Dissertations / Theses on the topic 'Autoimmune arthritis; Ankylosing spondylitis'

Background: Presenteeism is an economic concept that is difficult to identify, measure, and value. Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are three chronic auto-immune conditions that increase levels of presenteeism. Workplace interventions (WPIs) help individuals to manage their health condition at work. Existing methods used to quantify the impact of presenteeism are unable to adequately inform the employer of the productive benefits of WPIs. The overall aim of this thesis was to appraise current methods used to quantify presenteeism and to develop methods to value the impact of presenteeism suitable for use in economic evaluations (EE) of WPIs. Methods: Two systematic reviews were conducted: 1) to assess the extent to which self-report measure of presenteeism were underpinned by economic theory; and 2) to explore if, and how, productivity was quantified and included in EE of WPIs for musculoskeletal conditions (MSDs). Thematic analysis methods were used to analyse qualitative data collected from working individuals with RA, AS or PsA (n=22) that explored the extent to which measures of health status (EQ5D; SF6D) and capability (ICECAP-A) capture the impact on ability to work caused by RA, AS or PsA. Econometric methods were used to specify prediction models that included measures of health status, capability and presenteeism, using a sample of 542 working people with RA and AS. Results The first systematic review identified 24 self-report measures of presenteeism; all, except one measure were not underpinned by economic theory. The second systematic review identified 20 EE of WPIs for MSDs. Absenteeism was included in all studies (n=20); however, presenteeism was included in only four. The qualitative data confirmed measures of health status and capability had the ability to capture those aspects of RA, AS and PsA that impact an individual’s ability to work. The best performing prediction model used an OLS specification including SF6D, age and gender to predict presenteeism measured by the WPAI. Conclusion: The results suggest that HRQoL measures, specifically the SF6D, can be used to capture and predict levels of presenteeism caused by RA, AS and PsA.

A consecutive series of 280 Charnley low-friction arthroplasties, performed between 1966 and 1978, on 192 patients, who were less than 40 years of age at the time of operation, were followed up for an average duration of 20.1 years. Patients were divided into four groups based on underlying disease process, and only three patients (5 hips) could not be traced. Patients with rheumatoid arthritis had significantly lower rates of acetabular component loosening, migration and revision (all p< 0.05), and patients with developmental dysplasia of the hip had the highest rates as well as a significantly higher rate of combined clinical and radiological component failure (p < 0.05). Patients with degenerative arthrosis had the highest rates of femoral implant loosening, revision and failure (all p < 0.05), and patients with ankylosing spondylitis and rheumatoid arthritis had the lowest. Age (< 30 years or 30 to 40 years at operation), gender, heterotopic ossification, hypertrophy of the femoral cortex at the tip of the prosthesis or development of changes in the medial femoral calcar were not associated with an increased risk of component failure or revision (all p > 0.05). The average annual rate of wear of revised components, in each of the four groups and the series as a whole, was significantly higher than the rate in surviving original components (p < 0.04), and the development of osteolysis, and increasing wear of the acetabular component were associated with failure and revision of both the acetabular and femoral components (both p < 0.01). Cox regression analysis confirmed that increasing average annual acetabular wear was the most significant factor determining the outcome of the arthroplasty (p < 0.001). For each additional millimetre of wear observed, the risk of component failure or revision in any one year increased significantly (p < 0.02). The 25-year survivorship of implants with an average acetabular wear rate of less than 0.1 mm/yr (117 arthroplasties) was greater than 90% but no arthroplasties with a rate in excess of 0.2 mm/yr survived 25 years, and only 40% survived 20 years.

Background: Psoriatic arthritis (PsA) is a chronic heterogenous inflammatory arthritis with five phenotypes. The two least studied phenotypes are investigated in this thesis, including: psoriatic spondyloarthropathy (PsSpA) and psoriatic arthritis mutilans (PAM). The aims of this thesis were to determine the prevalence, clinical characteristics and radiographic characteristics of PsSpA and PAM in a cohort of PsA patients, and serum-soluble bone- turnover biomarkers of these phenotypes. Aims: Comparisons were made with PsA patients without axial disease (pPsA), and ankylosing spondylitis (AS) patients. Methods: A prospective single-centre cross-sectional study was conducted of PsA and AS patients. Serum on psoriasis-only patients (PsC) and healthy controls (HC) were also obtained. Multivariate clinical, radiographic, genetic and serum biomarker comparisons were made between these five groups of subjects. Results: The study enrolled 201 PsA and 201 AS patients, who were then reclassified as 118 PsSpA, 127 pPsA and 157 AS cases, alongside 200 PsC and 50 HC subjects. Several clinical biomarkers, imaging biomarkers, serum-soluble biomarkers and genetic biomarkers were identified that differentiate PsSpA from pPsA and AS. PsSpA affected a significant proportion of PsA patients, and was not a milder version of AS. PsSpA involvement was as disabling and clinically impactful as AS. PAM was found to be associated with PsSpA, and clinical biomarkers of PAM occurrence and radiographic progression were identified. Conclusions: In conclusion, this thesis indicates that PsSpA is on a spectrum of musculoskeletal disease, in between pPsA and AS; with PsSpA comprising a continuum itself, and with a phenotype expression related to disease duration. These findings may prompt the inception of an international-consensus classification system for PsSpA, for which there is a great clinical need. Given that PsSpA has its own discrete clinical and biomarker signature, its clinical management and research should be tailored from that of pPsA and AS. Ultimately this may further the effort for stratified and personalised medicine.

