Le Tumor Necrosis Factor alpha (TNF-[alpha]) est une cytokine pro-inflammatoire qui occupe un rôle central dans la physiopathologie de nombreuses pathologies inflammatoires et particulièrement l'arthrite. La neutralisation de cette cytokine par l'utilisation d'anticorps anti-TNF-[alpha] a montré son efficacité dans la polyarthrite rhumatoïde et est aujourd'hui le traitement de référence pour la prise en charge de cette pathologie. Cependant, un tiers des patients traités par anticorps anti-TNF restent réfractaires ou ne répondent pas à ce traitement. Dans ce contexte, il apparait nécessaire de développer des approches nouvelles ou complémentaires pour renforcer l'arsenal thérapeutique actuellement disponible. L'utilisation d'oligonucléotides triple hélice (TFO) permet de moduler l'expression génique de manière spécifique par interaction avec la double hélice d'ADN. Dans cette étude, nous avons évalué les potentialités anti-inflammatoires d'un TFO anti-TNF-[alpha] in vitro sur les synoviocytes et chondrocytes articulaires et in vivo dans deux modèles d'arthrite expérimentale. Ce TFO interagit avec le promoteur du gène du TNF-[alpha], et son activité inhibitrice a été comparée à celle d'une approche par ARN interférence in vitro. Dans les modèles d'arthrite aigue et chronique, l'injection intra-articulaire préventive de TFO anti-TNF-[alpha] permet une amélioration significative des symptômes arthritiques. Particulièrement, le traitement par le TFO diminue sensiblement l'inflammation synoviale et les lésions ostéocartilagineuses articulaires. Ces résultats sont les premiers à montrer la possibilité d'utiliser un TFO in vivo et offrent d'intéressantes perspectives thérapeutiques / Tumor necrosis factor alpha (TNF-[alpha]), a pro-inflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including arthritis. Neutralization of this cytokine by anti-TNF-[alpha] antibodies has shown its efficacy in rheumatoid arthritis and is now widely used. Nevertheless, some patients currently treated with anti-TNF-[alpha] remain refractory or become non-responder to these treatments. In this context, there is a need for new or complementary therapeutic strategies. Triplex forming oligonucleotides (TFO) can inhibit gene expression with high sequence-specificity by interacting with the DNA double-strand. In this study, we investigated if an anti-TNF-[alpha] TFO had a therapeutic activity on inflammatory processes in vitro and in vivo, as judged from effects on two rat arthritis models. This TFO interacted with the TNF-[alpha] gene promoter, and its inhibitory activity was verified and compared to that of siRNA in vitro. A local intra-articular preventive injection of TFO in both acute and chronic arthritis models significantly reduced the development of the disease. Furthermore, the TFO efficiently blocked synovitis and cartilage and bone destruction in the joints. The results presented here provide the first evidence that gene targeting by anti-TNF-[alpha] TFO modulates arthritis in vivo, thus providing proof of concept that it could be used as therapeutic tool for TNF-[alpha]-dependent chronic inflammatory disorders

L’arthrose (OA) est un rhumatisme chronique dont la prise en charge médicamenteuse repose principalement sur les antalgiques et anti-inflammatoires, ce qui justifie la nécessité de rechercher d’autres cibles thérapeutiques. L’une de ces cibles potentielles est la galectine 3, une lectine sécrétée dans l’articulation au cours de la pathogenèse de cette maladie. Cette lectine interagit avec ses ligands à la surface des cellules articulaires pour engendrer des réactions inflammatoires et des phénomènes de catabolisme matriciel au niveau des tissus ostéo-articulaires, ce qui en fait une cible intéressante dans le traitement de l’OA. Le 1e objectif de ce projet de recherche est de mieux caractériser les effets biologiques de la galectine 3 sur le métabolisme chondrocytaire. Le 2e objectif est de concevoir des formes tronquées de galectine 3 et le 3e objectif est de démontrer leur potentiel d’inhibiteur de la forme entière de galectine 3 dans l’articulation. Ces travaux montrent que la galectine 3 est un facteur pro-inflammatoire, pro-catabolique mais aussi anti-anabolique dans le chondrocyte. Plusieurs formes tronquées recombinantes de galectine 3 ont été produites. Des tests préliminaires d’activité biologique in vitro démontrent que dans certaines conditions, l’une d’elles inhibe partiellement les effets biologiques de la galectine 3 sur des chondrocytes humains. Ces travaux de recherche constituent la preuve de concept d’un projet global s’inscrivant dans une perspective de thérapie génique pour l’OA / Osteoarthritis (OA) is a chronic rheumatism which drug management is based on antalgic and anti-inflammatory drugs, which requires the need to search for other therapeutic targets. One of these potential targets may be galectin 3, a