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The role of ERAP1 and HLA-B27 in ankylosing spondylitis pathogenesisChen, Liye January 2013 (has links)
HLA-B27 and ERAP1 are the two strongest predisposing genetic factors to Ankylosing Spondylitis (AS). As a key aminopeptidase in MHC class I presentation, ERAP1 potentially contributes to AS pathogenesis through altering HLA-B27 peptide presentation. In this thesis, I first studied the effect of AS-associated ERAP1 variation on its enzyme activity in vitro. Trimming of two N-terminally extended HLA-B27 epitopes was decreased by K528R mutation; the effect of R725Q was however substrate-dependent. I also investigated the effects of ERAP1 silencing on the repertoire of peptides bound to HLA-B27 and on peptide presentation to Cytotoxic T lymphocytes (CTLs). In both HeLa.B27 and C1R.B27 cells, the proportion of 9mer peptides, the canonical MHC class I peptide length, was decreased by ERAP1 silencing whereas the percentage of longer peptides (11-13mer) was increased. Surprisingly, following ERAP1 silencing, C-terminally extended versions of 9mer and 10mer peptides were readily identified. In both HeLa.B27 and mouse fibroblasts expressing HLA-B27, ERAP1 silencing/knockout reduced recognition by KK10-specific HLA-B27-restricted CTLs following recombinant vaccinia viral infection or transfection with minigenes expressing KK10 precursors. Interestingly, KK10 CTL recognition following extended KK10 minigene transfection was reduced in the presence of the AS protective variant, K528R-ERAP1 compared to wildtype ERAP1. The effects of ERAP1 inhibition (Leucinethiol), silencing (siRNA & shRNA) and introduction in ERAAP-/- cells on cell surface HLA-B27 expression were also studied. My finding validates the role of ERAP1 and HLA-B27 interaction in AS pathogenesis indicated by GWAS. ERAP1 inhibition could potentially be used for treatment in AS and other ERAP1-associated diseases.
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A synovial fluid fingerprint for end-stage knee osteoarthritisJayadev, Chethan January 2014 (has links)
No description available.
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Upregulation of early inflammation to enhance fracture repairChan, James January 2014 (has links)
Fractures of bone are very common, affecting 2% of the population per annum. Whilst the majority heal uneventfully, 10-15% exhibit delayed or non-union. These complications tend to occur in patients who have sustained high-energy open fractures, which are limb-threatening injuries, or low-energy osteoporotic fractures, which are associated with high morbidity and mortality rates. Enhancement or acceleration of fracture repair would confer significant benefit to these patients as well as reduce the public health burden. Inflammation represents the earliest response following trauma and initiates a cascade of downstream events crucial for wound healing. However, the mechanism by which this occurs remains poorly defined. A detailed understanding of how these upstream events initiate fracture healing is a necessary step in the development of therapeutics to enhance this process. Our group previously reported that addition of low dose recombinant human TNF (rhTNF) at the fracture site accelerated fracture repair in a murine tibial fracture model. Here I show that local rhTNF treatment is only effective when administered within 24 hours of injury, when neutrophils represent the major inflammatory cell infiltrate. Endogenous TNF was expressed at the fracture site initially by neutrophils and after 3 days by monocytes/macrophages. Systemic administration of anti-TNF resulted in impaired fracture healing. The addition of rhTNF to the fracture environment in an air pouch model enhanced neutrophil recruitment, and promoted the recruitment of monocytes through CCL2 production. Conversely, inhibition of either neutrophils or the chemokine receptor CCR2 resulted in significantly impaired fracture healing. Fragility fractures represent a major unmet medical need and they are associated with permanent disability and premature death. Using a murine model of fragility fractures, rhTNF treatment improved fracture healing during the early phase of repair. Translated clinically, accelerated healing would permit earlier load bearing and reduce the morbidity and mortality associated with delayed patient mobilisation.
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The role of glutamate in rotator cuff tendinopathy : glutamate in rotator cuff tendinopathyDean, Benjamin J. F. January 2015 (has links)
Thesis questions: • Is the glutaminergic system altered in rotator cuff tendinopathy? • Is the glutaminergic system altered by common treatments? • Are glutaminergic changes related to pain symptoms? • What are the effects of glutamate and glutamate receptor modulation on tendon derived cells? Summary answers: • The glutaminergic system is altered in rotator cuff tendinopathy • Changes within this system are seen after common treatments • Specific glutaminergic changes are associated with the resolution of pain following shoulder surgery but do not predict the severity of pain symptoms • Glutamate has significant effects on tendon derived cells. What is known: It is known that extracellular glutamate concentrations are increased in both Achilles and patellar tendinopathy. It has also been previously shown that the glutamate receptors NMDAR1 and mGluR5 are upregulated in patellar tendinopathy. What this thesis adds: This thesis has shown for the first time that glutamate and NMDAR1 are increased in rotator cuff tendinopathy. Increases in cell proliferation, vascularity and HIF1α are seen after surgical rotator cuff repair and these features are not seen after glucocorticoid injection. There are significant differences between painful and pain-free rotator cuff tendons in terms of glutamate receptor expression (KA1, mGluR7 and mGluR2) and inflammatory cell numbers (CD45 and CD206). Exposure to 1.875mM glutamate for 72 hours results in reduced cell viability, decreased collagen (COL1A1 and COL3A1) and increased aggrecan gene expression; NMDAR antagonism with MK-801 attenuates the deleterious effect on cell viability but had no effect on the changes in matrix gene expression. Bias, confounding and other reasons for caution: The observational histological work was limited by the control tissue. Some control tissue was not age matched, while some of the pain-free control tendons were post-surgical intervention. Confounding factors include tendon structure, length of symptoms and previous treatments. Caution must be applied when discussing the in vivo implications of the in vitro work.
