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

Abstract Rheumatoid arthritis (RA) is a systemic autoimmune disease with symmetrical articular manifestations. The etiology of the disease is unknown. The prevalence of RA is approximately 0.5–1.0% in adults. In Finland, the annual incidence is 39/100 000. RA is about three times more common in females than in males. Most commonly the disease affects first the joints of feet and fingers. Chronic inflammation leads to erosions of cartilage, bone and tendons and may destroy the whole joint. The diagnosis of RA is mainly based on the clinical features of the disease. The American College of Rheumatology (ACR) 1987 revised classification criteria of RA have commonly been used for diagnosis. No specific diagnostic test is available. Rheumatoid factor (RF) has traditionally been used in the diagnosis, but only 70 to 80% of RA patients have RF in their serum. Other antibodies found in RA are the antiperinuclear factor (APF), the anti-keratin antibody (AKA) and the antibodies to cyclic citrullinated peptide (CCP), which recognize the citrulline-containing antigenic filaggrin protein. Citrulline is an amino acid that is post-translationally formed from arginine by peptidylarginine deiminase enzymes (PADs). Autoantibodies to citrullinated proteins are more specific for RA than RF. There is no filaggrin in joints, which indicates that the autoantibodies reacting with this protein most probably only reflect immunological cross-reaction. It has been postulated that autoimmunity against collagens might be involved in the pathogenesis of RA. There are antibodies binding to collagen in cartilage (type II collagen) and in bone (type I collagen). They have been tested by using collagen preparations rendered soluble by pepsin digestion. This digestion removes the carboxyterminal (C-terminal) telopeptides of collagen. Autoantibodies to the C-telopeptides of type I and II collagens were studied in this doctoral research. Autoantibodies to the citrullinated C-telopeptides of type I and II collagens were found in the serum of patients with RA. ELISA, CLIA and inhibition ELISA were used to detect these autoantibodies. Automatic CLIA gives a more than twofold number of positive findings compared to previous ELISA. Currently the best method for the detection of these autoantibodies is inhibition ELISA. These autoantibodies are specific for citrulline in the peptide sequence. Autoantibodies that bind the normal C-telopeptides of type I and II collagens were not inhibited by soluble normal or citrullinated telopeptides. However, the antibodies that bind only citrullinated telopeptides could be inhibited by corresponding citrullinated telopeptides. Autoantibodies binding the citrullinated telopeptides of type II collagen and anti-CCP predict synergistically the development of seropositive RA. / Tiivistelmä Nivelreuma (arthritis rheumatoides) on krooninen autoimmuunisairaus, jonka aiheuttajaa ei tunneta. Nivelreuman esiintyvyys aikuisilla on 0.5–1.0 prosenttia. Siihen sairastuu vuosittain 39/100 000 suomalaista aikuista. Naiset sairastavat nivelreumaa kolme kertaa yleisemmin kuin miehet. Sairaus alkaa tavallisesti päkiöistä ja sormien nivelistä. Nivelreuma aiheuttaa ruston, luun ja nivelsiteiden syöpymistä ja voi lopulta tuhota koko nivelen. Nivelreuman diagnoosi perustuu pääasiassa taudin kliinisiin piirteisiin. Yhdysvaltain reumajärjestön (American College of Rheumatology, ACR) vuonna 1987 esittämät luokittelukriteerit ovat yleisesti käytössä. Taudin toteamiseen ei ole spesifistä laboratoriotestiä. Perinteisesti käytettyä reumatekijää esiintyy 70–80 prosentilla potilaista. Muita nivelreumapotilaan seerumista esiintyviä vasta-aineita ovat antiperinukleriaaritekijä (APF), keratiinivasta-aineet (AKA) ja vasta-aineet sykliseen sitrullinisoituneeseen peptidiin (CCP). Sitrulliini on aminohappo, jonka peptidyyliarginiini deiminaasi -entsyymit (PAD) ovat muokanneet arginiinista posttranslationaalisesti. Sitrullinisoituneiden proteiinien autovasta-aineet ovat spesifisempiä nivelreumassa kuin reumatekijä. Nivelissä ei ole filaggriinia, mikä viittaa siihen, että todetut vasta-aineet perustuvat immunologiseen ristireaktioon. On epäilty, että nivelreuman patogeneesiin voisi liittyä autoimmuniteettia kollageeniin. Aiemmin tutkitut luun ja ruston (tyypin I ja II) kollageenivasta-aineet eivät ole olleet sitrullinisoituneita. Autovasta-ainetesteissä on käytetty pepsiinidigestiota, jolla kollageeni on saatu liukoiseksi. Pepsiinidigestio tuhoaa kuitenkin kollageenin karboksyyliterminaaliset (C-terminal) telopeptidit. Tässä väitöskirjatutkimuksessa tutkittiin tyypin I ja II kollageenien C-telopeptidejä. Nivelreumapotilaiden seerumissa todettiin