lectin secreted in the joint during the pathogenesis of the disease. This lectin binds its ligands on the surface of the joint cells and thus, leads to inflammatory reactions and matrix catabolism phenomena, making it an attractive target in the treatment of OA. The 1st aim of our research project was to characterize the biological activity of galectin 3 on chondrocyte metabolism. The 2nd aim was to design and produce several truncated forms of galectin 3. The 3rd aim was to evaluate the presumed galectin 3-inhibiting activity of the truncated forms. This objective is part from the perspective of gene therapy for OA. Our work shows that galectin 3 is a proinflammatory, procatabolic but also an antianabolic factor in chondrocytes. Several recombinant truncated forms of galectin 3 were designed and produced. Preliminary tests of biological activity in vitro show that the chosen truncated lectin partially inhibits the biological activity of galectin-3 on human chondrocytes under a few conditions. This work is the proof of concept of a broader project of which perspective is gene therapy in OA

Des cellules immunitaires appartenant à l’immunité de type 3 sont localisées dans muqueuse digestive. Les micro-organismes du microbiote stimulent le système immunitaire pour le maintenir en veille face à de potentiels agents infectieux, tout en ayant une tolérance pour les micro-organismes commensaux. Des études montrent des anomalies du microbiote intestinal chez les patients arthritiques. Ceci suggère une dérégulation de l’immunité mucosale digestive dans la survenue de l’inflammation articulaire. Des cellules de l’immunité mucosale pourraient migrer vers les sites articulaires, notamment grâce à des récepteurs aux chimiokines. Le facteur RORγt, indispensable à la différenciation des cellules de l’immunité de type 3, est régulé négativement par le récepteur PPARγ. A l’aide de modèle murins, nous avons étudié l’impact de l’invalidation de PPARγ et de l’inhibition du récepteur aux chimiokines CCR3 sur les relations entre sphères digestive et articulaire. Nous avons montré que l’induction d’une colite au cours d’une arthrite au collagène modifie le microbiote intestinal, retarde l’apparition et diminue la sévérité des lésions articulaires. Nous avons démontré que les souris PPARγ-/- développent spontanément une inflammation articulaire associées à des anomalies dans la répartition des cellules immunitaires de type 3 de la muqueuse digestive. Ces souris sont dysbiotiques avec une flore enrichie notamment en entérobactéries, mais non-arthritogène. Enfin, l’inhibition de CCR3 au cours du développement d’une arthrite au collagène retarde et diminue la sévérité de la pathologie et altère la répartition des leucocytes dans les articulations et de la muqueuse digestive / Numerous type 3 immune cells (Th17 and ILC3) are physiologically located in lamina propria of the intestine. Microbial agents within the gut shape the immune system to make it efficient against threats but peaceful with commensals. Recent studies demonstrated changes in gut microbiota composition (dysbiosis) in chronic inflammatory rheumatism. These results suggest a role for mucosal immunity alteration in articular inflammation occurrence. Indeed, some type 3 immune cells once activated by microbiota, are thought to migrate to joints, involving notably chemokines receptors. Transcription factor RORγt, the master regulator of type 3 immune cells, could be negatively regulated by nuclear receptor PPARγ. Using experimental murine models, we studied the consequence of PPARγ deficiency and consequence of the chemokine receptor CCR3 inhibition on the joint-gut axis. Firstly, we demonstrated that experimental colitis induces microbiota changes, delays and reduces collagen-induced arthritis severity. Secondly, we showed that PPARγ deficient mice display spontaneous joint inflammation associated with abnormal type 3 distribution within the gut. Dysbiosis with enrichment in facultative anaerobic Enterobacteriaceae was found in these mice. Fecal microbiota transfer demonstrated this microbiota is non-arthritogenic. Finally, we demonstrated that CCR3 inhibition has profound anti-arthritic potencies associated with changes in leukocytes distribution within the joint-gut axis

