) expression was measured by flow cytometry. Results. (a) The anti-CD4 antibody caused a selective and significant decrease in the number of CD4+ T cells. No inhibition of PHA or mitogenic antibody stimulated proliferation was observed. (b) The anti-CD25 antibody caused a significant decrease in the percent CD25+ cells. The antibody bound CD25 and prevented interaction of IL-2 and IL-2R. Anti-CD25 antibody caused a significant decrease in PHA or mitogenic antibody stimulated proliferation. Clinically, the anti-CD25 antibody caused a significant decrease in the number of tender and swollen joints. (c) Elevated serum IL-15 was measured in 10 out of 21 RA patients but not in controls. No correlation was observed between IL-15 and TNF-α, CD122 expression or disease activity. Conclusions. (a) Methotrexate did not alter the effects of the PRIMATIZED® anti-CD4 antibody. Changes in antibody development processes have yielded two antibodies with different functions. (b) Anti-CD25 induced decrease in CD25+ T cells was associated with clinical benefit. The exact mechanisms of action are not clear. (c) Serum IL-15 levels in RA may be a more sensitive indicator of inflammation than TNF-α and may be a valuable tool in diagnosis.

Die Rheumatoide Arthritis (RA) ist eine chronisch-entzündliche Systemerkrankung des Bindegewebes mit autoimmunem Charakter. In dieser Studie wurden 7 Kandidatengene, welche in zentrale Abläufe der Apoptose involviert sind (CFLAR, XIAP, NFKB1, RELA, BCL2L1, FAS, FASLG), selektiert. Innerhalb dieser Gene wurden 23 Einzel-Basen-Polymorphismen (single nucleotide polymorphisms bzw. SNPs) sowie ein Insertions-Deletions-Polymorphismus in 300 französich-kaukasischen Individuen (100 RA-Trio-Familien) mittels Einzelbasenverlängerung (Single Base Extension bzw. SBE) in einer massenspektrometrischen Analyse durch MALDI-TOF-MS (Matrix Assisted Laser Desorption/Ionization–Time Of Flight Mass Spectrometry) genotypisiert. Die Auswahl der zu untersuchenden genetischen Polymorphismen erfolgte dabei unter Berücksichtigung einer möglichen funktionellen Bedeutung, bekannter Assoziationen mit RA oder anderer Autoimmunerkrankungen, der Lage im Gen sowie der genetischen Variabilität. Die Ergebnisse der Genotypisierung wurden genutzt um die Polymorphismen bzw. Kandidatengene mit Hilfe verschiedener statistischer Methoden auf ihre Assoziation mit RA hin zu untersuchen. Die statistischen Analysen des SNPs CFLAR-rs7583529 zeigten hierbei einen nicht signifikanten Trend, wobei das minor Allel A gehäuft in RA Patienten vorkam. Das Ergebnis des Genotypen-Tests (Lathrop) für FAS-rs1800682 belegte einen protektiven Effekt für homozygote Träger des major Allels C (Lathrop pval = 0.045). Unterstützung für die gefundenen Trends bzw. Assoziationen von CFLAR-rs7583529 und FAS-rs1800682 boten Vergleiche mit Daten genomweiter Studien (NARAC/EIRA- und WTCCC-Studie). In den Assoziationsanalysen von BCL2L1-rs3181073 zeigte sich ein protektiver Effekt des minor Allels A (TDT pval = 0.008, OR = 0.51 [0.3 – 0.9], OR pval = 0.014). Der Risikoeffekt des major Allels C spiegelte sich entsprechend im Lathroptest wider, welcher eine signifikante Anreicherung des homozygoten C/C-Genotyps in den Fällen anzeigte (Lathrop pval = 0.021). Die gefundenen Assoziationen von FAS und BCL2L1 mit RA gehen mit der Hypothese konform, dass veränderte Abläufe sowohl im intrinsischen mitochondrialen (BCL2L1) als auch im extrinsischen (FAS) Weg der Apoptose in die Ätiologie der RA involviert sind. Die Ergebnisse dieser Arbeit sollten in einer zweiten unabhängigen Kohorte repliziert werden. In Folgestudien wäre es ebenfalls interessant, weitere SNPs der Kandidatengene zu genotypisieren, um die genetische Variabilität anhand der Haplotypen genauer zu analysieren. Sollten sich die o. g. Assoziationen bestätigen, sind im Weiteren funktionelle Studien bezüglich unterschiedlicher Genexpression oder verändertem Apoptoseverhalten von Zellen oder synovialem Gewebe von großem Interesse<br>Rheumatoid arthritis (RA) is a chronic inflammatory systemic disease of the connective tissue with autoimmune character. In this study, 7 candidate genes that are known to be involved in key processes of