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The role of HLA-B27 in the pathogenesis of spondyloarthritisMcHugh, Kirsty Anne January 2011 (has links)
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.
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Functional analysis of polymorphisms associated with osteoarthritis susceptibility that affect cis-regulationWilkins, James January 2008 (has links)
Osteoarthritis (OA) is a common, multifactorial disease that is characterized by focal degeneration of the smooth articular cartilage in any of the synovial joints. Although the underlying molecular mechanisms for OA are still not fully understood, epidemiological studies have evidenced a significant genetic component to OA susceptibility. Genome-wide linkage scans and large-scale association studies have had success in unraveling the genetic architecture underlying OA with the identification of a number of susceptibility genes. In this work, functional analyses are reported of OA associated polymorphisms within two susceptibility genes: BMP5 and GDF5, both members of the TGF-β superfamily of secreted proteins. The extent of differential allelic expression (DAE) of BMP5 in human mesenchymal tissues was first examined with significant differences in BMP5 allelic output observed (allelic ratios exceeding 4:1 in the tissues of some donors). Significant variability in allelic expression within the different tissues of donors was also observed, suggesting that polymorphism in cis-regulation of BMP5 expression is common and that there is a considerable effect of tissue specific elements on BMP5 expression. DAE was then used as a phenotype to map tissue-specific cis-regulatory polymorphisms with the identification of a single nucleotide polymorphism (SNP) located downstream of BMP5 that was significantly associated with DAE as well as OA, suggesting that variability in cis-regulation of BMP5 is important in OA susceptibility. Moreover, the functional effect of a previously identified OA associated microsatellite within intron 1 of BMP5 was investigated using luciferase reporter assays and electrophoretic mobility shift assays (EMSAs) with significant differences observed both in the ability of various microsatellite alleles to modulate BMP5 promoter activity and to bind GATA-3 nuclear proteins, further suggesting a role for variability in BMP5 expression in OA susceptibility that may in part be due to altered GATA-3 binding. Finally, functional characterization of a previously reported OA associated SNP in the 5′ UTR of GDF5 is presented in which EMSAs show differential binding of nuclear factors between the two SNP alleles, strengthening the possible functional contribution of this SNP to OA susceptibility. Overall, this work demonstrates that polymorphism in cis-regulation is likely to play a role in OA susceptibility.
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Systematic overviews of the randomised evidence for the effects of traditional non-steroidal anti-inflammatory drugs and selective inhibitors of cyclo-oxygenase-2 on vascular and upper gastrointestinal outcomesBhala, Neeraj January 2013 (has links)
<b>Background:</b> Comparative assessments of the vascular and upper gastrointestinal risks of different regimens of non-steroidal anti-inflammatory drugs (NSAIDs) are required. <b>Methods:</b> Meta-analyses were conducted, using individual participant data where possible, of placebo-controlled trials of a selective cyclo-oxygenase [COX]-2 inhibitor ('coxib') or traditional NSAID, or randomised trials of a coxib versus traditional NSAIDs. A prespecified subdivision of traditional NSAID regimens of those with antiplatelet activity (mainly naproxen) and those without (mainly diclofenac) was made. Primary outcomes were major vascular events (MVEs; nonfatal myocardial infarction, nonfatal stroke or vascular death) and upper gastrointestinal complications (UGICs; perforation, obstruction or bleed). <b>Findings:</b> Searches identified 788 trials: 200 comparisons of a coxib vs placebo (88,604 participants, mean follow-up 0.60 years), 206 comparisons of a traditional NSAID vs placebo (43,482 participants, 0.46 years) and 149 comparisons of a coxib vs traditional NSAID (137,466 participants, mean follow-up 0.95 years). Compared to placebo, allocation to a coxib increased the risk of MVEs (rate ratio 1.38, 95% CI 1.14-1.66), vascular mortality (1.58, 1.11-2.24) and UGICs (1.81, 1.17-2.81). Overall, in the population studied, coxibs were associated with three additional major vascular events (one fatal) and two (rarely fatal) upper gastrointestinal complications per 1000 person-years exposure. There was no evidence of heterogeneity by duration of follow-up, coxib type, dose (other than for celecoxib), or patient characteristics, for the primary outcomes. The risk of MVEs for traditional NSAIDs without antiplatelet activity (mostly diclofenac 75mg bd or ibuprofen 800mg tds) were comparable to coxibs (1.40, 1.15-1.72); but the risk of UGICs (1.98, 1.39-2.84) was significantly greater. For traditional NSAIDs with antiplatelet activity (mostly naproxen 500mg bd) there were no significant excess of MVEs (0.84, 0.66-1.08), but UGICs were substantially increased (4.06, 2.85-5.78). Both coxibs and traditional NSAIDs increased risk of hospitalisation for heart failure by about two-fold. <b>Interpretation:</b> The vascular and upper gastrointestinal risks of coxibs and high-dose tNSAID regimens can be predicted, allowing the choice of analgesia to be tailored for particular patients.
