Despite the development of new biomarkers predicting prognosis in rheumatoid arthritis (RA) remains challenging and targeting of powerful biologics difficult. The presence of ectopic germinal centres (GC) within synovium has long been recognised (ectopic lymphoneogenesis [ELN]) and data have suggested that they manufacture antibody (Ab). High affinity class switched Ab production occurs through class switch recombination (CSR) and somatic hypermutation (SHM) both critically dependent on activation induced cytidine deaminase (AID). However, whether ectopic GCs express AID has not been known. Nonetheless data associating ELN with disease severity suggest a role for ELN in RA pathogenesis and as a potential biomarker. A classification system for RA synovium, based on the concept of ELN has been proposed as: (i) aggregate, (ii) aggregate GC+ and, (iii) an unorganised infiltrate. However whether these distinct pathotypes and/or degree of aggregation equate to disease severity is unclear. Thus my first aim was to develop and validate a pathological scoring system for rheumatoid synovium capable of quantifying the degree of ELN. My second aim was to investigate whether the presence and/or degree of ELN within the synovial membrane correlated with both clinical phenotype and predicted erosive damage. I demonstrate that the aggregational score developed is highly reliable and that ELN within synovial tissue associates with a higher level of synovial inflammation but is not predictive of damage. My third aim was to investigate whether GCs within RA synovium were functional. I provide evidence of functionality by demonstrating that ectopic GCs invariably express AID, are surrounded by anti-CCP+ plasma cells, support ongoing CSR and the manufacture of anti-CCP Abs. My final aim was to characterise a cohort of synovial B cells consistently found surrounding ectopic GCs. I identify a novel B cell subset within RA synovium, interfollicular large B cells, (5)(5)(5) and demonstrate that interfollicular large B cells in lymph node express a somatically mutated IgH.

The ubiquitous ɣ-herpesvirus Epstein-Barr virus (EBV) infects B cells and modifies their differentiation programme leading to B cell activation and immortalization. Although different evidences support a link between EBV infection and rheumatoid arthritis (RA) and Sjogren’s syndrome (SS), the exact role of EBV in RA and SS pathogenesis remain elusive. Recently ectopic lymphoid structures (ELS) have been identified as preferential niches for EBV persistence and reactivation in patients with multiple sclerosis and myasthenia gravis. Independent studies demonstrated that around 50% of RA synovia and 30% of SS salivary glands are characterised by the development of functional ELS, capable to promote local differentiation of autoreactive plasma cells. In this PhD project I explored the potential role of EBV in RA and SS pathogenesis by analysing EBV infection in the RA synovium and SS salivary glands and its relationship with ELS, in situ autoreactive plasma cell differentiation, pathogenic autoantibodies production and cytotoxic immune response. In this work I demonstrated that: i) markers of EBV latent and lytic infection are consistently associated with the presence of ELS in the RA synovium and SS salivary glands; ii) latent EBV proteins are preferentially expressed by B cells, while viral reactivation occurs in plasma cells; iii) a large subset of autoreactive plasma cells is EBV lytically infected in the RA synovia and SS salivary glands; iv) antibodies specific for unmodified and citrullinated EBV peptides, known to cross-recognize ACPA, are produced within ectopic lymphoid structures as 8 demonstrated in vivo in human RA/SCID chimeras; v) SS salivary gland grafts transplanted into SCID mice release human IgG against EBV antigens, whose production correlates with the level of SS-associated auto-antibodies and vi) analysis of CD8+ and CD4+ T-cell localization and granzyme B expression indicated that EBV persistence in ELS-containing RA synovia and SS salivary glands may be favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. Overall, these results redefine a novel and pathogenically relevant role for EBV in B-cell dysregulation and chronic inflammation in RA synovium and SS salivary glands.