apoptosis (CFLAR, XIAP, NFKB1, REAL, Bcl2l1, FAS, FASLG) were selected. Within these genes, 23 single nucleotide polymorphisms (SNPs) and one insertion/deletion polymorphism were genotyped in a sample of 300 French Caucasian individuals (100 RA trio families) by means of Single Base Extension (SBE) and MALDI-TOF (Matrix Assisted Laser Desorption /Ionization–Time Of Flight) mass spectrometry analysis. The possible functional significance, known associations with RA or other autoimmune diseases, the location in the gene and genetic variability were taken into account during the selection of genetic polymorphisms. The SNP genotyping results were used to analyse associations of polymorphisms or candidate genes with RA by applying various statistical methods. Analysis of the SNP CFLAR-rs7583529 showed a non-significant trend toward increased frequency of the minor allele A in RA patients. The genotypic test (Lathrop) of FAS-rs1800682 revealed a protective effect for homozygous carriers of major allele C (Lathrop pval = 0.045). Data of genome-wide studies (NARAC/EIRA- and WTCCC study) provided further support for association of CFLAR-rs7583529 and FAS-rs1800682 like confirmed in this study. Association analysis of Bcl2l1-rs3181073 showed a protective effect of the minor allele A (TDT pval = 0.008, OR = 0.51 [0.3 - 0.9], pval OR = 0.014). The genotypic Lathrop-test in turn revealed a corresponding risk effect for homozygous C/C genotype carriers (Lathrop pval = 0.021). Within this study, associations of the apoptosis genes FAS and Bcl2l1 with RA were found out. These results further indicate that changes of the intrinsic mitochondrial (Bcl2l1) and extrinsic (FAS) apoptosis pathway are possibly involved in the etiology of RA. For confirmation, results of this study should be replicated in a larger independent cohort. It would also be of interest to analyze the genetic variability based on specific haplotypes of additional SNPs within candidate genes. If the aforementioned associations are confirmed, functional studies with regard to different gene expression or changed apoptosis initiation in cells or synovial tissue would be of interest

B-Lymphozyten leisten unterschiedliche Beiträge zur Pathophysiologie der Rheumatoiden Arthritis. Sie produzieren Autoantikörper, präsentieren Autoantigene und schütten verschiedene Zytokine, die am proinflammatorischen Prozess beteiligt sind, aus. Aufbauend auf diesen Ergebnissen wurden in den letzten Jahren Therapien entwickelt, die gezielt B-Lymphozyten ansteuern um direkt oder indirekt in den autoimmunen Krankheitsverlauf einzugreifen. Die zeitlich begrenzte B-Zell-Depletion mit Rituximab (anti CD20-Antikörper) hat dabei in den letzten Jahren einen hohen Stellenwert erlangt und wird im klinischen Alltag insbesondere bei der Behandlung von Patienten mit rheumatoider Arthritis angewandt. Rituximab induziert im peripheren Blut bemerkenswerte Veränderungen in der Homöostase der B-Zell-Subpopulationen. Nach Therapie mit dem anti-CD20 Antikörper Rituximab beginnt die Repletionsphase mit der peripheren Aussaat von transitionalen unreifen B-Zellen. Im weiteren Verlauf kommt es zu einer Normalisierung des naiven B-Zell-Pools. Das B-Zell Gedächtnis und in besonderem Maße die IgD+CD27+ Gedächtniszellen erholen sich nach Therapie nur langsam. In einer prospektiven klinischen Studie hat unsere Arbeitsgruppe gezeigt, dass die Gesamtzahl der Gedächtniszellen gut mit der Dauer der klinischen Antwort auf Rituximab korreliert. Es ist wenig über die speziellen molekularen Veränderungen innerhalb der Gedächtnis B-Zellen nach Rituximab Therapie bekannt. Um die Veränderungen im peripheren Blut zu verstehen untersuchten wir die somatische Mutationsfrequenz und das Muster der Ig-VH3 Gen Rearrangements, indem wir prä- und posttherapeutisch bei 18 Patienten einzelne B-Zellen isolierten und den individuellen B-Zellrezeptor durch eine Einzelzell RT-PCR amplifizierten und sequenzierten. Wir verglichen das Mutationsmuster nach erfolgreicher B-Zelldepletion in den neu rezirkulierenden Gedächtnis B-Zellen mit dem Mutationsmuster von vier Gesunden Blutspendern und sechs nicht-RA Patienten, die eine Hochdosis Chemotherapie mit anschließender