autovasta-aineita, jotka sitoutuvat sitrullinisoituneisiin tyypin I ja II kollageeneihin. Todettuja vasta-aineita voidaan osoittaa ELISA-, CLIA- ja inhibitio-ELISA-menetelmillä. Automaattisella CLIA:lla saadaan kaksi kertaa enemmän positiivisia löydöksiä kuin aiemmin kehitetyllä ELISA:lla. Tällä hetkellä paras menetelmä näiden autovasta-aineiden osoittamiseen on inhibitio-ELISA. Todetut vasta-aineet ovat spesifisiä peptidin sekvenssissä olevaan sitrulliiniin. Vasta-aineita, jotka sitoutuvat normaaliin tyypin I ja II kollageenien C-telopeptidiin, ei voida inhiboida liukoisella normaalilla eikä sitrullinoidulla telopeptidillä. Kuitenkin vasta-aineita, jotka sitoutuvat vain sitrullinisoituihin telopeptideihin, voidaan inhiboida vastaavalla liukoisella sitrullinisoidulla telopeptidillä. Autovasta-aineet, jotka sitoutuvat samanaikaisesti sitrullinisoituneeseen tyypin II kollageenin C-telopeptidiin ja anti-CCP:hen ennustavat seropositiivista nivelreumaa.

Magister Psychologiae - MPsych / This study explored women's perceptions of how their illness (Rheumatoid Arthritis- RA) affects their relationship with their intimate life partner. RA is a chronic, inflammatory, auto-immune illnes, which mainly affects the synovial membranes of multiple joints. This highly inflammatory poly-arthritis may lead to joint destruction, chronic pain, deformity and loss of functioning as unfortunate outcomes of the established illness. RA affects key life domains such as psychological well-being, social well-being, family and couple relationships, employment, loss of independence and restrictions in daily functioning. / South Africa

Rheumatoid Arthritis (RA) is the most common type of chronic inflammatory arthritis (Thompson, 1998). When appropriately prescribed, therapeutic exercise is useful in the care of patients with RA (Semble et aI., 1990). A pre-test - post-test randomized groups design was adopted for the study to compare the effects of a land- and water-based exercise programme in RA patients. A total of ten subjects, diagnosed with RA functional class I or II according to Steinbrocker, were assigned to either a group performing water-based exercises (W, n=4), a group performing land-based exercises (L, n=4), or a control group, who were requested to continue with their present sedentary lifestyle (C, n=2). For inclusion in the study, subjects were required to be on stable medication. Categories of dependent variables measured, were disease activity, haematology, functional and psychological status as well as physical status. There was a reduction in total swollen and tender joint counts in both experimental groups, but not the control group. The reduction was greater in group W than group L. Total tender joint count (DC) decreased by 53% (p<0.1O) and the total swollen joint count (SJC) decreased by 31% (p>0.05) in group W. In group L, the total TIC decreased by 4,7% (p>0.05) and the total SIC decreased by 8,5% (p>0.05). The haematological values remained globally unchanged in all three groups concerning the hemolglobin (Hb) values. There were changes in the erythrocyte sedimentation rate (ESR) in the groups, however changes were not significant (p>0.05). The ESR decreased by 29% in group Wand by 33% in group C. There was a slight increase in group L's ESR (11,9%) but values remained within the normal range. There was an improvement in the patients self-assessed disability and psychological status in the experimental groups while there was a deterioration in the control group's. Health Assessment Questionnaire (HAQ) scores improved by 15% in group W (p>0.05), 18% in group L (p>0.05) and deteriorated by 13% in group C (p>0.05). There was no change in the total Profile of Mood States (POMS) score of the control group, however, significant (p<0.05) improvements were observed in the experimental groups. There was a 163% improvement in group L's and a 990/0 improvement in group W's affective states. As far as physical condition is concerned, in genera~ there was an improvement in group Wand group L's physical condition, while there was no improvements noted in group C. Group W showed the following changes in physical condition: Body mass decreased by 9,2% (p>0.05). Mean blood pressure values remained unchanged. 