Submitted by admin tede (tede@pucgoias.edu.br) on 2016-08-17T13:44:18Z No. of bitstreams: 1 Laura Goncalves.pdf: 1283052 bytes, checksum: de5235904a586f99fa9976b0bb8e0e7e (MD5) / Made available in DSpace on 2016-08-17T13:44:18Z (GMT). No. of bitstreams: 1 Laura Goncalves.pdf: 1283052 bytes, checksum: de5235904a586f99fa9976b0bb8e0e7e (MD5) Previous issue date: 2008-08-29 / Patients suffering from rheumatoid arthritis have their quality of life compromised due to the restrictions in their daily life activities, which are caused by the physical limitations brought by the disease. The therapies have the purpose of relieving the pain, minimizing or preventing the preliminary and secondary disabilities caused by the disease and also to enable the patients to monitor themselves. The main objective of this research was to evaluate the effects of the cinesiotherapy on the quality of life of the women with the disease. The study based on a group of women with rheumatoid arthritis diagnosed after the age of 18 by a rheumatologist. Classified in functional degrees II and III, except for the patients with comorbities. The study proposal included three stages: two evaluations of the quality of life of the patient and the therapeutical momentum characterized by the cinesiotherapy applied. The evaluation process consisted of the application a patterned questionnaire to a group of thirty-six women (SF-36). This group was randomly divided into two groups of the same size and then submitted to espcific anamnesis. The women in the group of cases (group I) were evaluated using the information on the SF-36 questionnaire concerning their life quality. And then they went through a program of global cinesiotherapy. After that, these women were evaluated a second time about their quality of life at the end of the period (40 days). The women in the control group (group II) did not go through the physical therapy treatment, but they were evaluated a second time concerning their quality of life. The variables that could interfere with the prognosis and development of the disease were analyzed by the anamnesis: age, time of disease, rheumatoid factor, number of joints, functional degree, VHS, scholarship and medicines, comparing at the two groups, via the test qui-squared and test u-man whitney, using á 0,05 to prove the groups were homogenous. The data from the query S-F36 have been evaluated and submitted at the validation statistic , by using - in case that the test t student , the test u-man whitney and the test wilcoxon In order to be validated, the data in the questionnaires were statistically analyzed using á 0,05. For the evidence from the normality as of each of the variables , it uses - in case that the test as of adherence kologorov smirnov. Confirms - in case that as soon , via the analysis from the statistic significance, than it is to the handling of cinesiotherapy influenced positively the status of life of the patients of the group I(work group). Statistically speaking, we can state that the cinesiotherapy influenced positively the quality of life of the patients in group I, whose women with rheumatoid arthritis were in better life conditions than the ones in the control group. / Os pacientes com artrite reumatóide apresentam limitações físicas que restringem as atividades de vida diária, comprometendo a qualidade de vida. O tratamento fisioterapêutico tem como objetivo aliviar a dor, minimizar as deformidades, melhorar ou manter a mobilidade articular, impedir ou minimizar a incapacidade pela doença e educar os pacientes a se auto-gerenciar. O objetivo deste ensaio clínico, randomizado, controlado e simples cego é avaliar o efeito da cinesioterapia sobre a qualidade de vida de mulheres com artrite reumatóide. O estudo selecionou 40 mulheres com a doença, diagnosticadas após os 18 anos de idade, por um médico reumatologista; classificadas em grau funcional II e III, excluindo-se as pacientes com comorbidades. As mulheres foram distribuídas de forma aleatória, em dois grupos iguais e submetidas à anamnese específica. O grupo de trabalho (grupo I) foi avaliado quanto à sua qualidade de vida, através do questionário SF-36 e, posteriormente, submetido a um programa de cinesioterapia global, com nova avaliação sobre sua qualidade de vida, ao final do período (40 dias). O grupo controle (grupo II) não foi submetido à cinesioterapia, apenas avaliado quanto à qualidade de vida no início e no final do período de estudo. Foram analisadas as variáveis, através da anamnese: idade, tempo de doença, fator reumatóide, número de articulações, grau funcional, VHS, escolaridade e medicamentos, que poderiam interferir nos prognósticos e desenvolvimento da doença, comparando-as nos dois grupos, através do teste qui-quadrado e teste u-mann whitney, com á 0,05,para comprovação da homogeneidade dos grupos. Os dados do questionário S-F36 foram analisados e submetidos à validação estatística, utilizando-se o teste t-student, o teste u-man whitney e o teste wilcoxon para á 0,05. Para a comprovação da normalidade de cada uma das variáveis, utilizou-se o teste de aderência, kologorov smirnov. Confirma-se assim, através da análise da significância estatística, que o tratamento cinesioterapêutico influenciou positivamente a qualidade de vida dos pacientes do grupo I(grupo de trabalho). Concluímos então, que o grupo de mulheres com artrite reumatóide, submetidas à cinesioterapia, apresentou uma melhora significativa na qualidade de vida, em relação ao grupo controle.