Osteoporosis and fragility fractures are recognized complications of inflammatory rheumatic diseases. This is thought to result from the effects of chronic inflammation, relative immobility and corticosteroid use. A rare syndrome of osteoporosis in a patient with coeliac disease has been described which results from production of neutralizing antibodies to the bone protective protein osteoprotegerin (OPG). The aim of my thesis is to evaluate prevalence and clinical predictors of osteoporosis in a contemporary cohort of patients with rheumatoid arthritis (RA) and to investigate the role of OPG autoantibodies in the pathogenesis of osteoporosis in rheumatic diseases. In a retrospective cohort study, I found that the overall prevalence of osteoporosis in patients with RA was 29.9% which is in keeping with older reports that recorded a prevalence rate between 17% and 36%. In our contemporary cohort osteoporosis was significantly more common than in a gender and age matched control cohort (17.4%). Further analysis showed that only age and BMI were independent predictors of osteoporosis in RA. A predictive tool based on age and BMI was developed which had 91.4% sensitivity for the detection of osteoporosis in an independent RA population. I went on to screen for the presence of autoantibodies to OPG in patients with various rheumatic diseases. In a study of 75 patients with rheumatoid arthritis and 199 healthy controls OPG autoantibodies were detected in two controls (1%) compared with seven patients with RA (9.3%). The RA patients with detectable OPG antibodies had a longer disease duration, higher DAS28 scores and higher levels of the bone resorption marker CTX than RA patients who did not have autoantibodies. Purified IgG from patients with high levels of OPG antibodies blocked the ability of recombinant OPG to inhibit RANKL induced NFκB activation in a HEK293 cell based assay indicating that they were functional. In a further study of 134 patients with ankylosing spondylitis (AS), 16 patients (11.9%) had detectable OPG antibodies. The presence of OPG-Ab was independently associated with reduced hip bone mineral density and an increased risk of fractures in this population. In patients with a longer disease duration we have also observed that there was a higher discrepancy between spinal and hip BMD in OPG-Ab positive patients compared with OPG ab negative patients (p=0.003). In order to investigate if OPG antibodies affected measurement of serum RANKL concentrations as detected by ELISA using OPG as the capture reagent, I measured OPG ab and free RANKL concentrations in 55 rheumatic disease patients. Surprisingly there was a significant positive correlation between free RANKL and OPG Ab concentrations (r=0.430, p=0.001) which was the opposite to what I had expected. These findings reject the hypothesis that OPG ab block binding of synthetic OPG to RANKL in the ELISA. In conclusion, I have shown that osteoporosis is a common complication in RA and I have developed a new risk prediction tool for the use in clinical practice. I have also found that OPG antibodies are produced more commonly in patients with RA and AS than in healthy controls and that antibody levels correlate with bone resorption markers in RA and bone mineral density in AS patients. In vitro studies have shown that some OPG antibodies have functional effects on RANKL signalling. These findings raise the possibility that OPG antibodies may contribute to the pathogenesis of local and systemic bone loss in rheumatic diseases and signal the need to study the relationship between these antibodies and bone disease in large-scale longitudinal studies.

SAMMANFATTNING Hydroterapi har under en längre tid använts som behandlingsform, men det är brist på forskning inom området. Syftet med denna studie var att undersöka upplevelsen av hydroterapi hos personer med reumatisk sjukdom. En kvalitativ ansats anlades och enskilda semistrukturerade intervjuer användes. Deltagarna rekryterades genom bekvämlighetsurval via en kontakt på en reumatologklinik. Sex personer inkluderades: fem hade reumatoid artrit och en hade pelvospondylit. Resultatet tolkades med hjälp av en kvalitativ innehållsanalys, som visade på att deltagarna i denna studie var positivt inställda till hydroterapi. Deltagarna upplevde att hydroterapin ledde till minskad smärta, ökad rörlighet samt ett ökat välmående. Utöver de upplevda positiva effekterna återfanns även upplevda negativa effekter i form av träningsvärk och en ökad stelhet under sommaruppehållen. Det sociala samspel som uppstod i samband med hydroterapin upplevdes av de flesta deltagare ha haft positiv betydelse. Deltagarnas upplevelser kunde i diskussionen kopplas till operant och respondent inlärningsteori, där tänkbara konsekvenser och stimulin identifierades. Slutsatsen som drogs var att deltagarna upplevde hydroterapin som ett sätt att få tillbaka sin friska kropp genom att symtomen minskade. Då deltagarna upplevde att uppehåll orsakade försämring behövs hydroterapi som kontinuerlig behandlingsform för dem. Vidare forskning inom området bör fokuseras på enskilda patientgrupper samt inkludera ett större antal deltagare per patientgrupp. Nyckelord: hydroterapi, intervju, kvalitativ, pelvospondylit, reumatoid artrit.

The Human Leukocyte Antigen (HLA)-B27 is a Major Histocompability Complex (MHC) class I antigen that is strongly associated with development of a group of closely related arthritic diseases, collectively known as the spondyloarthropathies (SpA). However, the mechanism by which HLA-B27 confers this susceptibility is unclear. Studies have shown that HLA-B27 heavy chains can form classical heterotrimers associated with peptide and β2-microglobulin (B27HT), and also non-classical heavy chain homodimers (B27₂). B27₂ assemble intracellularly during maturation and are also expressed at the cell surface following endosomal recycling of B27HT. A pathogenic role for B27₂ has been proposed in two of the current theories of pathogenesis: the B27 homodimer theory and the B27 misfolding and UPR theory. Yet, determinations of the extent, distribution, and triggers of B27₂ expression, as well as the functional consequences of its receptor interactions in AS pathogenesis, have been hampered by the lack of a specific detection reagent. Therefore, to investigate the role of B27₂ in AS, we generated a novel antibody to B27₂ – HD6 – using phage display technology, which binds to in vitro refolded B27₂ but not B27HT complexes by ELISA. This thesis provides evidence that HD6-reactive molecules, which include B27₂, are expressed at the cell surface in both cell lines and in the context of a disease setting. Recognition is B27-specific and strongly correlated with the magnitude of B27 expression, which could account for the lack of staining in some cell subsets. Moreover, staining was comparable in cell lines expressing the disease-associated B*27:05 and the less disease-associated subtype B*27:09. In addition, I have shown cells expressing physiologic levels of B27, including EBV-transformed BCLs and AS patient PBMCs, are capable of expressing the HD6 epitope upon low pH treatment. Interestingly, these ‘acid-inducible HD6’ molecules were absent from cells lacking a functional PLC. Finally, I have shown that HD6-reactive molecules can derive from pre-existing folding B27 molecules at the cell surface, which may be inhibited by the addition of exogenous B27-binding peptides. These findings are consistent with a mechanism of pathogenesis involving the surface expression and recognition of B27₂ and/or other aberrantly folded forms of B27, as proposed in the homodimer theory. HD6 will be a powerful tool to address the potential pathogenic role of B27₂ in SpA and may additionally have therapeutic potential.