50-ft walk time improved by 18% (p<0.05). Right and left grip strength increased by 18% and 35% respectively, (p<0.05). Absolute VO2max increased by 28% and relative VO2max increased by 30% (p<0.05). Right knee flexor strength increased by 43% (p<0.05) and left knee flexor strength by 24% (p>0.05). Increases in right and left knee extensor strength were 32% (p>0.05) and 34% (p>0.05) respectively. Improvement in joint mobility was also noted. There was a significant (p<0.05) improvement in both right and left wrist extension range of motion(ROM). Right wrist extension ROM improved by 49% and left wrist extension ROM improved by 31%. Improvements were also noted in wrist flexion ROM however changes were not significant (p>0.05). There was an 12% and 19% increase in right and left wrist flexion ROM respectively. In addition, there was a 12% (p<0.05) increase in right knee flexion ROM and a 14% increase in left knee flexion ROM (p<0.05). Mean body mass and blood pressure remained unchanged. 50-ft walk test time improved by 15% (p<0.05). Right and left grip strength increased by 4,8% and 16.1% respectively (p>0.05). Relative VO2max increased by 16.6% and absolute VO2max by 31% (p<0.05). Right knee flexor strength increased by 22.1% and left knee flexor strength by 23.8% (p>O.05). Increase in right and left knee extensor strength was 9% and 2,4% respectively (p>O.05). Right wrist extension ROM increased by 20.7% and left wrist extension ROM increased by 15,7% (p>0.05). There was a significant (p<0.05) increase in left wrist flexion (7,6%), but right wrist flexion ROM decreased by 2.6% (p>0.05). Improvements in right and left knee flexion ROM were also significant (p<0.05), 9,2% and 7,4%, respectively. Group C showed the following changes in physical condition: Mean body mass increased by 2% (p>O.05), while blood pressure and 50-ft walk time remained globally unchanged. Left grip strength decreased by 16% (p>O.05) and right grip strength remained the same. Although not significant (p>O.05), there was a 11% decrease in relative VO2max and a 6,7% decrease in absolute VO2max. Muscle strength also showed deterioration in group C. Right and left knee flexor strength decreased by 1,8% and 12%, respectively (p>0.05). Left knee extensor strength remained unchanged while right knee extensor strength decreased by 9,7% (p>0.05). Right wrist extension ROM decreased by 4.7% and left wrist extension ROM increased by 6.7%, although the increase was not significant (p>O.05). While right wrist flexion ROM decreased by 1,3% and left wrist flexion ROM decreased by 21% (p>0.05). There were no significant (p>O.05) changes in group C's right and left knee flexion ROM. Right knee flexion ROM decreased by 1,2% and left knee flexion ROM increased by 1,2%. Based on the above results of the study, both exercise interventions are beneficial in the treatment of RA. Appropriate land-based exercises do not appear to enhance disease activity, however, the water-based exercise programme was superior in controlling the disease activity. Further research is required, using larger samples and evaluating the long-term effects of various exercise interventions. / Dissertation (MA (Human Movement Sciences))--University of Pretoria, 2006. / Biokinetics, Sport and Leisure Sciences / unrestricted

AimThe broad aim of this thesis was to explore the risk of malignancy in people with rheumatoid arthritis (RA), treated with anti-tumour necrosis factor (TNF) drugs.MethodsThis thesis used data from patients with RA registered with the British Society of Rheumatology Biologics Register-RA. The risk of cancer in biologic-naive patients treated with traditional disease modifying drugs (nbDMARD) was compared to that in the general population by calculating standardised incidence ratios (SIR). The influence of anti-TNF on cancer risk was then explored by comparing the risk in the anti-TNF cohort to that in the nbDMARD cohort using Cox proportional hazard models.ResultsThe risk of cancer was increased in the nbDMARD cohort by 28% compared to the general population (SIR 1.28, 95% confidence interval (CI) 1.10, 1.48). Risks of lung cancer (SIR 2.39, 95% CI 1.75, 3.19), Hodgkin lymphoma (SIR 12.82, 95% CI 4.16, 29.92) and Non-Hodgkin Lymphoma (SIR 3.12, 95% CI 1.79, 5.07) were increased compared to the general population and both prostate cancer and cancers of the female genital organs reduced; SIRs 0.35 (95% CI 0.11, 0.82) and 0.35 (95% CI 0.10, 0.90) respectively. There was no difference in the risk of cancer in patients treated with anti-TNF compared to nbDMARD, after adjusting for differences in baseline characteristics; Hazard ratio for lymphoma: 1.00 (95% CI 0.49, 2.05); cancers of the solid organs: 0.83 (95% CI 0.64, 1.07); and keratinocyte skin cancer: basal cell carcinoma 1.06 (95% CI 0.64, 1.75), squamous cell carcinoma 1.62 (95% CI 0.44, 5.90). ConclusionsSubjects with RA, treated with nbDMARD were at increased risk of cancer compared to the general population. In particular, lung cancer, lymphoma and KSC were increased. Treatment with the TNF inhibitors ETA, INF or ADA was not associated with a difference in relative risk of lymphoma, solid cancer or skin cancers when compared to nbDMARD.