A artrite induzida por pristane (PIA) em camundongos AIRmax homozigotos para o alelo R e S do gene Slc11a1 foi usada para avaliar a influência do polimorfismo deste gene na resposta imune, mais especificamente na ativação de macrófagos peritoneais durante a PIA. Estudos anteriores mostraram que a presença do alelo S do gene Slc11a1 aumentou a incidência e a severidade da PIA em AIRmaxSS, sugerindo que este gene ou outro próximo esteja interagindo com o loci da inflamação para modular a PIA. O tratamento com pristane nos animais AIRmaxSS induziu infiltrado intenso composto por linfócitos, monócitos/macrófagos e neutrófilos. Macrófagos AIRmaxSS apresentaram perfis de expressão gênica e celular exacerbados durante a PIA, com expressão/produção elevada de H2O2, NO, IL-1b, IL-6, TNF-a e várias quimiocinas. Entretanto, o alelo R do gene Slc11a1 foi capaz de regular a intensidade de ativação do macrófago de forma mais eficiente que o alelo S e controlar desenvolvimento da artrite. Houve acometimento do rim, pulmão e timo durante a PIA. Nossos dados sugerem que o gene Slc11a1 modula a ativação dos macrófagos envolvidos na suscetibilidade a PIA e estas linhagens representam um modelo murino alternativo para o estudo da artrite reumatoide. / Pristane-induced arthritis (PIA) in AIRmax mice homozygous for Slc11a1 R and S allele was used in this study to characterize the role of Slc11a1 polymorphisms on immune response, more specifically in the activation of peritoneal macrophages during PIA. Previous reports showed the presence of S allele of Slc11a1 increased the incidence and severity PIA in AIRmaxSS, suggesting that this gene or another closed-linked gene interacts with inflammatory loci to modulate PIA. Pristane treatment induced intense infiltration of lymphocytes, monocytes/macrophages and neutrophils in AIRmaxSS animals. AIRmaxSS macrophages demonstrated exacerbated cellular and gene expression profiles during PIA, with higher expression/production of H2O2, NO, IL-1b, IL-6, TNF-a and chemokines. However, Slc11a1 R allele could be regulating macrophage activation intensity more efficiently than the S allele and control the development of arthritis. There was involvement of kidney, lung and thymus during PIA. Our data suggest that the Slc11a1 gene modulates macrophage activation involved in PIA susceptibility and these lines represent an alternative murine model of rheumatoid arthritis.