The MHC class I allele, HLA-B27, is strongly associated with a group of inflammatory arthritic conditions collectively known as spondyloarthropathies (SpA). Ankylosing spondylitis (AS) shows the strongest association with 90-95 % of patients being HLA-B27 positive. The relationship between HLA-B27 and SpA has been known for over 30 years, however despite ongoing research, the reason for this association has not yet been elucidated. In more recent years, research has focused on intrinsic properties of the HLA-B27 allele, in particular its propensity to misfold, forming homodimers. It has been proposed that these homodimers could be associated with the disease process through the activation of an ER stress response known as the unfolded protein response (UPR), or through aberrant recognition at the cell surface. We have investigated whether the expression of HLA-B27 is associated with the activation of the UPR. We have studied the expression of BiP, and the cleavage of XBP1 and ATF6 using stable and transiently expressing cell lines. We have also investigated the formation of non-B27 homodimers using a human cell line stably expressing HLA-B8, and finally we have studied the expression of homodimers in exosomes, small immunomodulatory vesicles released from numerous cell types. The results presented here lead us to conclude that in vitro studies of the UPR are complicated, prone to a number of technical issues, and may therefore not be appropriate for gaining information that would be of significant use when comparing to the real disease scenario. Our data suggest that non-B27 dimers may be strongly influenced by both the overexpression of MHC class I heavy chains and also the redox environment within the cell. We have isolated a novel fully folded, beta-2m-associated, MHC class I homodimer in exosomes and have detected a novel HLA-A and HLA-B mixed heavy chain dimer. Our results suggest that these dimers form through interactions between the cysteine residues in the cytoplasmic tail and that these dimers form in exosomes because they contain lower levels of the important antioxidant glutathione when compared to whole cells. Together, these results define a new MHC class I structure present on exosomes at significant levels, which could potentially influence immune recognition by both antigen-specific T cell receptors and NK family receptors. The data also poses questions about whether these novel structures, when they involve HLA-B27, could influence the pathogenesis of spondyloarthropathies.

The Major Histocompatibility Complex (MHC) is a genomic region in chromosome 6 which has been consistently found to be associated with the risk of developing virtually all common autoimmune diseases. Although its importance in disease pathogenesis has been known for decades, efforts to disentangle the roles of the classical human leukocyte antigens (HLA) and other variants responsible for the susceptibility to disease have often met with limited success, owing to the complex structure and extreme heterogeneity of the region. In this thesis, I interrogate the MHC for association with three common autoimmune diseases, ankylosing spondylitis, psoriasis and multiple sclerosis, with the aim of confirming the previously-reported associations and of identifying novel ones. To do so, I employ a systematic, joint analysis of single nucleotide polymorphism (SNP) and HLA allele data, in a logistic regression framework, using a recently developed algorithm to predict the HLA alleles for samples where such information is unavailable. To ensure the reliability of the analysis, I apply stringent quality control procedures and integrate over the uncertainty of the HLA allele predictions. Moreover, I resolve the haplotype phase of individuals from the HapMap project to create reliable reference panels, used in both HLA prediction and in quality control procedures. By directly testing HLA subtypes for association with the disease, the power to detect such associations is increased. I present the results of the analysis on the three disease phenotypes and discuss the evidence for important novel findings amongst both SNPs and HLA alleles in two of the diseases. In the final part of this thesis, I introduce a novel, model-based approach to detect inconsistencies in the data and show how it can be used to flag problematic SNPs which conventional quality control procedures may fail to identify.

Rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis belong among serious disorders, that effect joints and connective tissue. The patients suffer from pain and stiffness. Disease modifying drugs are an important part in the management of rheumatical disorders. When disease modifying drugs are failing, than biologic treatment is applied. For biologic treatment are registred etanercept (Enbrel), adalimumab (Humira), rituximab (MabThera), abatacept (Orencia) and infliximab (Remicade). In the Czech Republic was established the National registry of rheumatic disorders. There is a need of long term observations of patients, who have biologic treatment, to evaluate safety and socioeconomic data of the biologic treatment.