Over the past decade results from a series of research studies have contributed to the development and evaluation of the Arthritis Self-Management Program (ASMP), a volunteer-led patient education program for persons with arthritis. To date, these studies have primarily focussed on program effectiveness, process, implementation, and dissemination. In these studies self-efficacy was identified as an important construct contributing to the program's effectiveness, however, the exact relationship between self-efficacy and health outcomes has not been determined. In this dissertation research I investigate the evidence of a causal relationship between self-efficacy and three program outcomes (a decrease in depression, less pain, and a higher self-rating of overall health status), and attempt to determine the nature of that relationship. The research methodology involved the use of structural equation modeling (SEM) with two longitudinal samples, one (n=122) of 1991 ASMP participants in British Columbia, the other (n=189) of 1992 ASMP participants in Ontario. In the analysis self-efficacy was paired separately with depression, pain and perceived health status. The results of the SEM failed to confirm a dominant causal relationship from self-efficacy to depression, or to pain. This may indicate that these variables have a reciprocal or "spiral" relationship or that both sets of variables may be caused by factors not considered in the analysis. The results of the SEM between self-efficacy and perceived health status did, however, show that higher self-rated health status leads to higher self-efficacy at a later time. The data did not show statistical significance for other causal patterns among these variables. The findings suggest that self-efficacy may play a moderator role in the complex relationship involving individuals with arthritis, their behaviors, and health outcomes. As well, the findings have implications for health promotion planning and research in that they reinforce the complex interplay of psychological and behavioral variables (probably influenced by social variables) in programs which attempt to give individuals greater control over their health. The efficacy and effectiveness of the ASMP has been established in previous studies. This study in no way calls these into question. It does, however, suggest that the mechanism by which these effective outcomes are achieved warrants further investigation. / Graduate and Postdoctoral Studies / Graduate

Inflammatory arthritis (IA) is a debilitating disease that is characterized by joint destruction. This destruction is caused by osteoclasts (OCLs) degrading bone within the synovium, however the exact cellular origins of these synovial OCLs is not well understood. We hypothesize that the synovial OCLs seen in IA are independently derived from two contributing cell lineages: the canonical source of OCL which are Hematopoietic Stem Cell (HSC) derived monocytes and the newly described Erythro-Myeloid Progenitor (EMP). To explore the contribution of these two lineages to synovial OCL, we used Cx3cr1CreERT2;Rosa26LSL-tdTomato mice to label EMP-derived cells, and Flt3Cre;Rosa26LSL-YFP to label HSC-derived cells. Using immunofluorescent histology, we found that synovial OCLs formed under arthritic conditions derive from both HSC and EMP-progenitors, suggesting the possibility that regulatory mechanisms unique to each developmental lineage promote OCL differentiation in arthritic joints. In support of these observations, in IA we detected two populations of mononuclear cells, as possible osteoclast precursors, that express both the OCL marker TRAP and the monocytic/macrophage marker CD68. These mononuclear TRAP+ CD68+ cell populations are found within the inflamed synovium and in bone periosteal surfaces of arthritic joints, and are mostly EMP derived, however, the HSC lineage significantly contributes to osteoclast formation, as suggested by our lineage-tracing strategy. We are currently investigating the dynamics of these cell populations during the early, peak, and resolution stages in an acute murine model of IA. To better understand regulatory differences between OCL derived from each of these lineages, we plan to isolate EMP- and HSC-derived OCLs directly from joints to study their precise phenotype, cytokine differentiation requirements, resorptive capacity and transcriptional activity, by flow cytometry, in vitro cell cultures and single cell RNA sequencing, respectively.