INTRODUÇÃO: Artrite reumatoide é uma doença autoimune que causa inflamação nas membranas sinoviais de articulações chamadas diartroses. As articulações da orelha média podem estar sujeitas às mesmas lesões reumáticas que as outras articulações do corpo. Perdas auditivas neurossensoriais e condutivas foram observadas na literatura, assim como o envolvimento da artrite reumatoide na orelha média, mas os resultados não estão em concordância. A imitância acústica de banda larga pode fornecer maior sensibilidade a mudanças sutis nas articulações dos ossículos. OBJETIVO: avaliar o efeito da artrite reumatoide no sistema auditivo com enfoque na avaliação da orelha média. MÉTODOS: O delineamento desta pesquisa é estudo de casos comparando três diferentes grupos: dois grupos de indivíduos com artrite reumatoide e um grupo controle pareado por gênero e idade. Foram incluídos 39 participantes com idades entre 26 e 51 anos, de ambos os gêneros. O grupo estudo foi separado em dois: AR1, com 15 participantes (artrite reumatoide há dez anos ou mais); e AR2, com 9 participantes (artrite reumatoide há cinco anos ou menos). Estes grupos foram recrutados do ambulatório de reumatologia do Hospital das Clínicas da FMUSP e o grupo controle com 15 participantes, foi composto por voluntários, usuários do Centro de Docência e Pesquisa da FMUSP. Os procedimentos utilizados foram: Imitanciometria com sonda de 226 Hz, audiometria tonal liminar, audiometria de altas frequências e imitância acústica de banda larga, cujos dados foram extraídos como bandas de 1/3 de oitava para reflectância de energia em um documento de extensão Excel. Para análise dos dados, utilizaram-se estatística descritiva, análise de variância, teste quiquadrado e Kruskal-Wallis. RESULTADOS: As médias de idade para AR1, AR2 e controle foram de 41,1, 38,6 e 39,9 anos, respectivamente, e não houve diferença significante entre grupos. As medidas de imitanciometria 226 Hz (timpanometria e reflexos acústicos ipsilaterais) e reflectância de energia não demonstraram diferença significante entre os grupos. Foram encontradas diferenças na comparação entre os grupos AR1 e AR2 e grupo controle para os testes audiometria tonal liminar, e AR1 com o grupo controle para audiometria de altas frequências e reflexos acústicos contralaterais. Não foi observada diferença entre os resultados da orelha direita e da esquerda. CONCLUSÕES: Os grupos de artrite reumatoide apresentaram o mesmo padrão de resposta para as avaliações de orelha média do grupo controle, levantando a possibilidade de apresentarem um mesmo comportamento de sistema ou de que as ferramentas utilizadas para avaliação não são sensíveis para detectar as mudanças sutis. Foi possível observar mudança nos limiares audiométricos e de altas frequências dos grupos com artrite reumatoide em comparação com o grupo controle, mostrando a importância de monitorização auditiva nessa população / BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease that causes inflammation of synovial membranes of the diarthrodial joints. Middle ear joints may be subject to the same rheumatic lesions as other joints in the body. Sensorineural and conductive hearing loss have been reported, as well as middle ear involvement, but the results are not in agreement. Wideband acoustic immittance (WAI) may provide better sensitivity to subtle changes in the ossicular joint. PURPOSE: To evaluate the effect of RA in the auditory system focusing on the middle ear assessment with WAI. METHODS: This was a cross-sectional study comparing audiological behavior of 3 different groups: two groups of individuals with rheumatoid arthritis and a normal control (NC) group matched 1:1 based on age and gender. Thirty-nine participants, raging age from 26-51 years old, male and female. The study groups were pooled into two groups: RA1 with 15 participants (patients who had had RA for more than ten years) and RA2 with nine participants (those who had had RA for less than five years). The study groups were recruited from rheumatology clinic of Hospital das Clinicas (FMUSP) and were compared with 15 normal control, which consisted in volunteer students or employees from Centro de Docencia e Pesquisa (FMUSP). Standard 226 Hz probe tone tympanometry, pure-tone audiometry, extended high-frequency audiometry and WAI were performed in all subjects. The results from WAI were extracted as 1/3 octave bands for energy reflectance as an Excel file. Descriptive statistics, chi-square test, mixed model ANOVA and Kruskal-Wallis test were used to investigate the differences in all performed tests across the three groups. RESULTS: Mean ages for RA1, RA2 and NC were 41.1, 38.6 and 39.9 years old respectively and the difference between groups was not statistically significant. Comparison of standard immittance measures (such as 226 Hz probe tone tympanometry and ipsilateral acoustic reflex) and 1/3 octave band energy reflectance did not reveal significant difference between groups. This study found difference in standard audiometry thresholds comparing RA1 and RA2 to NC group, extended high-frequency audiometry and contralateral acoustic reflex comparing RA1 group to NC group. There was no significant difference between right and left ears. CONCLUSIONS: RA groups showed the same middle ear response pattern than the NC group, there was either no significant difference between the middle ears of RA patients and healthy individuals, or WAI is not appropriate for differentiating these two groups. Pure-tone audiometry and extended high-frequency audiometry showed different behaviors between the three groups. The actual incidence of hearing loss in RA remains to be defined. The results showed the importance of monitoring potential audiological manifestations in patients with RA