autologer oder allogener Stammzelltransplantation erhalten hatten. Zunächst haben wir die Zusammensetzung der Gedächtniszellen im peripheren Blut analysiert. Der Phänotyp der peripheren prä-switch (IgD+CD27+) und post-switch (IgD-CD27+) Gedächtniszellen zeigte keine quantitativen Unterschiede in RA-Patienten im Vergleich zu Gesunden. Bei der direkten Analyse des B-Zell Immunglobulin Rezeptors fanden sich jedoch zwischen klassengeswitchten und ungeswitchten Gedächtnis B-Zellen signifikante Unterschiede in der Anzahl der Mutationen in der variablen Region der Ig Rezeptors. Die Population der IgD+CD27+ Gedächtniszellen beinhaltete sowohl nicht mutierte, wenig mutierte und stark mutierte (Median= 9 Mutationen pro Sequenz) rearrangierte Ig- Rezeptoren, wohingegen die IgD-CD27+ Gedächtniszellen einen durchgehend hoch mutierten (Median = 18 Mutationen pro Sequenz) Rezeptor aufwiesen. Der Unterschied zwischen beiden Gruppen war signifikant (Mutationsfrequenzen 3.83±0.19% vs. 7.1±0.53%; P=0.0001). Grundlegende Veränderungen wurden bei den rezirkulierenden ungeswitchten Gedächtniszellen (IgD+CD27+) nach vorübergehender B-Zell Depletion mit Rituximab festgestellt. Diese Zellen wurden bis 6 Jahre nach Rituximab beobachtet und zeigten eine stark verzögerte Zunahme an Mutationen im Ig-Rezeptor. Ein Jahr nach einmaliger Gabe von Rituximab waren 84% der einzelnen zirkulierenden IgD+/CD27+ B-Zellen unmutiert. Zu diesem Zeitpunkt fanden sich keine stark mutierten Ig-VH3 Gen Rearrangements (P=0.0001). Mit zunehmendem Abstand zur B-Zell depletierenden Therapie konnten in der Repopulationsphase zunehmende Zahlen an Mutationen in den B-Zell Ig Rezeptoren festgestellt werden. Beispielsweise waren während des 2. Jahres der Regeneration (P=0.0001) 7.8%, sowie nach 4 Jahren nur 14% der Ig Rezeptoren mutiert. Sogar 6 Jahre nach Behandlung, waren VH Mutationen in IgD+ Gedächtniszellen noch deutlich vermindert. Selbst nach dieser Zeit fanden sich in der prä-switch Gedächtnispopulation nur 27% hochmutierte Sequenzen während vor der passageren B-Zelldepletion 52% ein hohe Zahl an Mutationen trugen (P=0.0001). Die posttherapeutische Analyse der CDR3 Länge der regenerierten IgD+ Gedächtniszellen ergab eine erhöhte CDR3 Länge, die signifikant mit der Anzahl der nicht mutierten VH Genrearrangements während der Repletionsphase korreliert. Interessanterweise regenerierten Patienten nach Hochdosis Chemotherapie und allogener Stammzelltransplantation ihre IgD+ Gedächtniszellen mit einer deutlich höheren Anzahl an Mutationen. Ein Jahr nach Transplantation zeigten die Ig Rezeptoren schon 22% hoch mutierte und 42% unmutierte VH Rearrangements. Das zeigt, dass eine gegen CD20 gerichtete Behandlung nicht nur eine Verzögerung der Produktion der ungeswitchten Gedächtniszellen zur Folge hat, sondern darüber hinaus einen signifikanten Effekt auf die Mutationsrate im präswitch Gedächtnis B-Zellpool besitzt. Im Gegensatz zum Mutationsmuster der IgD+ Gedächtniszellen regenerierten die klassengeswitchten Gedächtniszellen nach anti-CD20 Depletion im peripheren Blut mit quantitativ normalen Mutationen im Ig Rezeptor. Interessanterweise fand sich allerdings eine Änderung der exprimierten Isotypen mit deutlicher Dominanz IgA exprimierender B Zellen. Weitere Analysen der klassengeswitchten Gedächtnis B-Zellen zeigen außerdem eine Therapie induzierte qualitative Veränderung dieses B-Zellpools. So waren posttherapeutisch die Mutationen in bestimmten T-Zell abhängigen Mutationshotspots, dem RGYW/WRCY Motiv, signifikant vermehrt (Mutationstargeting vor Therapie 27% vs. 43% nach Rituximab, P=0.0003). Dies weist darauf hin, dass die Mechanismen der Affinitätsreifung im klassengeswitchten B-Zellgedächtnis vor und nach B-Zelldepletion unterschiedlich funktionieren. Der Mutationsmechanismus selbst ist allerdings in diesen Zellen quantitativ nicht eingeschränkt. Zusammenfassend zeigt unsere Arbeit zum erstem mal, dass es nach einer passageren B-Zelldepletion mit anti-CD20 Antikörpern zu einer über Jahre hinweg nachweisbaren ausgeprägten Verzögerung in der Aquisition von somatischen Mutationen in rearrangierten VH Genen der IgD+ Gedächtniszellen kommt. Demgegenüber erholt sich das