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Development of biochemical tools to characterise human H3K27 histone demethylase JmjD3Che, Ka Hing January 2013 (has links)
Covalent modifications of histone tails play essential roles in mediating chromatin structure and epigenetic regulation. JmjD3 is a JumonjiC domain containing histone demethylase, belongs to the KDM6 subfamily, and catalyses the removal of methyl groups on methylated lysine 27 on histone 3 (H3K27), a critical mark to promote polycomb mediated repression and gene silencing. The importance of JmjD3 has been implicated in development, cancer biology and immunology. In this thesis, I report the recombinant production of active human JmjD3, development of two in vitro screening assays, a cell-based assay, and structural determination of JmjD3 in complex with the inhibitor 8-hydroxy-5-carboxyquinoline (8HQ). A highly selective and potent small molecule inhibitor GSK-J1 was subsequently identified. The inhibitor is active in HeLa cells and promotes a dose-dependent increase of global H3K27 methylation. The inhibitor GSK-J1 was used in two different cell assay systems related to inflammation and differentiation, to understand how H3K27 demethylation controls cellular functions. By inhibiting H3K27me3 demethylation, it is demonstrated that tumor necrosis factor (TNF) and other pro-inflammatory cytokines are regulated by H3K27 demethylase inhibition in M1- type macrophages derived from healthy volunteers and rheumatoid arthritis patients. It is also shown that inhibition of H3K27me3 demethylation abrogates cellular fusion of M2- type macrophages. During RANKL induced osteoclast differentiation, JmjD3 is up-regulated and promotes the expression of the key transcription factor NFATc1. By inhibiting JmjD3, NFATc1 expression is reduced and osteoclastogenesis is inhibited. This mechanism demonstrates a novel anti-resorptive principle of potential utility in conditions of excess bone resorption such as osteoporosis, bone erosion in inflammatory arthritis or cancer of the bone. These experiments further resolve the ambiguity between scaffold and catalytic function associ- ated with the H3K27 demethylase in these biological systems, and demonstrate that its enzymatic activity is crucial for epigenetic regulation of macrophage and osteoclast function.
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Genetics of ankylosing spondylitisKaraderi, Tugce January 2012 (has links)
Ankylosing spondylitis (AS) is a common inflammatory arthritis of the spine and other affected joints, which is highly heritable, being strongly influenced by the HLA-B27 status, as well as hundreds of mostly unknown genetic variants of smaller effect. The aim of my research was to confirm some of the previously observed genetic associations and to identify new associations, many of which are in biological pathways relevant to AS pathogenesis, most notably the IL-23/T<sub>H</sub>17 axis (IL23R) and antigen presentation (ERAP1 and ERAP2). Studies presented in this thesis include replication and refinement of several potential associations initially identified by earlier GWAS (WTCCC-TASC, 2007 and TASC, 2010). I conducted an extended study of IL23R association with AS and undertook a meta-analysis, confirming the association between AS and IL23R (non-synonymous SNP rs11209026, p=1.5 x 10-9, OR=0.61). An extensive re-sequencing and fine mapping project, including a meta-analysis, to replicate and refine the association of TNFRSF1A with AS was also undertaken; a novel variant in intron 6 was identified and a weak association with a low frequency variant, rs4149584 (p=0.01, OR=1.58), was detected. Somewhat stronger associations were seen with rs4149577 (p=0.002, OR=0.91) and rs4149578 (p=0.015, OR=1.14) in the meta-analysis. Associations at several additional loci had been identified by a more recent GWAS (WTCCC2-TASC, 2011). I used in silico techniques, including imputation using a denser panel of variants from the 1000 Genomes Project, conditional analysis and rare/low frequency variant analysis, to refine these associations. Imputation analysis (1782 cases/5167 controls) revealed novel associations with ERAP2 (rs4869313, p=7.3 x 10-8, OR=0.79) and several additional candidate loci including IL6R, UBE2L3 and 2p16.3. Ten SNPs were then directly typed in an independent sample (1804 cases/1848 controls) to replicate selected associations and to determine the imputation accuracy. I established that imputation using the 1000 Genomes Project pilot data was largely reliable, specifically for common variants (genotype concordence~97%). However, more accurate imputation of low frequency variants may require larger reference populations, like the most recent 1000 Genomes reference panels. The results of my research provide a better understanding of the complex genetics of AS, and help identify future targets for genetic and functional studies.
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