Iron plays a potential role in oxidative stress-mediated injuries and pathologies e.g. rheumatoid arthritis (RA). Four decades ago it was suggested that iron may have a crucial role in the progression of inflammation in RA. Indeed, free radicals generated by iron can cause damage to lipids, proteins, carbohydrates, and DNA. It is this destructive process that is believed to occur in rheumatoid joints. However, none had differentiated between the role of iron in both acute and chronic phases of the disease and the origin of this 'labile' iron. Since RA cells are chronically exposed to oxidative stress, we have therefore chosen Jurkat cells to be our cell model. We used the parental (J16) cell line was used to mimic the acute phase of oxidative stress and the H2O2-resistant (HJ16) cells to mimic the chronic phase. By using hydrogen peroxide (H2O2) as the oxidising agent, we aim to study the role of iron in acute and chronic phase of oxidative stress and to know its origin. In the present study, we found that both antioxidants and H2O2-induced labile iron are modulated when cells are chronically exposed to H2O2. HJ16 cells contain higher total intracellular glutathione levels and glutathione peroxidase activity than J16 cells while the superoxide dismutase and catalase activity are similar. Haem oxygenase-1 (HO-1) was not detectable nor was it induced in these cell lines; HO-2 on the other hand was expressed but not induced. Although they had the same ‘basal’ LIP and L-Ft levels, J16 cells contain more than 7-fold higher H-Ft levels than in HJ16 cells. It was also found that H2O2-induced labile iron is directly correlated with necrotic cell death. These results are consistent with the conclusion that both antioxidant defence mechanism and labile iron status are modulated in cells chronically exposed to H2O2. We have also shown that the ‘basal’ and ‘H2O2-induced’ NFκB activation was higher in the HJ16 cells. We have also provided a link between labile iron release, lysosomal membrane damage and the ensuing necrotic cell death following H2O2 treatment.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease that causes inflammatory polyarthritis, typically of the small joints. Obesity, a serious global epidemic, has been shown to increase systemic inflammatory biomarkers, several of which are related to RA pathophysiology. Associations have been observed between obesity and worsened RA disease activity outcomes in crosssectional studies. Limited longitudinal studies investigated the effects of weight change on RA disease activity measures. Surgical interventions for weight loss in RA patients showed marked improvement in RA disease activity measures and outcomes but typical weight change in a clinical setting has not been investigated. OBJECTIVE: To investigate the impact of typical weight change on RA disease activity measures. METHODS: We conducted a retrospective cohort study on 178 RA patients seen in typical clinical practice that met the inclusion criteria for the study, which included patients with a minimum of two clinical disease activity assessments (CDAI) with corresponding body mass index (BMI) measures. Medical record review was conducted for each clinic visit where CDAI and BMI were measured, and at each of these visits, sociodemographic, lifestyle, medication usage, questionnaire data, RA characteristics, laboratory values, and comorbidities were collected. Linear regression was used to analyze the association between ΔBMI and ΔCDAI, defined at the dates of minimum and maximum BMI for each subject, adjusting for confounders including sex, age, disease duration, smoking status, serologic status, and steroid usage. Logistic regression was performed to evaluate whether ΔBMI was associated with low/remission RA disease activity according to accepted CDAI cutoffs. RESULTS: Unadjusted linear regression was performed on all 178 subjects to analyze the overall trend within the sample population. For every 1 kg/m2 increase in BMI, CDAI increased by 0.49 points, but these results were not statistically significant (p=0.155, 95%CI -0.176, 1.097). Subjects were stratified into BMI gain, stable, and loss groups. Within the BMI loss group (defined as those whose BMI decreased by more than 1 kg/m2), a significant association was found with ΔCDAI (β= -2.61 [p=0.028, 95%CI -4.91, -0.298]). Unadjusted linear regression on the BMI gain and stable groups was found to be not statistically significant. This association remained significant after adjusting for sex, age, disease duration, smoking status, serologic status, and steroid usage (β=-2.499 [p=0.044, 95%CI -4.94, -0.061]). There was no association between ΔBMI and low/remission RA disease activity (OR 0.990, (95%CI 0.855, 1.146). When stratified by BMI gain, stable, and loss groups there was no significant association with low/remission RA disease activity. CONCLUSION: These results suggest that weight loss may be associated with improved disease activity among patients with RA seen in a typical clinical setting. Weight loss has the potential to be a non-pharmacologic intervention to improve RA disease activity. Prospective studies of weight loss and RA disease activity are necessary to replicate these results.

