Dissertations / Theses on the topic 'Ankylosis spondylitis'

Background. The Spondyloarthritis (SpA) are a group of a multifactorial diseases characterised by a complex interplay between an inherited background and environmental factors that lead to immune response dysregulation and inflammation of the joints, mainly the sacro-ileal. Different from rheumatoid arthritis, there are no specific biomarkers for disease activity in the SpA that could be used in clinical practice. New biomarkers discovery could be helpful for early diagnosis, monitoring of disease activity, as well as for prognosis, outcome measures, and for assessing treatment efficacy. In SpA patients, macrophages infiltrating the inflamed joints, derive from circulating monocytes, express not only inflammatory cytokines, like TNF-α, IL-1β or TGF-β, but also enzymes causing tissue destruction and remodelling, like metalloproteinases. Metalloproteinases (MMPs), MMP3 in particular, have been reported to be highly expressed in synovial tissue and in peripheral blood of SpA patients. Recent studies have showed that MMP8 and MMP9, in particular, are produced by peripheral blood mononuclear cells (PBMCs) if they are stimulated by calprotectin (S100A8/S100A9 heterodimer). The SpA synovial tissue is characterized by an increased vascularization and an infiltrate composed of nucleated polymorphs, macrophages and lymphocytes. In these cells calcium signals are essential for various cellular functions, including proliferation, differentiation, apoptosis, and gene transcription. The aims of this work are to investigate whether the TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 and MMP9 mRNA expression levels and intracellular calcium ([Ca2+]i) fluxes variations in PBMCs might be associated with SpA. Methods. The study population comprised 64 patients with a diagnosis of SpA (39 males and 25 females; mean age±standard deviation: 39.5±13.2 years) and 100 healthy controls (58 males and 42 females; mean age±standard deviation: 46.68.5). Among patients, 26 (40.6%) had diagnosis of Ankylosing Spondylitis (AS), (modified New York criteria) and 38 (59.3%) had a diagnosis of Psoriatic Arthritis (PsA) (CASPAR criteria). Blood samples were collected and complete blood count, CRP, ESR, uric acid, ALT and glucose were evaluated. Relative quantification (Real Time PCR) of TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 and MMP9 mRNA were performed. Intracellular calcium ([Ca2+]i) fluxes were studied in patients and controls monocyte cells by a fluorescent microscope. Results. The mRNA expression levels in PBMCs of TNF-α, IL-1β, TGF-β were similar in AS and PsA patients when compared to controls. The variations of TNF-α, TGF-β and IL-1β were correlated each other. TNF-α mRNA expression levels also show a significant correlation if patient’s relatives with SpA where found (t=-2.5386, p=0.013). MMP8 and MMP9 mRNA expression levels did not vary between controls and patients, nor they were related to disease clinical activity indices. S100A9 mRNA expression did not vary, the expression of S100A8 (F=3.29, p=0.039) was reduced in PsA patients. S100A8 and S100A9 expression levels were significantly correlated with circulating inflammatory cells and S100A8 was correlated with CRP and ESR. Monocytes from healthy controls had evident and frequent ([Ca2+]i) oscillations, while SpA patients monocytes did not. The percentage of cells exhibiting ([Ca2+]i) oscillations profile was significantly lower in AS with respect to controls (F=6.15, p=0.003). The percentage of monocytes with intracellular calcium oscillations and the studied molecules were not correlated with the type of therapy or of drug used. Conclusions. SpA associates with a reduced expression of the inflammatory S100A8 calcium binding protein and with a decreased intracellular calcium fluxes in patients' cells compared to healthy subjects, suggesting that the presence of the disease affects the "on-off" mechanisms that regulate the concentration of intracellular calcium.
