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Novel anti-arthritic drugs are often assessed in murine collagen-induced arthritis (CIA), which is a widely used pre-clinical model of rheumatoid arthritis. However, CIA studies are lengthy, development of arthritis is not synchronised and not all animals develop disease. Work conducted in this thesis addressed some of these issues by developing short-term mechanistic models of collagen II (CII) immunity. Drug effects on Cll-induced hypersensitivity, anti-CII antibodies and ex vivo CII stimulated CD4⁺ T cell proliferation in mice 14 days post-CII sensitisation were assessed and compared to their anti-arthritic effect in CIA.

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Introduction: The endocannabinoid signalling system has been implicated in a broad spectrum of pathological and physiological processes including the control of inflammation and pain. Recent pre-clinical studies have demonstrated the therapeutic potential of cannabis-based drugs in the treatment of inflammatory musculoskeletal conditions, including rheumatoid arthritis. Studies in rodent models suggest that the endocannabinoid system modulates bone remodelling, via actions at the cannabinoid CB1, CB2 receptors and the TRPVl ion channel. The effects of the endocannabinoid system in inflammatory processes and bone homeostasis suggest that they may impact on the pathogenesis of Charcot osteoarthropathy, a non-infective, chronic destructive condition of the bone initiated by neuro-traumatic stimuli, most prevalent in patients with diabetic peripheral neuropathy. Neuropathy-induced changes in the endocannabinoid system may contribute to bone breakdown in Charcot, dependent or independent of the RANKL/RANK/OPG signalling pathway which is known to regulate bone remodelling. The aims of this thesis were to quantify and compare the levels of endocannabinoids and components of the endocannabinoid receptor system in human trabecular bone from Charcot and non-Charcot subjects, to identify a functional endocannabinoid system in human osteoclasts in culture, and to determine the effect of TRPVl modulation on osteoclastogenesis. Methods: Bone biopsies and blood were taken from patients with Charcot osteoarthropathy or those undergoing elective foot surgery. Histological features of the Charcot and non-Charcot bone were compared following staining with haematoxylin. Plasma levels of the inflammatory marker, CRP, and bone turnover markers (ALP, CTx) were measured with routine biochemical analysis. Plasma levels of RANKL, OPGJ and inflammatory cytokines (TNFa, IL-l, IL-6, IL-7) were measured using enzyme-linked immunosorbent assay. Levels of endocannabinoids in plasma and bone were analysed by liquid chromatography-tandem mass spectrometry (LC- MS/MS). Osteoclasts were differentiated from U-937 cells (human leukaemic monocyte lymphoma cells) using TPA (O.Lpq/rr»). followed by calcitriol (10- BM) or TNFa (3ng/ml). Tartrate-resistant acid phosphatase (TRAP) staining and resorption of calcium phosphatase were used to identify mature osteoclasts. Components of the endocannabinoid system, together with osteoclast markers (TRAP, cathepsin K) and RANKL/OPG were detected at the mRNA level in human trabecular bone and osteoclasts with quantitative PCR. Modulation of ATP(lOOIJM)-induced calcium responses by CB1 and CB2 receptor agonists (ACEA (lIJM), JWH133 (3IJM), CP55940 (3IJM» were used to demonstrate the presence of functional cannabinoid receptors. Exposure of osteoclasts to the TRPVl receptor antagonist capsazepine (3IJM) and agonist AEA (lIJM) was used to study the role of TRPVl in osteoclastogenesis. Results: Histology of the Charcot bone showed a loss of bone microarchitecture, loss of marrow space and features of increased bone turnover. Higher levels of ALP, CTx and IL-6 were detected in Charcot plasma, compared to controls, indicating an inflammatory response and an increase in bone turnover. An increase in OPG level, but not RANKL was found in Charcot plasma. The endocannabinoids 2-AG, AEA and related compounds PEA and OEA were detected in human trabecular bone (not AEA) and plasma although no significant differences were evident between Charcot and control samples. Components of the endocannabinoid system, with the exception of CB2 receptors, were detected at the mRNA level in human trabecular bone. Lower levels of the endocannabinoid-related nuclear receptor PPARy (known to be anti-inflammatory) were found in Charcot bone compared to that of controls. Osteoclasts differentiated from U-937 cells showed positive TRAP staining, were multinucleated and calcium phosphate-resorbing from day 5 of culture. mRNA expression of components of the endocannabinoid system were significantly up-regulated with osteoclast maturation. The application of CB1/2 receptor agonists modified ATP-induced calcium responses in osteoclasts. Exposure of osteoclasts to capsazepine, but not AEA, Significantly attenuated the number of TRAP-positive osteoclasts. Effects of capsazepine were attenuated by AEA. Conclusion: Work in this thesis has confirmed the link between Charcot and an increase in bone turnover (increased ALP and CTx levels), and the presence of an inflammatory response (increased IL-6 levels). It also demonstrated the presence of active endocannabinoid signalling, in Charcot and non-Charcot trabecular bone, and osteoclasts. Marked histological changes were observed in Charcot bone. The lack of genetic data and the sporadic and rare nature of this condition renders it difficult to establish the mechanisms of the disease. Marked differences were seen in the effect of osteoclast maturation on the endocannabinoid system. Importantly, the process of osteoclastogenesis was seen to be halted by TRPVl channel inhibition suggesting that pharmacological interventions targeting TRPVl could be used in the control of osteoclastogenesis. These data add to the growing evidence that the endocannabinoid signalling system plays an important role in bone homeostasis, and that alterations in the components of this system may contribute to pathological conditions of the bone, such as osteoporosis, osteoarthritis, and Paget's disease.