The skeleton is a dynamic organ that undergoes a continual process known as bone remodeling. Bone remodeling is necessary to maintain structural integrity, heal micro-fractures caused by from daily wear and tear, and to store and release essential ions and minerals. Remodeling is a highly regulated process, with bone resorption precisely balanced by bone formation under homeostatic conditions. In the setting of rheumatoid arthritis (RA), an inflammatory condition affecting joints, this balance is lost and bone around inflamed joints is eroded. These so-called "bone erosions" compromise joint function, causing disability. Osteoclasts, multinucleated cells of hematopoietic origin, are the only cells known to resorb bone. Osteoclasts are found at erosion sites in human joints, and data from mouse models of inflammatory arthritis suggest that osteoclasts are required for erosions to form in bone. The canonical pathway of osteoclast differentiation requires stimulation of myeloid precursors by the cytokine Receptor Activator of NF-Kappa B ligand (RANKL) through its receptor, RANK. In the inflamed joint, RANKL expression can be induced on mesenchymal lineage cells by inflammatory cytokines such as tumor necrosis factor alpha (TNF alpha). Surprisingly, our lab observed bone erosions and osteoclast formation in a mouse model of RA in the absence of RANK. Thus we hypothesized that in addition to RANKL expression, the cytokine milieu in RA may directly stimulate osteoclast formation. It was recently reported that the inflammatory cytokines TNF alpha and interleukin-6 (IL-6) in combination stimulate osteoclast differentiation, independent of exogenous RANKL. We have reproduced these results and shown that these osteoclast-like cells form entirely independently of RANK signaling. However, TNF alpha/IL-6 induced osteoclast formation still requires the transcription factor Nuclear Factor of Activated T cells (NFATc1), a master regulator of RANK-mediated osteoclast differentiation, as well as co-stimulatory signaling provided by the immunoreceptor tyrosine based activation motif (ITAM)-containing DNAX-activating protein (DAP12) molecules. We also showed that TNF alpha/IL-6 induced osteoclast formation requires activity of IL-6 receptor (IL-6R), as osteoclast formation can be inhibited through co-culture with an IL-6R blocking antibody (MR16-1). Finally, using an in vivo mouse model of RA in RANK-deficient mice, we tested whether blocking IL-6R with MR16-1 antibody protects against the formation of periarticular bone erosions. Our results suggest that a RANK-independent pathway of osteoclast formation contributes to inflammatory bone erosions. Targeting this pathway may improve outcomes for RA patients.

Objectives: The purpose of this study is to advance the understanding of secondary conditions experienced by persons aging with the long-term disabilities of polio and rheumatoid arthritis and the consequences of these declines in health and function on disability bed days. Additionally, it explores the effects of the timing and severity of onset of disability characteristics on the frequency and consequences of secondary conditions. A life course conceptual framework with the Institute of Medicine’s model of disablement is used to frame and anchor disability and life events. Methods: In-depth structured in-home interviews were conducted on 216 individuals with polio and 186 individuals with rheumatoid arthritis. They consisted of objective and subjective self-reports of current status and prior condition. The survey was a regional crosssectional, group comparison design with a cross-sequential sampling and data analytic framework. Scale development for data reduction was utilized to obtain parsimonious measures for the models. Linear regression was then performed to test the models for three outcome variables (number of chronic secondary conditions, increases in functional limitations and number of disability bed days in six months) in a theorized order for the polio and RA samples individually. Results: There was partial support for within-sample hypotheses for both polio and rheumatoid arthritis regarding interrelationships and disability bed days in past six months. No significant differences were found across subsamples for the effects of timing and severity of onset of disability characteristics, predicting chronic secondary conditions, predicting increase in functional limitations, and the number of disability bed days in six months. Similarities were found between the two samples when examining subgroup predictors on the three outcomes above. Chronic secondary conditions predicted (p<.05 for both subsamples) increase in functional limitations and increase in mobility was a significant predictor (p<.001 for both subsamples) of increase in functional limitations. Discussion: There were limited findings for these data. Judgment must be withheld with respect to the hypotheses. The analyses did not yield enough predictive strength to make comparisons possible across subsamples. Likewise, in examining similarities, only general, descriptive statements could be made. The subjective nature of disability is an immense challenge in cross disability research for comparability within disabilities and across disabilities.