OBJETIVOS: Avaliar as alterações clínicas da superfície ocular, citologia de impressão (CI) e sintomas de olho seco (OS) dos pacientes com Espondilite Anquilosante (EA) e Artrite Reumatoide (AR). Classificar a intensidade de OS. Avaliar prospectivamente os parâmetros clínicos, laboratoriais e da superfície ocular dos pacientes com EA e AR submetidos a tratamento com drogas anti-TNF. MÉTODOS: Estudo prospectivo envolvendo inicialmente (pré-tratamento) 36 pacientes com EA e 20 pacientes com AR comparados com grupo controle de 39 voluntários saudáveis para o grupo de EA e 24 voluntários saudáveis para o grupo de AR. Do total inicial, 14 pacientes consecutivos com EA e 20 pacientes consecutivos com AR foram submetidos a terapia anti-TNF. Foram realizados os seguintes exames: teste de Schirmer I, tempo de rompimento do filme lacrimal, tingimento com corantes vitais e questionário dos sintomas de OS (Ocular Surface Disease Index-OSDI), citologia de impressão (CI) conjuntival, avaliação laboratorial inflamatória: velocidade de hemossedimentação e proteína C reativa (VHS e PCR) e atividade da doença pelas medidas de Bath Ankylosing Spondylitis Activity Index e Bath Ankylosing Spondylitis Functional Index (BASDAI e BASFI respectivamente) na EA e Disease Activity Score 28 (DAS 28) para AR. Além disso, avaliou-se a qualidade de vida pelo Health Assessment Questionnaire (HAQ) para ambas as doenças. As avaliações foram realizadas pré-tratamento e repetidas aos 3 meses (3M) e 12 meses (12M) após o início da terapia. RESULTADOS: Na avaliação pré-tratamento com drogas anti-TNF, os pacientes com EA apresentaram OS de intensidade leve a moderada (80,5% versus 43,6%, p=0,01) e maior escore de alteração da CI (55% versus 12,8%, p=0,007) associados a provas de atividade inflamatórias elevadas (p < 0,001) quando comparados com controles saudáveis. A avaliação longitudinal do tratamento com terapia anti-TNF demonstrou melhora da produção aquosa lacrimal (pré-tratamento: 13,7 ? 11,3 mm, aos 3M: 18,3 +- 11,1 mm e aos 12M: 19,3 +- 9,0 mm, p=0,04) assim como da CI conjuntival (pré-tratamento: 78,6% alterada, aos 3M: 57,1%, e aos 12M: 35,7%, p=0,03). Houve, paralelamente, melhora dos parâmetros inflamatórios e da atividade da doença (p < 0,05). No grupo de pacientes com AR, no momento pré-tratamento com drogas anti-TNF, foram observados maior frequência (75% versus 4%, p < 0,001) e intensidade leve de OS (65% versus 4%, p < 0,001) associados a sintomas moderados (escore OSDI 24,0 +- 17,6 versus 7,5 +- 14,3, p=0,001) quando comparados com controles saudáveis. Esses pacientes também apresentaram maior frequência de disfunção das glândulas de meibômio (55,0% versus 8,3%, p=0,001), maior escore de alteração da CI conjuntival (1,0 +- 0,6 versus 0.0 +- 0,2, p=0,001) e menor densidade de células caliciformes (431,3 +- 209,5 células/mm2 versus 804,8 +- 383,2 células/mm2, p < 0,001) quando comparados com o grupo controle. A análise prospectiva dos pacientes com AR tratados com drogas anti-TNF mostrou um aumento dos valores do teste de Schirmer (prétratamento: 11,8 +- 6,7 mm, aos 3M: 21,0 +- 10,4 mm, e aos 12M: 23,0 +- 9,7mm, p < 0,001), melhora da CI conjuntival (pré-tratamento: 1,0 +- 0,6, aos 3M: 0,8 +- 0,6, e aos 12M: 0,5 +- 0,5, p=0,005) e da densidade de células caliciformes (pré-tratamento: 429 +- 211,7 células/mm2, aos 3M: 908 +- 291,4 células/mm2, e aos 12M: 1265,4 +- 430,6 células/mm2, p=0,001). Os marcadores de atividade inflamatória sistêmicos (VHS e PCR) também melhoraram ao longo do tratamento (p=0,005 e p=0,006, respectivamente). CONCLUSÃO: Os pacientes com EA e AR avaliados nesse estudo apresentaram prevalência elevada de OS de intensidade leve a moderada associada à alteração da citologia conjuntival. A recuperação precoce e manutenção de longo prazo na produção aquosa da lágrima e na CI conjuntival, em especial, das células caliciformes, nos pacientes submetidos a terapia anti-TNF, pode refletir a melhora da condição inflamatória. Esse resultado histológico pode ter influência como biomarcador da inflamação na superfície ocular
OBJECTIVES: Evaluate ocular surface parameters, impression cytology (IC) and dry eye (DE) symptoms of patients with Ankylosing Spondylitis (AS) and Rheumatoid Arthritis (RA). Classify DE severity grade. Analyse prospectively clinical and laboratory, as well as ocular surface parameters of AS and RA patients, submitted to anti-TNF therapy. METHODS: This prospective study initially (baseline) enrolled 36 AS patients and 20 RA patients who were compared to a control group of 39 and 24 healthy volunteers for the AS group and RA group, respectively. From the initial group, 14 consecutive AS and 20 consecutive RA patients received anti-TNF therapy. They underwent the following exams: Schirmer I test, tear break-up time, vital dyes staining of the ocular surface, a questionnaire for dry eye symptoms- Ocular Surface Disease Index (OSDI), and conjunctival IC. Laboratory tests for inflammatory activity were assessed by erythrocyte sedimentation rate and C- reactive protein (ESR and CRP). The Bath Ankylosing Spondylitis Activity Index and Bath Ankylosing Spondylitis Functional Index (BASDAI and BASFI, respectively) in AS and Disease Activity Score 28 (DAS 28) in RA analyzed disease activity parameters. Besides, the Health Assessment Questionnaire evaluated the quality of life in both group of diseases. These measurements were taken at baseline (BL) and repeated at 3 months and 12 months (3M and 12M, respectively) after the beginning of anti-TNF therapy. RESULTS: At the baseline moment, AS patients presented mild to moderate DE (80.5% vs 43.6%, p=0.01) and a higher score of altered IC (55% vs 12.8%, p=0.007) associated with the systemic inflammatory activity (ESR and CRP, p < 0.001) when compared to healthy volunteers. The longitudinal evaluation of anti- TNF treatment showed an improvement of aqueous tear production (BL: 13.7 +- 11.3 mm, 3M: 18.3 +- 11.1 mm and 12M: 19.3 +- 9,0 mm, p=0.04). The IC also improved (BL: 78.6% altered IC, 3M: 57.1% and 12M: 35.7%, p=0.03). There was a parallel amelioration of systemic inflammatory markers and disease activity (p < 0.05). Concerning the RA group of patients, at the baseline moment, there was a higher frequency of DE (75% vs 4%, p < 0.001) as well as mild DE severity grade (65% vs 4%, p < 0,001) associated with moderate symptoms of DE (OSDI score: 24.0 +- 17.6 vs 7.5 +- 14.3, p=0.001) when compared to healthy volunteers. This group of patients also presented higher frequency of meibomian gland dysfunction (55% vs 8.3%, p=0.001), a worse score of IC (1.0 +- 0.6 vs 0.0 ? 0.2, p=0.001) and lower goblet cells count (431.3 +- 209.5 cells/mm2 vs 804.8 +- 383.2 cells/mm2, p< 0.001) when compared to the control group. The prospective analysis of RA patients treated with anti-TNF drugs demonstrated an increase of Schirmer\'s test (BL: 11.8 +- 6.7, 3M: 21.0 +- 10.4, 12M: 23.0 +- 9.7, p < 0.001) and an improvement of cytological grade (BL: 1.0 +- 0.6, 3M: 0.8 +- 0.6, 12M: 0.5 +- 0.5, p=0.005) and goblet cells density (BL: 429,0 +- 211.7 cells/mm2, 3M: 908,0 +- 291.4 cells/mm2, 12M: 1265.4 +- 430.6 cells/mm2, p=0.001). The systemic inflammatory markers (ESR and CRP) also improved throughout the treatment period (p=0.005 and p=0.006, respectively). CONCLUSION: Patients with AS and RA enrolled in this study presented a higher prevalence of mild to moderate DE associated with altered IC. The prompt and maintained aqueous tear and conjunctival cytology recovery, especially the goblet cells, in patients submitted to anti-TNF therapy seem to represent the improvement of inflammatory condition. This histological outcome may have an influence as a biomarker of ocular surface inflammation