klassengeswitchte B-Zellgedächtnis mit uneingeschränkter Zahl von Mutationen im Ig Rezeptor. Diese Resultate zeigen, dass anti-CD20 gerichtete Therapien in besonderem Maße IgD+ Gedächtniszellen beeinflussen. Der Selektionsdruck durch Antigene und/oder die Selektion der Ig Rezeptoren erscheint unter diesen Bedingungen speziell bei IgD-Gedächtnis B-Zellen reduziert. Die Daten unterstützen die Hypothese, dass prä-switch Gedächtnis B-Zellen im Vergleich zu post-switch Gedächtnis B-Zellen andere Bedingungen für die Aktivierung der Mutationsmaschinerie benötigen. Die Resultate eröffnen neue Wege für das Verständnis der Pathophysiologie der B-Zell Gedächtnisentwicklung und können helfen neue zielgerichtete Therapien zur Behandlung von Autoimmunerkrankungen zu konzipieren<br>Diverse roles of B cells in the pathophysiology of rheumatoid arthritis are now well established. B cells contribute to autoimmunity by producing autoantibodies, processing autoantigen and the production of different cytokines which are involved in the inflammatory cascade. Therefore approaches to target B lymphocytes directly or indirectly are developed for clinical practice to treat autoimmune diseases including rheumatoid arthritis. Transient B cell depletion by rituximab (anti-CD20 antibody) has gained prime importance in recent years. Meanwhile anti-CD20 mediated transient B cell depletion therapy is now used with clinical efficiency in the treatment of patients with rheumatoid arthritis. Rituximab induces noteworthy changes in the homeostasis of peripheral B cell subpopulations during the repletion phase with emerging immature B cells in peripheral blood followed by normalization of the naïve B cell pool and a longterm delay in memory B cell subsets in patients with rheumatoid arthritis. Particularly IgD+CD27+ memory B cells repopulate very slowly during B cell regeneration. In a prospective clinical study, our laboratory has shown that the overall number of memory B cells correlates well to the duration of clinical response to rituximab. Little is known about the particular molecular changes in the memory B cell repertoire after rituximab therapy. To better understand peripheral memory B cell subsets, we explored in detail the somatic mutational frequency and pattern of Ig-VH3 gene rearrangements by using a single B cell sorting technique followed by nested PCR before and up to 6 years after rituximab therapy in 18 RA patients. We compared rituximab inflicted dynamics of mutational acquisition to memory B cell repopulation in 4 healthy donors and 6 non RA patients undergoing high dose chemotherapy followed by autologous or allogeneic stem cell transplantation (SCT). Firstly we analyzed the peripheral composition of memory B cell subsets. The phenotypic analysis of peripheral pre-switch (IgD+CD27+) and post-switch (IgD-CD27+) memory B cells did not reveal any quantitative differences in RA patients prior to B cell depletion therapy compared to healthy donors. However extending those studies in directly analysing the B cell immunoglobulin receptor from individual B cells of RA patients and healthy controls brought interesting results. Pre-switched and post-switched memory B cells showed a highly significant difference in the amount of mutations/sequence. The population of IgD+CD27+ memory B cells is comprised of non-mutated, low and highly mutated (median= 9 mutations/ sequence) rearranged Ig receptors whereas the IgD-CD27+ memory B cell compartment shows quite uniformly highly mutated (median 18 mutations/ sequence) sequences indicating a significant difference between these two groups (mutational frequencies 3.83±0.19% vs. 7.1±0.53%; P=0.0001). Profound changes were noted in the re-emerging pre-switch memory B cells (IgD+/ CD27+) after transient B cell depletion with rituximab. These cells showed over a time period of 6 years after treatment with rituximab significantly delayed acquisition of mutations in Ig receptors on the single B cell level. One year after a single course of rituximab 84% of single repopulating IgD+/CD27+ B cells were unmutated and no highly mutated Ig-VH gene rearrangements were found(P=0.0001). Over time increasing numbers of mutations could