Patienten mit JIA erhielten weniger Lebendimpfungen als gesunde Kontrollpersonen, für Totimpfstoffe waren die Immunisierungsraten vergleichbar. Ebenso zeigten die Antikörper Konzentrationen zwischen den JIA Patienten und den gesunden Kontrollpersonen keine signifikanten Unterschiede. Bei Untersuchungen innerhalb der JIA Patienten Gruppe zeigte lediglich der Abstand zwischen der letzten Auffrischungsimpfung und der Blutentnahme signifikante Ergebnisse. / Vaccination status of patients with Juvenile Idiopathic Arthritis. Our study shows that children with JIA receive fewer vaccinations with live vaccines, for all other vaccinations vaccination rates are similar to healthy controls. Vaccinated JIA patients develop a similar antibody response as healthy controls. Only the time between the last booster vaccination and time point of blood withdrawal within the patients group show significant results.

Serious cardiovascular side effects are reported from synthetic cyclooxygenase-2 (COX-2) selective nonsteroidal anti-inflammatory drugs, the most common medication for rheumatoid arthritis (RA) and osteoarthritis (OA). Natural products from herbal medicine are inspirational source of safe and effective remedy due to its distinguished chemical diversity. Nanomagnetic ligand fishing using enzyme-anchored-magnetic nanoparticles (MNPs) is an advanced selective bioseparation strategy based on macromolecular target-ligand binding, which can screen enzyme inhibitors from complex mixtures. "Snow lotus" herbs have been clinically applied as safe and effective treatment for arthritis throughout centuries in Asia. Some major chemicals from the herbs have been found with anti-COX-2 activities. It is therefore hypothesized that novel and safe COX-2 selective inhibitors can be separated from a most representative snow lotus herb via ligand fishing using COX-2-functionalized MNPs (COX-2-MNPs), and that the efficacy and safety of the screened COX-2 ligands can be verified by subsequent evaluation. Saussurea laniceps Hand.-Mazz. (SL), S. medusa Maxim. (SM) and S. involucrata (Kar. et Kir.) Sch.Bip. (SI) are three authenticated sources of "snow lotus" herbs. An ultra-high performance liquid chromatography hyphenated with diode array detector and quadrupole time of flight-mass spectrometry (UPLC-DAD-QTOF-MS) method was developed to analyze 49 herbal samples for species analysis and overall quality evaluation. With 25 simultaneously identified constituents, of which 12 were quantified, the chemical determination, four-dimensional principle component analysis (4D-PCA), and orthogonal hierarchical cluster analysis (2D-HCA) showed a distinctive bioactive component profile of SL from the other two species, and explained the therapeutic potency of SL. As a result, SL has been chosen as a model herb to screen for novel and safe COX-2 selective inhibitors. With systematic uniform experimental designs and statistical modeling, COX-2-MNPs with high magnetic moments and outstanding enzyme activity have been synthesized. Four COX-2-selective compounds, namely, chlorogenic acid, syringin, umbelliferone, and scopoletin, were separated from the herbal extract using fine-tuned fishing protocol and were identified by UPLC-DAD-QTOF-MS. All the four ligands were proved with evidently lower in vitro and in vivo cardiotoxicity than celecoxib, a known selective COX-2 inhibitor. Some of them exerted potent anti-inflammatory activities on cells, and their optimum combination ratios were investigated. Among the ligands, scopoletin showed most evident therapeutic potential in rats with adjuvant-induced arthritis and anterior cruciate ligament transection (ACLT)-induced OA, respectively, by alleviating clinical statuses, immune responses, and joint pathological features. An equal mixture of scopoletin and syringin brought possible synergistic remedial effects on rat OA. Molecular docking results explained the structure-specific enzyme-binding affinities of the ligands; the ligands' inhibition on COX-2 may involve direct interaction as well as upstream signaling pathways. In conclusion, promising candidates of COX-2 selective inibitors, e.g. scopoletin, have been screened and validated on a nanomagnetic ligand fishing platform using COX-2-MNPs from the extract of SL, a most representative snow lotus herb with distinctive chemical composition and outstanding therapeutic efficacies. The quality evaluation strategy of snow lotus herbs combining chemical determination and multidimensional chemometric analysis can be applied in other multi-original herbal medicines. The nanomagnetic ligand fishing platform of compound bio-separation and multi-model bio-evaluation should be equally valuable for uncovering other therapeutic chemicals in different natural sources.