Introduzione. Le Spondiloartriti (SpA) sono un gruppo di malattie multifattoriali caratterizzate da una complessa interazione tra fattori genetici ed ambientali che determinano una disregolazione del sistema immunitario e l’attivazione di processi infiammatori a livello articolare, in particolar modo nelle articolazioni sacro-iliache. A differenza dell’artrite reumatoide, nelle SpA non esistono dei biomarcatori specifici di attività di malattia che vengono utilizzati nella pratica clinica. Pertanto, la ricerca di nuovi biomarcatori potrebbe essere d’ausilio per una diagnosi precoce e per un adeguato monitoraggio dell’attività di malattia, oltre che essere impiegati come fattori prognostici, misure di outcome e strumenti di valutazione dell’efficacia di trattamento.Nei pazienti con SpA, i macrofagi che infiltrano le articolazioni e che derivano principalmente dai monociti circolanti non esprimono solo citochine infiammatorie come TNF-α, IL-1β o TGF-β ma anche enzimi coinvolti nel rimodellamento tissutale come le metallo proteinasi di matrice (MMPs). La metalloproteinasi di matrice 3 (MMP-3), infatti, è riconosciuta come una molecola altamente espressa nel tessuto sinoviale e nel sangue periferico dei pazienti con SpA. Studi recenti hanno evidenziato che le metallo proteinasi di matrice 8 e 9 (MMP8 e MMP9) vengono prodotte dalle cellule mononucleate derivate da sangue periferico (PBMCs) quando vengono stimolate da calprotectina (eterodimero formato dalle proteine S100A8 e S100A9).Vi è poi una crescente evidenza del ruolo patogenetico nelle Spa svolto dalle cellule appartenenti all’immunità innata quali macrofagi, mastociti e neutrofili; il tessuto sinoviale dei pazienti con SpA infatti è caratterizzato da una elevata vascolarizzazione e quindi da una forte infiltrazione delle cellule immunitarie. In queste cellule i segnali di calcio intracellulare sono essenziali nella regolazione di numerose funzioni cellulari incluse proliferazione, differenziazione, apoptosi e trascrizione genica. Lo scopo di questo lavoro è stato quello di analizzare i livelli di espressione genica delle molecole TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 e MMP9 e di valutare se le variazione dei flussi di calcio intracellulare ([Ca2+]i) nei PBMCs potrebbero essere associati alla presenza di SpA. Metodi.La popolazione studiata comprendeva 64 pazienti con diagnosi di SpA (età media ± deviazione standard: 39.5 ± 13.2 anni; 39 maschi, 25 femmine) e 100 controlli sani (età media ± deviazione standard: 46.6 ± 8.5 anni; 58 maschi, 42 femmine). Tra i pazienti, 26 (40.6%) presentavano spondilite anchilosante (AS) e 38 (59.3%) artrite psoriasica (PsA), con diagnosi formulata sulla base dei criteri rispettivamente di New York e CASPAR. Per ciascun soggetto arruolato, sono stati raccolti i dati demografici e fisiologici, la storia clinica e familiare. Sono stati raccolti poi, campioni di sangue, al fine di valutare l’emocromo e la VES, e di determinare i livelli di PCR, acido urico, prealbumina, alanina aminotransferasi (ALT) e glucosio. Per ciascun soggetto è stata effettuata un’analisi di espressione genica relativa (Real Time PCR) di TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 e MMP9. La determinazione dei flussi di calico intracellulare ([Ca2+]i) nei pazienti e nei controlli è stata effettuate mediante microscopio ad epifluorescenza. Risultati. I livelli di espressione genica relativa nei PBMCs delle citochine infiammatorie of TNF-α, IL-1β, TGF-β erano simili nei pazienti con AS e PsA se comparati ai livelli di espressione nei controlli sani. Le variazioni dei livelli di espressione di TNF-α, TGF-β e IL-1β correlavano tra di loro. I livelli di espressione di TNF-α però risultavano correlati in maniera diretta, nei pazienti, con la presenza di una familiarità per la malattia (t=-2.5386, p=0.013). I livelli di espressione di MMP8 e MMP9 non risultavano essere associati con la diagnosi di SpA e non correlavano con gli indici clinici di attività di malattia.Anche i livelli di espressione di S100A9 non risultavano essere associati con la diagnosi di SpA mentre i livelli di espressione di S100A8 (F=3.29, p=0.039) erano ridotti nei pazienti con PsA.I livelli di espressione di S100A8 e S100A9 correlavano in maniera significativa con il numero di cellule infiammatorie circolanti e S100A8 correlava con i valori di PCR e VES. Dall’analisi dei dati ottenuti dai controlli sani e dai pazienti risultava evidente come la maggior parte dei monociti dei soggetti di controllo presentassero pulsazioni regolari di calcio intracellulare a differenza delle cellule ottenute da pazienti.I pazienti affetti da AS presentavano una ridotta percentuale di monociti con oscillazioni dei flussi di calcio intracellulare rispetto ai controlli sani (F=6.15, p=0.003). La percentuale di monociti con variazione di calcio intracellulare e l’ espressione delle molecole studiate non risultavano essere correlati ne con il tipo di terapia ne con il tipo di farmaco utilizzato. Conclusioni. In conclusione, I risultati di questo studio hanno evidenziato che nei pazienti con SpA vi è una ridotta espressione della proteina legante calcio S100A8 e vi è un decremento dei flussi di calcio intracellulare rispetto ai controlli sani, suggerendo che la presenza della malattia influenza i meccanismi "on-off" che regolano la concentrazione di calcio intracellulare.