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

This study examined Gujarati and Caucasian patients with RA, with respect to disease activity, genetic factors, treatment, socioeconomic and psychological status.;61 Gujarati and 61 Caucasian subjects underwent a structured interview, including detailed social history, disease history, drug history, pain on VAS, HAQ, WHO Self Reporting Questionnaire, list of Threatening Life Events and an examination including a swollen joint count.;Caucasian patients had higher swollen joint counts (10.39 vs. 8.07, p=0.05), more nodulosis (46% vs. 16%, p=0.0005) and more sero-positivity for RF (66% vs. 45%, p=0.02). Gujaratis had longer EMS (1.36 hrs vs. 0.86 hrs, p=0.03), greater pain on VAS (5.1 vs. 3.7, p=0.0008) and greater disability on HAQ (1.9 vs. 1.2, p=0.0001). Gujaratis had an earlier age of onset (42.0 yrs vs. 46.3 years, p=0.01). There were no differences in DMARD therapy, but Gujaratis perceived their treatment to be less effective (p=0.0009). 76% of patients had tried complementary therapies.;Gujarati patients had a lower frequency of HLA DRB1 shared epitope (0.77 vs. 1.12 copies/patient, p=0.01). Caucasians expressed HLA DRB1*04 (37% vs. 12%, p=0.001) and DRB1*01 (15% vs. 1% p=0.0007). Gujaratis expressed HLA DRB1*10 (21% vs. 3%, p=0.0009).;59% Gujaratis were not working because of ill health, compared with 31% Caucasians. Gujaratis had a larger social network (3.15 vs. 1.89, p=0.0004) and more social services support (75% vs. 54%, p=0.01). 44% Gujarati patients were vegetarian, and few dark or smoked. Gujarati patients were significantly depressed (9.44 vs. 5.16 on SRQ, p<0.0001), and ethnicity was an independent risk factor for depression (p<0.0005) when other variables were adjusted for.;Ethnicity was an independent predictor for disability (p=0.001) despite adjusting for markers of disease severity and activity. Asian patients had an odds ratio of 8.20 (p=0.006) of having a HAQ >=2. Ethnicity was not a predictor for SJC.

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In rheumatoid arthritis (RA), formation of new blood vessels (angiogenesis) is required to provide oxygen and nutrients to actively proliferating synovial tissue. The resultant neovasculature s dysfunctional and fails to vascularise the innermost synovium thus perpetuating synovitis. The aim of this thesis was to investigate the relationship between synovial tissue oxygen levels (PO2) and microvascular density in inflammatory arthritis by direct in vivo measurements of PO2 and thus establish whether angiogensisrestores tissue oxygenation through the analysis of Hypoxia-inducible factor (HIF) and Vascular endothelial growth factor (VEGF) which are up regulated in hypoxic conditions.