be detected i-e 7.8% during 2nd year of regeneration (P=0.0001), 14% after 4 years (n=2). Nevertheless even 6 years after rituximab, VH mutations in IgD+ memory B cells were still reduced with 27% highly mutated sequences compared to 52% pre therapy(P=0.0001). Post-therapy analysis of CDR3 length of regenerated IgD+ memory B cells revealed increased CDR3 length which also correlates well with elevated number of non-mutated VH gene rearrangements observed during repletion phase. In comparison patients undergoing high dose chemotherapy followed by allogeneic stem cell transplantation repopulated IgD+ memory cells earlier with higher numbers of mutations in IgD+ memory B cells. One year after transplantation Ig receptors showed already 22% highly mutated and 42 % unmutated VH rearrangements. These findings indicated that anti-CD20 mediated B cell depletion seems not only to delay the production of pre-switch memory B cells but also significantly affects the acquisition of mutations in the IgD+ memory B cell pool. In contrary to the mutational pattern of IgD+ memory B cells after rituximab class switched memory B cells repopulate in the periphery with quantitatively normal mutations in their Ig receptors. Although the numeric replenishment of these recirculating class-switched memory B cells was also reduced after rituximab, we found no delay in quantitative acquisition of mutations also an increased proportion of IgA expressing B cells in this memory B cell subset was detected. Our data showed that post-therapy mutational targeting in RGYW/WRCY motifs were significantly increased as compared with that of pre-treatment (27% before rituximab vs. 43% after therapy, P=0.0003) indicating that affinity maturation may operate differently in class-switched memory B cells before and after B cell depletion. These results indicate a normal development process with an unimpaired mechanism of mutational acquisition in class-switched memory B cells. These data argue for different requirements to undergo somatic hypermutations in IgD+ memory B cells in comparison to class switched memory B cells. To conclude, our work has demonstrated for the first time a delayed acquisition of somatic hypermutations at single Ig receptor VH gene rearrangements of IgD+ memory B cells in comparison to class-switched memory B cells. These results demonstrate that IgD+ memory B cells are particularly susceptible to anti-CD20 treatment in patients with rheumatoid arthritis. In addition antigenic pressure and/or selection are substantially reduced by rituximab therapy which is basically not seen in the class-switched memory compartment. These data are in line with the hypothesis that IgD+ memory B cells have distinct requirements for activating their mutational machinery compared to class-switched memory B cells which recover normal mutations during regeneration phase. The results have implications in understanding the pathophysiology of memory B cell in rheumatoid arthritis and may be helpful in designing new targeted therapies

Rheumatoid arthritis (RA) patients develop a chronic inflammation in synovial joints and often in other tissues. Since efforts to isolate microbial agents from the involved sites have failed to confirm an infectious origin, it is generally agreed that RA is an autoimmune disease. However, the mechanisms initiating and maintaining the abnormal immune response remain poorly understood. The number ofNKT cells is reduced in several human autoimmune disorders and in animal models of autoimmunity. The purpose of this study was to investigate whether the frequency of CD161+ NKT cells in RA correlates with clinical disease parameters. As a group, RA patients had approximately three times less CD161+ CD4+ T cells in their PB than age and sex matched controls, as expected. In contrast, the number ofCD161+ CD4+ Tcells in the SF was 2.5-3 times higher than in the PB of the same patient. There was no obvious correlation between the frequency of these cells and clinical parameters. Despite reductions in absolute CD161+ cell numbers in RA PB, the abundance of the mRNA coding for this protein was higher in the patiept group than in controls. Furthermore, CD161 