RA is a chronic and disabling disease with no known cure. The disease has a strong genetic component and modern genetic studies have successfully identified over 100 loci associated with the onset of RA. Despite the number of associations identified, the full genetic component of RA is not known, and for the majority of the loci the causal variant remains unknown. The overall aim of this study was to utilise well-powered genetic data, in order to identify novel loci, refine genetic associations, and generate robust evidence for the causal SNP and causal gene at a selected RA locus. An initial analysis was undertaken utilising 3870 RA cases and 8430 controls from the UK-ImmunoChip, a study designed for comprehensive fine-mapping of confirmed RA susceptibility loci. Analysis of the UK-ImmunoChip data identified a novel finding with the TYK2 locus, and proved informative to refining association signals, illustrating the utility of fine-mapping and implicated SNPs with putative regulatory function. The UK-ImmunoChip was subsequently expanded to incorporate samples from five additional cohorts in a study led by Dr. Stephen Eyre. In additional to novel loci discovery, this study provided evidence for SNPs putatively associated with RA (P smaller or equal to5E-05 < 5E-08). In a combined meta-analysis of 17,581 cases and 20,160 controls, convincing evidence was obtained for two novel RA loci, BACH2 and RAD51B.The newly identified genes implicate two novel pathways in RA (B-cell differentiation and DNA repair) and add to the growing number of loci associated with multiple AIDs. These findings are important to aid comprehensive pathway analysis and add to the knowledge of RA risk genes. The third most associated RA locus in both serological subtypes of disease, with an uncharacterised protein, ANKRD55, was subsequently selected for in-depth characterisation. Utilising genetic and haplotypic analysis the association at this locus was refined to a single signal, with four SNPs in strong LD (r2 > 0.8). Through bioinformatic analysis, two SNPs rs6859219 and rs10065637 showed evidence for functional activity, with evidence of being located in an enhancer element, supported by histone marks, DNAse hypersensitivity, evidence of transcription factor binding and eQTL. The use of RNA and ChIP experiments have established a testable hypothesis that the presence of the putative causal variants rs6859219 and rs10065637, act to weaken the strength of the enhancer element in which they are located, (evidenced by diminished H3k4me1 modification), which in turn down-regulates the transcriptional output of the target gene ANKRD55 (evidenced by eQTL in both whole blood and CD4+ T cells).In summary this study has led to the identification of three novel loci, highlighted the importance of fine-mapping and developed a successful systemic strategy for the characterisation of the 5q11 risk locus associated with RA.

Abstract   Introduction Failure in the mechanism of self-tolerance in T or B cells can lead to autoimmunity. One of the autoimmune diseases is rheumatoid arthritis (RA), which is a chronic inflammatory disease of unknown cause and is characterized by systemic inflammation, autoantibodies and joint destruction. Serology is crucial for the classification of this disease. The first autoantibody found in RA patients was Rheumatoid factor (RF). However, anti-citrullinated peptide antibodies (ACPAs), a relatively new group of autoantibodies found in 70-90% of RA patients, are diagnostically more specific than RF. Type II collagen (CII) is the most abundant protein in human cartilage. In RA patients, immunity against CII leads to cartilage degradation and loss of joint function. Already from the 1970s, antibodies to CII (anti-CII) were found in RA sera, suggesting that CII autoimmunity might be pathogenetically important. Previous studies from our group show that a subgroup of patients with high levels of anti-CII at the time of diagnosis at the same time have high levels of inflammation in the joints. This is probably caused by anti-CII immune complexes (IC) inducing pro-inflammatory cytokines from macrophages. Although anti-CII positive patients have high inflammatory activity early on, as anti-CII levels decrease during the first year, the associated inflammation also diminishes. Thus, anti-CII positive patients have a rather good prognosis. Moreover, it is assumed that since anti-CII positive patients have a better prognosis than ACPA positive, patients with elevated anti-CII at the time of diagnosis might benefit from different and milder treatment. Previous studies from the group were performed on stored patient samples from the time before modern treatments with biologic agents (1995-2005). In this study, we aimed to investigate patients belonging to a more recent RA cohort, diagnosed between 2005-2014, with the aim to investigate whether patients with the anti-CII-associated RA phenotype would respond differently depending on the use of different modern RA therapies. Patients and Methods The primary cohort consisted of 2335 RA patients and 480 non-RA controls from the Epidermiological Investigations in Rheumatoid Arthritis (EIRA) case-control study. As we run into methodological problems two subgroups with 62 and 40 RA patients from the previous anti-CII studies were investigated when modifying the ELISA procedure, as well as a group of earlier investigated patients with non-specific ELISA reactivity. Totally 2776 RA patients were investigated. All investigated patients fulfilled the American College of Rheumatology classification criteria. To measure the anti-CII levels in RA patients and healthy controls anti-CII ELISA was performed. During the experiments, several different sources of CII from human, rat and bovine origin, and two different alternative coating buffers were used. The optical density (OD) was measured at 450 nm and anti-CII concentrations were calculated against the standard curve from an RA patient with high anti-CII levels.   