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2016
In many inflammatory diseases, such as rheumatoid arthritis (RA) and ankylosing spondylitis (AS), bone is a target for immune cells unbalancing bone remodeling. RA typically presents as a symmetric polyarthritis, affecting more women than men and is linked with the presence of autoantibodies in the serum such as anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF). Human leukocyte antigen (HLA)-DR genes are strongly associated with the disease. Chronic inflammation in RA leads to cartilage and bone destruction, which is typically recognized as erosive disease on x-rays. In contrast, AS is characterized by axial disease involving the sacroiliac joints and the spine. AS affects more men than women and is strongly associated with HLA-B27 haplotypes. The long-term outcome is characterized by ankylosis of the spine and sacroiliac joints. While RA is a disease characterized by destruction of bone and cartilage, the predominant finding in AS is bone formation rather than its destruction. In this work, we hypothesize that the inability of osteoclasts (OC) or its precursors to respond to osteoclastogenic stimuli in AS patients contributes to the excessive bone formation characteristic of this disease. Therefore, the aim of this thesis was the characterization of OC circulating precursors (monocytes) in RA and AS, as well as their ability to differentiate in resorbing OC when cultured in vitro. Moreover, we also aimed to understand the effect of therapies, such as methotrexate (MTX) and tumor necrosis factor inhibitors (TNFi), in the OC precursors in RA and AS patients. We first investigated whether cytokines were dysregulated in very early rheumatoid arthritis patients with less than 6 weeks of disease duration (VERA) before and after treatment with corticosteroids and MTX. Pro-inflammatory cytokines were quantified in the serum of VERA and established RA patients and compared with other very early arthritis (VEA) and healthy controls. Patients were also analyzed after therapy. Synovial fluid (SF) from RA and osteoarthritis (OA) patients was also analyzed. VERA patients had increased serum levels of cytokines promoting Th17 polarization (IL-1β and IL-6), as well as IL-8 and Th17-derived cytokines (IL-17A and IL-22) known to promote the chronicity of inflammation. These cytokines are also associated with the promotion of osteoclastogenesis. In established RA this pattern was more evident within the SF. Early treatment with MTX or corticosteroids led to clinical improvement but without an impact on the cytokine pattern. VERA patients already display increased levels of cytokines related with Th17 polarization and osteoclastogenesis, a deregulation also found in SF of established RA, suggesting that a cytokine-milieu favoring Th17 and OC activity is an early event in RA pathogenesis. We then aimed to assess the effect of MTX on circulating OC precursors and OC differentiation in RA patients. RA patients were assessed before therapy and at least 6 months after the introduction of MTX therapy and results controlled with healthy donors. We determined receptor activator of NF-κB (RANK) ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines and in vitro OC differentiation were also performed. We found that serum RANKL, classical activation monocytes markers (C-C chemokine receptor 2 - CCR2, CD86, HLA-DR) and RANK were increased in RA patients with active disease compared to healthy donors, and after MTX exposure these parameters normalized to control levels. Although we found no differences in OC number, cells differentiated from RA patients showed higher resorption activity than from healthy donors. Again, after MTX treatment, osteoclasts resorption activity was normalized. The results of this work suggested that MTX plays an important role in downregulating OC function through the decrease in RANK surface expression in monocytes. We then proceeded to study the effect of TNFi in osteoclastogenesis in RA patients. RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Results were controlled with healthy donors. After TNFi therapy, RANKL surface expression was downregulated in B lymphocytes and the frequency of circulating OC precursors was also decreased. Cells from TNFi treated patients had decreased osteoclast numbers and resorption activity as well as decreased expression of specific genes important for osteoclastogenesis, like tumour necrosis factor receptorassociated factor (TRAF6), fos-related antigen 2 (FRA-2) and for bone resorption like cathepsin K. Therefore, we suggest that in RA TNFi decreases bone resorption through the direct reduction of the number of circulating precursors and the inhibition of intracellular signalling pathways acting through TRAF6. After exploring OC precursor behavior in untreated and treated RA patients we aimed to characterize bone remodeling and pro-osteoclastogenesis inflammatory environment, monocytes phenotype and in vitro OC differentiation in AS patients. xli Patients with active AS without any ongoing therapy and age and gender matched healthy donors were recruited. We observed that pro-inflammatory cytokine levels were higher in a cohort of untreated AS patients when compared to healthy donors, but CD51/CD61 expression (integrin αvβ3 or vitronectin receptor, important for osteoclast attachment to the bone matrix) was downregulated in the classical OC precursors. No differences in the in vitro osteoclastogenesis or bone resorption was observed when compared to healthy donors, however we found low expression of colony stimulating factor 1 receptor (CSF1R), RANK and nuclear factor of activated T cell c1 (NFATc1) in AS osteoclast precursors that consequently led to a decreased expression of important resorption genes such as cathepsin K. These findings showed us that despite the high levels of proinflammatory cytokines present in AS patients, circulating monocytes have low OC specific gene expression supporting our hypothesis of an impaired response of OC precursors to pro-osteoclastogenic stimuli in AS patients. In an effort to understand the effects of TNFi therapy in circulating OC precursors and their differentiation ability from AS patients, we followed up a cohort of patients before and after therapy. Results were controlled with healthy donors. We found that IL-17A and IL-23 circulating levels decreased after TNFi treatment. OC number was decreased in AS patients before treatment when compared to control. However, no differences in OC precursor frequency or in the number OC in culture were found after treatment. RANK, CSF1R and NFATc1 expression was downregulated in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiated from AS TNFi-treated patients showed higher resorption activity than cells from patients before treatment. These results showed us that in AS patients, TNFi treatment reduces systemic proosteoclastogenic stimuli but when OC precursors are exposed to TNFi therapy they have increased in vitro activity in response to osteoclastogenic stimuli. From this work we were able to understand some of the mechanisms by which MTX and TNFi therapies act on circulating OC precursors in RA and AS patients. Although RA and AS are two chronic immune mediated diseases their effect on bone metabolism is different. The work here discussed shows that RA and AS OC precursors have a different behaviour in vitro, even coming from a similar pro-inflammatory milieu. Our findings support the hypothesis that OC from AS patients have impairment in their activity when compared both to RA patients or healthy controls. This difference can be partially explained by an intrinsic inability to respond to osteoclastogenic stimuli and by downregulation of key OC differentiation and activity genes in AS patients.
Projeto,“Differences in Bone Cell Activity Between Rheumatoid Arthritis and Ankylosing Spondylitis” financiado pela Merck Sharp & Dohme Corp - Merck_P08574.