message expression showed a bimodal distribution. High CD161 abundance correlated with the lack of erosions, while low levels - with erosive changes. These observations held both in newly diagnosed patients (disease duration < 6 months) and the established RA group (disease duration> 3 years). This study confirms the loss of CD161+ CD4+ T lymphocytes in the PB ofRA patients. However, the frequency of these cells was comparatively higher in the SF. Significantly increased mRNA expression by PB CD161+ cells may indicate an activation-induced intemalisation of this receptor from the cell surface that, due to differences in the immunological microenvironment, does not happen in the SF. CD161 upregulation appear to be a disease-specific event in RA. Furthermore, the correlation between low CD161 expression levels and formation of erosions in RA may be used to predict longterm disease outcome in patient

Rheumatoid arthritis (RA) is a complex autoimmune disease affecting approximately 1% of the population. Multiple factors contribute to the development of RA, with genetic factors accounting for around 60% of the disease risk. Over the last few years, genome-wide association studies (GWAS) have successfully been used to identify regions of the genome predisposing to complex disease. There are now 101 confirmed RA risk loci, but for the vast majority of these loci the causal gene and causal variant remain unidentified and therefore, their function in disease is unexplored. The majority of genetic variants, or single nucleotide polymorphisms (SNPs), associated with disease map to non-coding enhancer regions, which may regulate transcription through long-range interactions with their target genes. The aims of this project were to identify the causal genes within an RA locus, pinpoint the causal variants and elucidate the mechanisms by which the variants modify gene function. Capture Hi-C (CHi-C) was carried out with the aim of identifying long range interactions between disease-associated SNPs and genes in four related autoimmune diseases. Many long-range interactions were identified which implicated novel candidate genes, interactions involving multiple genetic loci which had a common target, and interactions with loci which had previously been implicated in disease. Complex interaction patterns were observed in many of the disease associated loci, particularly in the 6q23 locus which is associated with a number of autoimmune diseases and is the focus of the present thesis. Within the 6q23 locus, associated SNPs lie a large distance from any gene (>180kb) making it difficult to pinpoint the exact causal gene. Results from CHi-C and chromosome conformation capture (3C-qPCR) experiments indicated that restriction fragments containing disease associated intergenic SNPs could display genotype-specific interactions with genes associated with autoimmunity (IL20RA and IFNGR1). Interactions could also be detected with long non-coding RNAs (lncRNAs), The lead SNP in the 6q23 region is in tight LD with eight other SNPs which are equally likely to be causal. Bioinformatics analysis suggested that the most plausible causal SNP in the 6q23 intergenic region was rs6927172, as it maps to an enhancer in both B-cells and T-cells, is in a DNaseI hypersensitivity cluster, shows transcription factor binding and is in a conserved region. Chromatin immunoprecipitation (ChIP) demonstrated binding of chromatin marks of active enhancers (H3K4me1 and H3K27ac) and the transcription factors BCL3 and NF-κB to the rs6927172 SNP target site in Jurkat T-cells and GM12878 B-cells, suggesting the risk allele could be associated with increased regulatory activity. In conclusion, these results show that CHi-C can help identify novel GWAS causal genes with the potential to suggest novel therapeutic targets. For example IL20RA is already a target for a monoclonal antibody which has been shown to be effective in treating RA in clinical trials. This project has also provided compelling evidence that the autoimmune risk variant in the 6q23 locus, rs6927172, is within a complex gene regulatory region, involving multiple immune genes and regulatory elements, such as lncRNAs.