Results My first analysis of the EIRA cohort showed that anti-CII are higher in RA patients than in controls, but could not confirm the association with acute onset RA. This was an unexpected finding and changed the focus of this master thesis project, to modify the measurement of anti-CII. Re-investigation of EIRA I showed that a proprietary coating buffer is important in the assay. Moreover, when different samples from RA patients were tested with bovine, rat and three different lots of human CII, correlation tests with clinical measures showed that bovine collagen and a new lot of human CII- prepared by the supplying company solely for this project- showed the strongest associations. Thereafter the EIRA cohort was re-investigated with two ELISAs, using bovine and human CII coated with the proprietary buffer. At the time of thesis writing almost all of the EIRA samples have been re-analysed, and results from both the modified ELISAs show the awaited clinical associations to early inflammation.   Conclusion Keeping the integrity of triple helical collagen is very important for the identification anti-native CII in RA patients. Our results show that the use of the proprietary coating buffer appears to be instrumental in this assay, irrespective of what source of CII was used. The new lot of human CII shows significant associations with the clinical measures, but associations are somewhat stronger with bovine CII. After finalising the re-investigations, we will be able to conclude which of the two analyses is most appropriate, and the corresponding dataset will then be merged with data from the first part of the EIRA study investigated previously by other group members. As anti-CII analysis shows the association to disease activity and prognosis, it can be used for predicting prognosis of RA and choosing the appropriate therapy in newly diagnosed RA patients, which might be clinically useful for rheumatologists. Our hypothesis is that as anti-CII positive patients have strong early inflammatory response but good long-term prognosis, they might benefit from other and perhaps short-term treatment compared to other RA patients.  If this is correct, our finding can have impact on the economy as it can define the patients who will not need expensive long-term medications. As modern anti-rheumatic therapies carry the risk of infections, such individualized therapies might also benefit anti-CII positive patients.

No description available.

O objetivo deste estudo foi adaptar e validar o Foot Health Status Questionnaire (FHSQ) avaliando suas propriedades de medida. Este instrumento foi traduzido, traduzido de volta para o inglês, avaliado por comitê multidisciplinar e submetido a pré-teste, gerando o FHSQ-Br. O FHSQ-Br foi submetido a teste de campo em um grupo de estudo composto por 65 pacientes com Artrite Reumatóide (AR) para avaliar a confiabilidade teste-reteste, a consistência interna e a validade do construto. A validade do construto foi testada correlacionando os escores do instrumento com dados clínicos e laboratoriais usados para avaliar a AR. Este estudo demonstrou que o FHSQ-Br é um instrumento confiável, consistente e válido, útil na avaliação da saúde dos pés, sendo passível de adaptação para diferentes culturas / The purpose of this study was to conduct a cross-cultural adaptation and validation of the Foot Health Status Questionnaire (FHSQ) evaluating its measurement properties. All ten domains of the FHSQ were translated into Portuguese by two Brazilian translators creating Version 1. This version was back-translated by two native English-speaking teachers who made suggestions for Version 1, creating Version 2. A multidisciplinary committee was formed to test the instrument’s semantic, idiomatic, experiential and conceptual equivalences. After being reformulated and approved by the committee, Version 3 was pre-tested on a group of patients from the Rheumatology Service of the Hospital das Clínicas. They answered this version and made suggestions for the better understanding of the instructions, questions and response option. The FHSQ-Br was then created. The translated and adapted version was submitted to field test on a study group composed of sixty-five Rheumatoid Arthritis (RA) patients to evaluate test-retest reliability, internal consistency and construct validity. The construct validity of the FHSQ-Br was tested correlating the scores to clinical and laboratory parameters commonly used to assess RA (Health Assessment Questionnaire; Numbered Rating Scale for foot pain; foot X-rays; erythrocyte sedimentation rate and C-reactive protein). The cultural adaptation of the FHSQ was successfully accomplished, since patients suggested changes in only three items of the instrument during the pre-test phase. In the field test, the intra-class correlation coefficients showed high reliability for both intra- and inter-observer correlations. Internal consistency coefficients were statistically significant (p<0.05) for all domains. As for the evaluation of the construct validity, each domain revealed correlations with a specific group of parameters, according to what the domains were intended to measure. The FHSQ was cross-culturally adapted generating a reliable, consistent and valid instrument. This study has proven the FHSQ-Br to be a useful tool to evaluate foot health in systemic diseases and is easily adaptable to different cultures