碩士
中山醫學大學
醫學研究所
98
Background：Imbalance of immune tolerance had been found to associate with the occurrence of autoimmune diseases. The CD4+ and CD8+ T cells had also been revealed more predominant expression in ankylosing spondylitis (AS) patients than healthy controls, and such expression may relate to the imbalance in the negative signal of activated T cells. Especially, cytotoxic T lymphocyte antigen-4 (CTLA-4) might play a role in the inhibition of activated T cells in the initial period of autoimmune tolerance. Programmed cell death-1(PD-1) binds its ligands, programmed cell death-1 ligand 1(PD-L1) and programmed cell death-1 ligand 2 (PD-L2), and induced negative signals that might maintain the balance of immune tolerance during late and sequential periods. However, molecular roles of autoimmune tolerance-related genes in AS development are unclear. Therefore, we designed a hospital-based case-control study to evaluate the association between CTLA-4, PD-1, PD-L1, and PD-L2 genetic polymorphisms and AS occurrences and clinical manifestations. Methods and Materials：A total of 325 AS patients and 325 age and gender-matched controls were recruited in our study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied to identify the CTLA-4 A+49G, PD-1 G-536A, PD-L1 A8923C, and PD-L2 C47103T genotypes. The disease activity and functional status of AS patients were evaluated by Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), and Bath Ankylosing Spondylitis Global (BAS-G). Results：Our results showed that those who carried PD-1 G-536A G allele had a 1.42 fold (95% confidence interval; 95% C.I. = 1.14-1.76) significant risk of AS than those with A allele. Those who carried PD-L2 T allele also had a significantly lower risk of AS development than those with C allele (relative risk; RR = 0.01; 95% C.I. = 0.001-0.070). In addition, those who simultaneously carried PD-1 GG or GA, PD-L1 CC, and PD-L2 CC genotypes had a significant combined effect in AS occurrence (RR = 7.17; 95% C.I. = 1.38-37.31). Compared to the carriers with CTLA-4 GG and PD-1 AA combined genotypes, the carriers with CTLA-4 AA or AG and PD-1 GG or GA genotypes had a 2.30 fold (95% C.I. = 1.27-4.16) risk for disease development. Conclusion：These results show that CTLA-4, PD-1, PD-L1, and PD-L2 genes may associate with the imbalance of autoimmune tolerance in AS patients.

Abstract Graeme Kohler BSc. (Health Education), MA (Health Promotion) School of Health and Human Performance, Dalhousie University Objective: To understand the experiences of young men living with Ankylosing Spondylitis (AS). Methods: Using an interpretive phenomenological research approach, two semi-structured interviews were conducted with 7 male informants ranging from ages 22 – 37. All of the informants lived in Nova Scotia and had been diagnosed with AS for at least one year. Thematic analysis was used to analyze the data. Results & Conclusions: Informants displayed a strong affinity to hegemonic masculine behaviours. The overriding theme was I’m a Man. The four emerging themes were: Trying to maintain normalcy, Do what I like to do, I have to work, and I don’t really ask for support. Several barriers to support and health care access were identified that have implications for health promotion, the men themselves, and various AS care providers.

Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
O aparecimento dos medicamentos biológicos no mercado nacional veio revolucionar o tratamento de doenças graves e de mau prognóstico, tendo repercussões tanto a nível pessoal como social e, por isso, o seu potencial inovador em saúde marcou o início de uma nova era de tratamentos.Associado ao poder de inovação destas terapias surgiram preços elevados, podendo ter sido criada uma dificuldade ao seu acesso.Com o objetivo de minimizar estas dificuldades, muitos destes medicamentos estão ao abrigo de um regime excecional de comparticipação, em que o Serviço Nacional de Saúde financia a 100% estes regimes de tratamentos, levando à isenção de encargos por parte do doente. Contudo, numa tentativa de monitorizar o consumo destes medicamentos biológicos, constantes na Portaria no. 48/2016 de 22 de março, e suas alterações, tornou-se obrigatório um registo mínimo para a dispensa deste tipo de medicamentos. É com base nestes registos que se procedeu à elaboração do presente estudo que tem como objetivo principal a caraterização do consumo hospitalar em Portugal dos medicamentos biológicos identificados para o registo mínimo e indicados para o tratamento da artrite reumatoide, artrite psoriática, artrite idiopática juvenil poliarticular, psoríase em placas e espondilite anquilosante.O estudo inicial de base populacional pretende mostrar o consumo real destes medicamentos nos anos de 2016 e 2017, permitindo avaliar discrepâncias a nível regional, diferenças de consumo para medicamentos indicados para a mesma doença e diferenças nos padrões de prescrição entre o serviço público de saúde e o privado. Palavras-chave: medicamentos biológicos; consumos hospitalares; psoríase em placas; artrite reumatóide; espondilite anquilosante; artrite psoriática; artrite idiopática juvenil poliarticular.
The emergence of biological medicines on the national market has revolutionized the treatment of serious and bad prognosis diseases, having repercussions on both personal and social levels, and that is why its innovative health potential, marked the beginning of a new era of treatments.Associated with the innovative power of these therapies, high prices and difficulties in their access may have been created.In order to minimize these difficulties, many of these medicines are under an exceptional reimbursement system, whereby the National Health Service finances 100% of this treatment and so the patient doesn’t have to pay anything.However, in order to control the consumption of these biological medicines, present at the Ordinance no. 48/2016 of March 22, a minimum registration for dispensing this type of medicines has become mandatory. Based on these records, the present study was designed to characterize the hospital consumption in Portugal of the biological medicines covered by the minimum registration and indicated for the treatment of rheumatoid arthritis, psoriatic arthritis, polyarticular juvenile idiopathic arthritis, plaque psoriasis and ankylosing spondylitis.The initial population-based study aims to show the real consumption of these medicines in 2016 and 2017, allowing assessing to regional discrepancies, differences in drug uses and different prescribing patterns between public and private health services.Keywords: biological medicines; hospital consumptions; rheumatoid arthritis; psoriatic arthritis; polyarticular juvenile idiopathic arthritis; plaque psoriasis and ankylosing spondylitis.

v anglickém jazyce Introduction: No information was known about frequency of common inflammatory disorders in rheumatology in the Czech Republic. Aims of the study: To estimate the standardized annual incidence (INC) and point prevalence (PREV) of six diseases (rheumatoid arthritis-RA, juvenile idiopathic arthritis-JIA, gout, psoriatic arthritis-PsA, ankylosing spondylitis-AS, reactive arthritis-ReA) in a population-based study in two regions of the Czech Republic (CR). Methods: INC: Incident cases were registered on condition that the definite diagnosis was confirmed according to existing classification criteria during the study period. PREV was studied on the basis of identification of established diagnoses at a time point. Crude rates were standardized for age and sex. Results: Both INC and PREV are shown per 100.000 inhabitants. RA INC:31 (95%CI 20-42), PREV:610 (95%CI 561-658). Gout-INC:41 (95%CI 28-53), PREV:300 (95% CI 266-334). JIA-INC: 13 (95% CI 1-20), PREV:140 (95%CI 117-280). PsA-INC:3,6 (95% CI 1-8), PREV:49 (95%CI 40-60). AS-INC:6 (95% CI 3-11), PREV:94 (95% CI 94-109). ReA-INC:9 (95% CI 6-15), PREV:91 (95% CI 78-106). Conclusion: This is the first population-based study estimating annual incidence and prevalence rates of the most common rheumatological disorders in the Czech...