Nossa hipótese é de que os efeitos pró-inflamatórios iniciais da ativação do receptor ativado por protease 2 (PAR2) na articulação temporomandibular (ATM) sejam mediados por mecanismos neurogênicos. A análise por imunofluorescência revelou um alto grau de imunorreatividade ao PAR2 em aferentes primários trigeminais da ATM. Além do mais, a imunorreatividade ao PAR2 também foi observada na camada íntima da sinóvia, além de co-localizar com o marcador neuronal PGP9.5 e o neuropeptídeo substância P. A injeção intra-articular de agonistas PAR2 na ATM induziu um aumento dependente da dose no extravasamento plasmático, influxo de neutrófilos e indução de alodinia mecânica. O bloqueio farmacológico de receptors NK1 inibiu o aumento no extravasamento plasmático, influxo de neutrófilos e alodinia induzido pela ativação do PAR2. Em conclusão, a ativação do PAR2 é pró-inflamatório na ATM, via mecanismos neurogênicos envolvendo receptores NK1, sugerindo que o PAR2 é um importante componente da resposta imunológica inata na ATM. / We hypothesised that the early pro-inflammatory effects of proteinase-activated receptor 2 (PAR2) activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR2 in retrogradely labelled trigeminal ganglion neurons. Furthermore, PAR2 immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance P-containing peripheral sensory nerve fibres. The intra-articular injection of PAR2 agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx and induction of mechanical allodynia. The pharmacological blockade of NK1 receptors abolished PAR2-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR2 activation is pro-inflammatory in the TMJ, through a neurogenic mechanism involving NK1 receptors. This suggests that PAR2 is an important component of innate neuro-immune response in the TMJ.

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting 1% of the population. The aetiology of rheumatoid arthritis is unknown, although there are multiple postulated theories. In 1950, Philip Hench won the Nobel prize for treating patients with rheumatoid arthritis with cortisone. He also treated 6 patients with adrenocorticotropic hormone (ACTH) with good results. ACTH is a melanocortin. The melanocortin system describes the five melanocortin receptors, their ligands, agonists and antagonists and the accessory proteins. The aim of this study was to explore the melanocortin receptors in rheumatoid arthritis synovium. Methods HA-tagged stable cell lines were created for MC1R, MC3R and MC5R. Multiple antibodies were tested for their utility using Western Blot, immunohistochemistry and flow cytometry. Samples of synovium from 28 patients with RA were tested using RTPCR for the presence of MC1R and MC3R. Gene expression was correlated with clinical characteristics, cytokine (RTPCR) expression and immunohistochemical score. Results The stable cell lines expressed MC1R, MC3R and MC5R respectively. Of the antibodies tested none were found to be of utility in detecting MC1R or MC3R .The MC1R RQ values in rheumatoid synovium appear to split into two groups, high and low. The medians of the two groups are significantly different (p=0.0005). There is almost a 5 cycle, or 64 fold, difference in gene expression between the medians of the two groups (1.59 v 6.23). Of note no MC3R positive samples were CD138 high (i.e. no MC3R positive samples had a significant plasma cell infiltrate) (p=0.006). Categorical analysis using Fishers Exact test revealed an association between MC1R high samples and CD68 lining high scores, (i.e. MC1R high samples also had a high macrophage score in the lining of the sample) (p=0.02). MC1R low samples were associated with not being on combination therapy, 15 this did not quite reach significance (p=0.07). Linear regression analysis confirmed these associations for MC1R. PCA analysis did not show any grouping of samples according to any of the variables tested, likely due to sample size. Conclusion MC1R and MC3R are found in human synovium. Current commercial antibodies are not of utility in detecting MC1R or MC3R. Synovial samples can be split into high and low MC1R gene expression groups. MC3R was either present or absent. High expression of MC1R was associated with a high macrophage score and MC3R expression was associated with a low plasma cell score. MC1R and MC3R expression in RA synovium could be used as biomarkers of disease state or severity as well as a target for therapy.