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_H17 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.

Background: For effective provision of healthcare, knowledge of disease epidemiology is of paramount importance. Within Ankylosing Spondylitis (AS), however, several areas are poorly understood, including information regarding disease prevalence and natural history, and the quality of life (QoL) of patients. This thesis aimed to address these areas, specifically to: a) determine the prevalence of AS, b) identify determinants of poor QoL, and c) describe the natural history of the disease. Methods: The Scotland and Ireland Registry for Ankylosing Spondylitis (SIRAS) was used to fulfill these aims and collects clinical and patient-reported information on all AS patients seen within Scottish secondary care. AS prevalence was calculated as the number of patients, per 10,000 adults, while QoL was described using the Ankylosing Spondylitis Quality of Life (ASQoL) questionnaire and factors associated with poor QoL assessed using Poisson regression. To assess the natural history of AS, the mean scores of commonly collected disease measures (including disease activity) were plotted yearly, from diagnosis. Results: The prevalence of AS was calculated to be 4.7 per 10,000. Poor QoL was associated with being retired/unemployed due to ill health, poor physical function, high disease activity, pain, fatigue, poor spinal mobility and being prescribed a biologic therapy. Regarding the natural history of AS; of the clinical measures studied, all remained relatively stable over time at the group level; however differences were demonstrated, when stratifying by gender, initial disease activity and by biologic use. Discussion/Conclusions: This study has added to the knowledge of disease in three main ways: accurate prevalence estimates aid the distribution of healthcare, the factors associated with reporting poor QoL may provide novel targets for future intervention and the reported consistency in the natural history of the disease may be used to reassure patients who present with low disease activity while initiating aggressive therapy for those with initial high activity.

The aim of this thesis was to examine the role of diet in ankylosing spondylitis (AS). Patients were examined in: i) a postal questionnaire survey of dietary habits and gastrointestinal (GI) symptoms; ii) a study on biomarkers of diet and disease activity; iii) a comparison of cardiovascular risk factors with the general population using data from the Västerbotten Intervention Programme (VIP), and; iv) a 21-week omega-3 fatty acid supplementation study regarding the effects on disease activity. The postal survey (111 respondents) revealed no correlation between dietary habits and disease activity measured by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). However, GI problems, and in particular GI pain, were prevalent in patients with AS irrespective of NSAID usage.Gastrointestinal pain was predicted by higher BASDAI and a higher consumption of vegetables. Overall, 30 (27%) of the patients experienced an aggravation of gastric symptoms when consuming certain foods. In the study of biomarkers (n=66) no correlation was found between diet and disease activity as assessed by BASDAI. There were, however, positive correlations between BASDAI and the content of arachidonic acid (AA) in plasma phospholipids (rs=0.39, p<0.01) and the estimated activity of the enzyme delta-5-desaturase (rs=0.37, p<0.01). This may reflect a process involved in the inflammation associated with AS that requires further investigation. Comparing data from the VIP for patients (n=89) and controls showed no significant differences regarding diet, physical activity or smoking. Nonetheless, more pronounced correlations between blood lipids and diet were identified among patients than in controls. Furthermore, the levels of cholesterol and triglycerides were lower in patients compared with controls. Lastly, in the supplementation study, a high-dose of long-chain omega-3 fatty acids (4.55 grams/day) was found to lower disease activity, as measured by BASDAI, whereas low-dose treatment (1.95 grams/day) caused no change. In conclusion, within a group of Swedish AS patients we found no correlation between ordinary dietary habits and disease activity. Diet in western populations of patients with AS may, however, be of importance for gastric symptoms and for cardiovascular risk factors. The finding of a lowered disease activity in patients on high-dose supplementation with long-chain omega-3 fatty acids indicates that a radical dietary shift may influence disease activity. The findings of a positive correlation between disease activity and plasma AA, and the decreased levels of blood lipids imply the need for further studies into fatty acid metabolism in AS.