Οι αντι-TNFα βιολογικοί παράγοντες χρησιμοποιούνται ευρέως στην καθ’ ημέρα κλινική πράξη για την αντιμετώπιση συστηματικών φλεγμονωδών νοσημάτων όπως είναι η ρευματοειδής αρθρίτιδα, η αγκυλοποιητική σπονδυλίτιδα, η ψωριασική αρθρίτιδα και η νόσος του Crohn. Σύντομα, όμως, έγινε αντιληπτό ότι οι ασθενείς υπό θεραπεία με ανταγωνιστές του TNFα συχνά αναπτύσσουν στον ορό τους αντιπυρηνικά αντισώματα (ΑΝΑ) κυρίως τύπου IgM, ενώ ένα μικρό ποσοστό των ασθενών αυτών αναπτύσσει κλινικό σύνδρομο που θυμίζει ιδιοπαθή ΣΕΛ. Tα Β λεμφοκύτταρα θεωρείται ότι κατέχουν κεντρικό ρόλο στην παθογένεια του ΣΕΛ. Xαρακτηρίζονται από επίταση των φωσφορυλιωμένων πρωτεϊνών σε υπολείμματα τυροσίνης μετά από διέγερση του Β αντιγονικού υποδοχέα (BCR) και από μειωμένη έκφραση της κινάσης Lyn, ενός ενζύμου που ανήκει στην οικογένεια των Src κινασών και διαδραματίζει διπλό ρόλο, ευοδωτικό και ανασταλτικό, στην οδό σηματοδότησης του BCR. Επιπλέον, τα Β λεμφοκύτταρα στον ΣΕΛ χαρακτηρίζονται από μειωμένη έκφραση του δείκτη CD21 (υποδοχέας του συμπληρώματος τύπου 2), ενώ υπάρχουν αναφορές για αυξημένη έκφραση του δείκτη επιφανείας CD20. Για να μελετήσουμε τους μηχανισμούς της αυτοανοσίας που επάγεται από τη χρήση των αντι-TNFα παραγόντων εξετάσαμε εάν τα Β λεμφοκύτταρα των ασθενών υπό αντι-TNFα αγωγή αποκτούν φαινοτυπικά ή/και λειτουργικά χαρακτηριστικά που να προσομοιάζουν με αυτά που έχουν περιγραφεί για τα Β λεμφοκύτταρα στον ΣΕΛ. Συγκεκριμένα μελετήσαμε τα επίπεδα της Lyn, Syk, SHP-1, της φωσφορυλιωμένης Syk στη θέση τυροσίνης 348 (ΡΥ348-Syk), τα επίπεδα των τυροσινικά φωσφορυλιωμένων πρωτεϊνών καθώς και τους δείκτες επιφανείας CD20, CD21 και CD5 σε 29 ασθενείς με συστηματικά ρευματικά νοσήματα πριν και μετά την έναρξη θεραπείας με αντι-TNFα βιολογικούς παράγοντες. Διαπιστώσαμε ότι τα επίπεδα της Lyn, αλλά όχι της Syk ή της SHP-1, αυξάνονται μετά τη θεραπεία με αντι-TNFα παράγοντες, ιδίως σε ασθενείς με σπονδυλοαρθροπάθειες. H ενζυματική δραστηριότητα της Lyn διατηρείται μετά την έναρξη αγωγής με αντι-TNFα, όπως διαπιστώθηκε από την κατά 2.9 φορές αύξηση των επιπέδων της PY348-Syk, η οποία χαρακτηρίζει την ενεργοποιημένη μορφή της Syk και αποτελεί ειδικό στόχο της Lyn. Επιπλέον, βρήκαμε ότι τα μη-διεγερμένα Β λεμφοκύτταρα ασθενών που λαμβάνουν ανταγωνιστές του TNFα εμφανίζουν επίταση των φωσφορυλιωμένων πρωτεϊνών σε θέσεις τυροσίνης. Το CD20, ένας δείκτης επιφανείας που εκφράζεται αποκλειστικά στα Β λεμφοκύτταρα και συμμετέχει στη διατήρηση των ενδοκυτταρίων επιπέδων Ca++ μετά από διέγερση του BCR αυξάνεται μετά από θεραπεία με αντι-TNFα βιολογικό παράγοντα κυρίως στους ασθενείς με ρευματοειδή αρθρίτιδα, ενώ τα επίπεδα του CD21 παρέμειναν αμετάβλητα. Επίσης, διαπιστώσαμε επέκταση του πληθυσμού των CD5+ B λεμφοκυττάρων, ενός υποπληθυσμού των Β κυττάρων που αποτελεί γνωστή πηγή φυσικών αυτοαντισωμάτων, κατά τη διάρκεια θεραπείας με αντι-TNFα. Τα ευρήματά μας υποδηλώνουν ότι τα Β λεμφοκύτταρα των ασθενών που λαμβάνουν θεραπεία με ανταγωνιστές του TNFα εμφανίζουν λειτουργικές και φαινοτυπικές διαταραχές οι οποίες μπορεί να σχετίζονται με την επαγωγή αυτοανοσίας, αλλά διαφέρουν από τις διαταραχές που έχουν περιγραφεί για το Β λεμφοκύτταρο στον ΣΕΛ και μπορεί να αναπαριστούν ένα διαφορετικό μοντέλο αυτοανοσίας. Περαιτέρω μελέτες για την αποσαφήνιση των μηχανισμών αυτοανοσίας από την χρήση των αντι-TNFα παραγόντων θα μπορούσαν να συμβάλλουν στην κατανόηση των μοριακών μηχανισμών και της παθογένειας άλλων αυτοάνοσων φλεγμονωδών νοσημάτων.
Biologic agents against ΤΝFα have been widely used for the management of systemic inflammatory disorders, such as rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and Crohn’s disease. However, it soon became apparent that patients under ΤΝFα blockade commonly develop serologic autoimmune manifestations. Emergence of ANA, usually of the IgM isotype, is common in anti-TNFα treated patients and a few of these patients may develop a SLE-like syndrome. B cells are implicated in the pathogenesis of SLE and are characterized by enhanced tyrosine phosphorylation of proteins following BCR ligation and reduced expression of Lyn, a Src-family kinase abundantly expressed in B cells with both stimulatory and inhibitory properties on the BCR-signaling pathway. Reduced levels of B-cell surface CD21 (complement receptor type 2) and increased expression of CD20 have also been described for lupus B cells. To dissect the mechanisms of anti-ΤΝFα induced autoimmunity we examined the phenotype and function of B cells prior to and following treatment with ΤΝFα antagonists. Levels of Lyn, Syk, SHP-1, tyrosine 348 phospho-Syk (PY348-Syk) and tyrosine phosphorylated proteins were evaluated in 29 patients before and following treatment with TNFα blockers. B-cell surface expression of CD20, CD21 and CD5 were also assessed. Following treatment, B cell cytoplasmic levels of Lyn, but not Syk or SHP-1, significantly increased following treatment with anti-ΤΝFα agents, particularly in patients with spondyloarthropathies. Increased Lyn levels following treatment correlated with increased Lyn enzymatic activity as evidenced by a 2.9-fold increase of PY348-Syk levels, which comprises a Lyn-specific target. Moreover, unstimulated peripheral B cells from anti-ΤΝFα treated patients displayed a tendency towards increased tyrosine phosphorylated proteins following treatment. CD20, a B-cell restricted signaling-associated molecule implicated in the regulation of intracellular calcium levels following BCR ligation, significantly increased in patients with rheumatoid arthritis following treatment with anti-ΤΝFα agents whereas levels of CD21 remained unchanged. Circulating CD5+ B cells, a B-cell subpopulation and a known source of natural autoantibodies, were also significantly expanded during treatment. Our findings suggest that B cells in anti-ΤΝFα treated patients display functional and phenotypical aberrations that may be associated with the induction of autoimmunity, but do not acquire a typical idiopathic SLE profile. These aberrations are distinct from those previously described for lupus B cells and may thus represent a different model of autoimmunity. Further studies regarding the mechanisms underlying the induction of autoimmunity by TNFα antagonists may enhance our understanding on the molecular backgrounds and the pathogenesis of other autoimmune inflammatory disorders.