Sjögren's syndrome (SS) and rheumatoid arthritis (RA) are characterised by breach of self-tolerance with high affinity circulating autoantibodies and peripheral B cell disturbances in the naïve and memory B cell compartments. In addition, both SS and RA develop functional ectopic B cell follicles in the respective target organs, i.e. the salivary glands and the joint synovium, whereby autoreactive B cell undergo antigen selection and affinity maturation. However, the exact stage at which errors in B cell tolerance checkpoints accumulate is unknown. In this PhD project, I amplified and sequenced Ig VH and VL gene transcripts from single B cells which were FACS sorted either from the peripheral blood of SS patients or from the RA synovium. Healthy donors (HD) were used as controls. Subsequently, I cloned and expressed recombinant monoclonal antibodies displaying identical antigenic specificity of the original B cells. Finally, I tested the poly- and autoreactivity profile of these antibodies against SS and RA-associated autoantigens. In SS, I analysed 353 VH and 293 VL sequences and obtained 114 recombinant antibodies from circulating naïve (n=66) and memory (n=48) B cells of 4 SS patients and compared their autoreactive and polyreactive profile to 45 naïve clones from 2 HD. Analysis of the VH and VL gene usage showed no significant differences between SS and HD. Conversely, I observed accumulation of circulating autoreactive naïve B cells in SS as demonstrated by Hep-2 cells, ENA, Ro/SSA and/or La/SSB reactivity. The elevated frequency of autoreactive naïve B cells in the circulation of SS patients supports the existence of early defects in B cell tolerance checkpoints in this condition In RA, I analysed the Ig gene repertoire and the VH gene somatic mutation rate of 139 VH and 175 VL sequences of synovial CD19+ B cells which demonstrated evidence of antigen selection and hypermutated alpha > gamma > mu VH chains with presence of intra-synovial clonal diversification. Recombinant antibodies from synovial B cell clones were then screened for reactivity towards citrullinated antigens with a plan for a wider analysis using autoantigen microarrays. Overall, these results highlighted the existence of B cell abnormalities and loss of tolerance for self-antigens both in the peripheral and/or lesional compartment of SS and RA. Further analysis of the fine specificity and pathogenicity of recombinant antibodies from autoreactive B cells will be invaluable in order to dissect the mechanisms and the antigens driving the development and the persistence of autoimmunity in RA and SS.

Psoriatic arthritis is an inflammatory arthritis affecting a fifth of patients with skin psoriasis. Inflammation of the joints and tendons causes pain, stiffness, reduced function and disability. Work disability is increasingly recognised as an important, patient centred, functional measure of disease yet little is known about work disability in psoriatic arthritis. The overall aim of my thesis is to examine patient reported work disability in psoriatic arthritis by undertaking the following; • A systematic review of the relevant literature • Classification of a cohort of patients to study • Validation of a commonly used work outcome measure used in other rheumatic diseases • Selection of a suitable measure of structural damage to inflamed joints for investigating the associations of work disability in longitudinal observational studies. The results of the systematic review identified limited data reporting high levels of work disability associated with a wide variety of disease and non-disease related factors. The review also identified the lack of a validated outcome measure for use in psoriatic arthritis. I report the classification of a large single centre longitudinal cohort of patients with psoriatic arthritis and evidence supporting the retrospective application of a psoriatic arthritis classification criterion. Subsequently I report a preliminary validation study of the work productivity and activity impairment questionnaire to measure work disability in psoriatic arthritis and a further study comparing the existing measures of structural damage in psoriatic arthritis. Finally I developed and supervised a multicentre observational study to examine the associations of work disability in psoriatic arthritis. The study identified reduced work effectiveness to be associated with measures of disease activity, whereas unemployment was associated with recent disease onset, greater age and worse physical function. The study will provide a valuable cohort for prospective study of work disability and the effect of medical treatment and will form part of my planned post-doctoral studies.