The Spondyloarthritides (SpA) are a group of genetically and pathophysiologically related diseases. Ankylosing spondylitis (AS), the prototypic SpA family member, is a systemic inflammatory disease primarily affecting the axial skeleton, characterised by sacroiliitis and bone formation, promoting joint inhibition. AS is highly heritable; approximately 90% of AS susceptibility is defined by an individuals’ genetic background, to which the MHC class I molecule HLA-B27 contributes approximately 30%. This association was discovered 40 years ago, yet the pathogenic role of HLA-B27 remains elusive. Dendritic cells (DCs) belong to the myeloid lineage and, the principal antigen presenting cells (APCs) of the immune system, activate naïve T cells and contribute to the balance between activation and suppression of the immune response. If affected by HLA-B27, DCs are therefore likely to contribute to the T cell-mediated aspects of AS pathogenesis. Studies in our laboratory, using HLA-B27 transgenic (HLA-B27 TG) rats, have revealed HLA-B27-mediated effects on DC populations. The affected DCs induce abnormally high levels of IL-17 production from T cells; CCR6+ IL-17-secreting cells appear to be important in driving pathology both in the HLA-B27 TG rats and in AS patients. We therefore aimed to perform the first characterisations of the phenotype and functions of DCs and other myeloid populations purified directly from AS patients, to understand their role in AS pathogenesis. Analyses of circulating myeloid populations revealed that AS patients have a reduced proportion of the CD1c-expressing blood DCs, offset by an increase in CD14- CD16+ mononuclear cells. Interactions between CD14- CD16+ mononuclear cells and CD4+ T cells generated high levels of IL-6 secretion, required for the generation of Th17 cells. CD14- CD16+ mononuclear cells also induced T cells to express CCR6, and may therefore contribute to pathology by promoting Th17 responses. Interestingly, our data also indicate that APCs of mucosal origin may make a significant contribution to the systemic inflammation observed in AS patients. These observations give new insights into the pathogenic mechanisms in AS.

Background: Ankylosing spondylitis (AS) is an incurable, fluctuating, long-term condition for which prescribed exercise is central to management. However, many people with AS do not undertake prescribed exercises, the reasons for which are poorly-understood. Aims: The project sought to develop a grounded understanding of the exercise beliefs of people with AS, the exercise behaviours they adopt, and the decision making processes they undergo when choosing exercise behaviours. Design: Adopting a constructivist approach, 23 semi-structured interviews and 7 focus groups were undertaken with people with AS in the North West of England. In-depth data were analysed using open, axial and selective coding to inform the development of a conceptual model of exercise behaviour in AS. Ethical approval and informed consent were obtained. Key findings: 48 people participated in interviews and focus groups, with a disease duration ranging from 6 months to 29 years, and age range 19-62 (mean age 44.8 years ) there were 37 men and 11 women. Participants described the need to predict and respond to a changing disease trajectory and utilised a number of informed strategies for long- and short-term exercise management. Participants described a process of ongoing appraisal of their AS status and used approaches similar to cost—benefit analyses to make decisions about exercise behaviour. In the context of the patient journey, four discrete exercise behaviours (no prescribed exercise, other exercise, two tier behaviour, prescribed exercise), and four responses to changing disease status (no behaviour change, transient change, goal oriented change, non exercise change) were identified. Some key determinants of exercise behaviour were also revealed. Conclusion: This study has led to the grounding of participant perspectives on exercise in AS, rendering the proposed model both relevant and understandable to people with AS, and may have use in helping people understand and plan their future exercise behaviour in AS and forms a baseline for future study.

The purpose of this two stage exploratory study was to investigate the psychosocial, and physical problems associated with a chronic form of arthritis, Ankylosing Spondylitis (AS), following the format of `Problem Specification' suggested by the competency-based model of coping (Goldfried and D'Zurilla, 1969). The first stage goal identified problem thoughts, feelings, and situations associated with AS, by thirteen AS sufferers, two physicians, two physical therapists, and ten significant others. The method for obtaining first stage information was an author constructed structured telephone interview. The second stage goal was the development of an author constructed survey instrument, the Inventory of Problems for Ankylosing Spondylitis (IPAS) completed by Iii individuals with AS, to identify problem frequency, and severity, both for the past twelve months, and for the total time a problem had been present. The second stage included a measure of social support, the Family Relationship Index, consisting of three scales from the Family Environmental Scales (Moos and Moos, 1986).Four areas of exploration were undertaken by this study which included: 1) Taxonomy of AS Problems - identification of AS problems 2) Frequency and Severity - how are identified problems rated and rank ordered 3) Social Support - how does social support affect responses to identified problems, and demographics 4) Demographics - how do such factors as age or gender affect responses to identified problems.The descriptive analysis confirmed that problems in the IPAS are problems experienced by the sample of AS participants. Items sorted into three categories of physical, relationship, and intrapersonal content areas were subjected to post hoc principle component analyses, yielding forty-five factors. Principle component analyses were conducted on first order factors within the three content areas yielding 13 second order orthogonal factors of AS problem themes which included work discrimination, and relationship difficulties with physicians. Significant findings were found for health ratings and second order factors `uncontrollability' and `stress'ratings of health status associated with lower levels of `uncontrollability' and `stress'. No significant results were found for social support and second order factors. The validity and reliability of the IPAS are suggested for future study, and current findings must be interpreted cautiously. Further research is recommended.
Department of Counseling Psychology and Guidance Services

Background: Whilst many isolated educational interventions have been evaluated, less is known about the practical steps patients take to learn about their condition and how to live with it, the factors that influence their learning, or even the ability and inclination of health professionals to provide relevant education. Understanding the experience of education for people with ankylosing spondylitis (AS) will help to develop resources in the future and tailor existing resources for individual patients. Methods: Focus groups and a survey of UK Rheumatology health professionals were used to describe current practice and professionals’ perspectives of education for people with AS. Patients’ perspectives of learning were reported through focus groups, serial semi-structured interviews with 10 ‘new’ patients with AS, and further interviews with 12 ‘review’ patients. Finally, consensus methods were employed to review the findings. Results: A detailed description of education and learning for people with ankylosing spondylitis has been constructed, based on the current provision of education and the perspectives of both patients and relevant health professionals. Analysis of the interviews with patients led to the development of the Established Patient Model, which describes a search for information in four stages. The model indicates that patients do not strive to be experts on their condition, but instead reach a self-defined level of adequate knowledge based on their background and the disruption to their lives caused by AS. Conclusions: This thesis details how, when and why people with AS learn about their condition, and the content and delivery methods they value and choose. Equally, I have identified variations in the delivery of education by Rheumatology Departments. Understanding these issues allows changes to the provision and organisation of educational resources to be suggested. These potentially complement and facilitate patients’ learning, allowing clinicians to recommend educational resources which are likely to be acceptable and useful.

The findings of this study provide an original contribution to knowledge in ankylosing spondylitis (AS) and have important implications for eiThancing clinical practice. The results demonstrate the existence and significance of associations between disease and psychological status in AS, and also demonstrate the value of using longitudinal, repeated measures approach to study this long-term condition. This study is also the first to demonstrate the value of using a mixed methods approach to investigate this issue in AS. Although existing literature on prospective longitudinal cohort studies in AS is very limited (other than for studies which involve clinical trials of medications and other interventions), this project demonstrates the feasibility of sustaining such a study over an 18-month period and of recruiting large numbers of participants to both the quantitative and qualitative phases. The results are based upon a hospital-ascertained cohort of 89 adults. Both the quantitative and qualitative phases produced important new findings: 1. In this cohort, mean BASMI, BASFI and BASDAI scores remained consistent throughout the 18-month period. People with BASDAI scores higher than 4 at the beginning of the study continued to score higher than 4 throughout. 2. BASMI, BASFI and BASDAI scores correlated significantly with anxiety, depression and internality scores, but not with levels of belief in chance or powerful others, throughout the study. This demonstrates that AS disease status is closely linked to some, but not all, psychological measures. 3. There was no effect of co-existent psoriasis or iritis on either disease or psychological status, but BASMI and BASFI (but not BASDAI) scores were significantly related to age. 4. Factors which appear to influence the associations between disease and psychological status are highly complex, often differing between individuals, and usually determined by other co-morbidities and life circumstances besides AS. These results suggest that the major implication for clinical practice would be the development of a more comprehensive and integrated assessment framework for AS set within the context of a biopsychosocial model. Envisaged would be a major programme of work to critically assess and validate potential components of such a framework with the aim of determining efficacy, feasibility and acceptability of such an approach.

Ankylosing Spondylitis (AS) is a chronic inflammatory disorder of the spine which leads to progressive spinal fusion and deformity. With improvements in MRI, this condition is now being recognized earlier. The treatment of this condition so far is limited to physiotherapy, NSAIDs and anti-TNF therapy. The assessment of response to therapy is largely subjective using clinical outcome measures such as the Bath Ankylosing Spondylitis disease activity index (BASDAI). This thesis describes the search for an objective measure of response to therapy in AS. It does so by studying two separate patient cohorts- one receiving anti-TNF therapy and the other receiving a novel oral phosphodiesterase-4 inhibitor, apremilast, in a clinical trial setting. In addition to various clinical outcome measures and laboratory biomarkers, it also explores novel volumetric analysis of bone oedema lesions on MRI and its correlation to clinical indices. The results of this study indicate that apremilast improves clinical indices of response in AS and also modulates bone biomarkers. However, it may do so differently to anti-TNF agents with plasma sclerostin and RANKL: OPG possibly playing important roles in its mechanism of action. This study highlights the fact that different laboratory biomarkers may be modulated differently by different drugs. The novel volumetric analysis developed using Dynamika software showed promise with good correlation to established methods of scoring scans such as Berlin scoring. In particular, a novel biomarker, the product of the volume of the lesion and its intensity correlated well with changes in BASDAI in the anti-TNF cohort. However, there are a number of issues, notably inter-observer variability as well as time required to carry out the analysis, that need to be resolved. This could be done by developing automated regions of interest using this software on the basis of intensity of the lesions, hence providing an objective measure of response to therapy in AS.

Ankylosing spondylitis (AS) is an inherited chronic immune-mediated disease, with a complex genetic and immunological basis. This thesis examined the effect of ankylosing spondylitis-associated genetic changes on DNA methylation and gene expression in isolated immune cell subsets. This study confirmed several pathways associated with disease, such as CD8+T-cell activation, and posed new evidence for the mechanisms of disease. The final chapter developed a signature for distinguishing individuals who have AS from healthy individuals by leveraging genetic profiling, gene expression and DNA methylation. This is the largest study of gene expression or DNA methylation in ankylosing spondylitis to date.

Background: Spinal fractures related to ankylosing spondylitis (AS) are often associated with serious complications. Therefore, knowledge of the incidence, best treatment, outcome, and prevention would assist in improving current guidelines. Objectives: This thesis aims at (1) analysing the complications and mortality of surgical treatment, (2) mapping the incidence and treatment modalities for these patients in Sweden, as well as (3) investigating the putative preventive effect of biological disease modifying anti-rheumatic drug (bDMARD) therapy on spinal fractures related to AS. Methods: Merged multiple national registries were used to identify predictors of mortality and spinal fractures in patients with AS. Beyond that a finite element model (FEM) was designed to simulating a cervicothoracic fracture related to AS. Results and Conclusions: During the last two decades an increase of the incidence of vertebral fractures in patients with AS was observed. With the introduction of bDMARD treatment of AS was revolutionised and quality of life and function improved.  It seems that the improved quality of life and function in these patients does not correlate with a reduced fracture risk. Still, for the first time a beneficial effect of bDMARD with regard to spinal fracture occurrence was provided. The risk of spinal fractures was not reduced, but the debut of a spinal fracture was delayed with bDMARD. Since for this study the observation interval was only a decade, a future follow-up should revisit the effect of bDMARD on spinal fractures related to AS. Furthermore, it was shown that posterior stabilisation is an effective method for restoring stability without the necessity of additional external fixation. Most likely the early rehabilitation reduced pulmonary complications, which in turn reduced early mortality of these fractures. The FEM could be used to identify the most appropriate implant configuration, since no well-established cadaver models exist. Clinical Trial Registration: ClinicalTrials.gov, Identifier NCT02840695.

This research aims to understand how 3-D visualisation can support the assessment of a chronic arthritic condition. The focus is on the assessment and associated feedback of information by the clinician. The assessment of any chronic disease is an integral part of the treatment. In the case of Ankylosing Spondylitis (AS), it is particularly important as it helps to determine whether or not pharmaceutical and/or surgical interventions are required for a patient. The assessment process for a patient with AS is currently overseen by the physiotherapist. There is growing awareness that the current protocols and means of taking measurements are both inaccurate and inconsistent. Therefore, the objective of this research is to investigate whether utilising 3-D visualisation technology can enhance the current assessment and feedback process by augmenting assessment instruments and by visualizing the function of a subject in ways afforded by a 3-D visualisation tool. From a consideration of the physical therapy process, a simplified assessment and feedback model is developed and used to understand where a new tool might add value. A 3-D visualisation research tool is prototyped, to include both subjective and objective aspects of current assessment instruments. The Bath Indices for AS were used to test the limits of the prototype as these are acknowledged as the gold standard of assessment for this condition. To verify the prototype, experiments were carried out, collecting motion data and measurement data from a healthy adult in a laboratory environment. To validate the prototype, a qualitative evaluation was undertaken using a pilot study, focus group and individual interview methods. Participants are experts in this condition and comprised physiotherapists in both service and academia. The results of the evaluation suggested that 3-D visualisation and a prototype for evaluation, as developed in this research, would enhance assessment and feedback from the physiotherapist's perspective. Furthermore, it would be beneficial to the treatment of AS with respect to obtaining subjective and objective measures, and supporting exercise activity.

HLA-B27 is a Major Histocompatibility Complex (MHC) class I molecule which exhibits a strong association with the inflammatory arthritic disorder Ankylosing Spondylitis (AS). It has been postulated that the tendency for HLAB27 to misfold and form disulphide linked heavy chain homodimers may contribute to AS pathogenesis. However, it remains elusive how these aberrant forms occur and to which extent they participate in AS development. I have analysed the contribution of conserved cysteine residues at positions (p) 101, 164, 203 and 259 and unpaired cysteines at p67, 308 and 325, in the formation of ER resident heavy chain homodimers. I demonstrated that HLA-B27 dimerisation involves a hierarchy of disulphide bonding. Moreover, my study indicates that heavy chain-dimers are composed of multiple species, with each possibly exhibiting varying degrees of folding/unfolding. I found that HLA-B*27:05 adopts novel in vivo conformations within the ER lumen, which have yet be to identified for other HLA alleles. Detection of these conformations depends on cysteine residue exposure to the ER environment what can explain the propensity for heavy chains to misfold. Finally, I investigated the role of the MHC class I antigen presentation pathway in HLA-B27 dimerisation. I found that HLA-B27 retains its tendency to dimerise despite the presence of β2m and high affinity peptide and both β2m-free (unfolded) and β2m+peptide-associated (folded) homodimers can associate with the Peptide Loading Complex. Moreover, the tendency for HLA-B27 to dimerise is influenced by p116 within the peptide binding groove, which is a natural polymorphism within AS-associated and non-AS–associated HLA–B27 subtypes. These studies may have implications for inflammatory disease since they begin to provide a biochemical understanding for the ability of HLA-B27 to misfold.

This thesis comprehensively applies genome-wide association study, Mendelian randomization analyses, and cytokine proteomics to investigate the genetic basis and immunopathogenic mechanisms of acute anterior uveitis (AAU). Multiple susceptibility loci, environmental risk factors, and potential biomarkers are identified, and a polygenic risk score for AAU developed. These findings provide novel insights into the immunogenetics in AAU, and contribute to clinical translational studies.

RESUMO: Introdução: A espondilite anquilosante (EA) é uma doença inflamatória crónica caracterizada pela inflamação das articulações sacroilíacas e da coluna. A anquilose progressiva motiva uma deterioração gradual da função física e da qualidade de vida. O diagnóstico e o tratamento precoces podem contribuir para um melhor prognóstico. Neste contexto, a identificação de biomarcadores, assume-se como sendo muito útil para a prática clínica e representa hoje um grande desafio para a comunidade científica. Objetivos: Este estudo teve como objetivos: 1 - caracterizar a EA em Portugal; 2 - investigar possíveis associações entre genes, MHC e não-MHC, com a suscetibilidade e as características fenotípicas da EA; 3 - identificar genes candidatos associados a EA através da tecnologia de microarray. Material e Métodos: Foram recrutados doentes com EA, de acordo com os critérios modificados de Nova Iorque, nas consultas de Reumatologia dos diferentes hospitais participantes. Colecionaram-se dados demográficos, clínicos e radiológicos e colhidas amostras de sangue periférico. Selecionaram-se de forma aleatória, doentes HLA-B27 positivos, os quais foram tipados em termos de HLA classe I e II por PCR-rSSOP. Os haplótipos HLA estendidos foram estimados pelo algoritmo Expectation Maximization com recurso ao software Arlequin v3.11. As variantes alélicas dos genes IL23R, ERAP1 e ANKH foram estudadas através de ensaios de discriminação alélica TaqMan. A análise de associação foi realizada utilizando testes da Cochrane-Armitage e de regressão linear, tal como implementado pelo PLINK, para variáveis qualitativas e quantitativas, respetivamente. O estudo de expressão génica foi realizado por Illumina HT-12 Whole-Genome Expression BeadChips. Os genes candidatos foram validados usando qPCR-based TaqMan Low Density Arrays (TLDAs). Resultados: Foram incluídos 369 doentes (62,3% do sexo masculino, com idade média de 45,4 ± 13,2 anos, duração média da doença de 11,4 ± 10,5 anos). No momento da avaliação, 49,9% tinham doença axial, 2,4% periférica, 40,9% mista e 7,1% entesopática. A uveíte anterior aguda (33,6%) foi a manifestação extra-articular mais comum. Foram positivos para o HLA-B27, 80,3% dos doentes. Os haplótipo A*02/B*27/Cw*02/DRB1*01/DQB1*05 parece conferir suscetibilidade para a EA, e o A*02/B*27/Cw*01/DRB1*08/DQB1*04 parece conferir proteção em termos de atividade, repercussão funcional e radiológica da doença. Três variantes (2 para IL23R e 1 para ERAP1) mostraram significativa associação com a doença, confirmando a associação destes genes com a EA na população Portuguesa. O mesmo não se verificou com as variantes estudadas do ANKH. Não se verificou associação entre as variantes génicas não-MHC e as manifestações clínicas da EA. Foi identificado um perfil de expressão génica para a EA, tendo sido validados catorze genes - alguns têm um papel bem documentado em termos de inflamação, outros no metabolismo da cartilagem e do osso. Conclusões: Foi estabelecido um perfil demográfico e clínico dos doentes com EA em Portugal. A identificação de variantes génicas e de um perfil de expressão contribuem para uma melhor compreensão da sua fisiopatologia e podem ser úteis para estabelecer modelos com relevância em termos de diagnóstico, prognóstico e orientação terapêutica dos doentes. -----------ABSTRACT: Background: Ankylosing Spondylitis (AS) is a chronic inflammatory disorder characterized by inflammation in the spine and sacroiliac joints leading to progressive joint ankylosis and in progressive deterioration of physical function and quality of life. An early diagnosis and early therapy may contribute to a better prognosis. The identification of biomarkers would be helpful and represents a great challenge for the scientific community. Objectives: The present study had the following aims: 1- to characterize the pattern of AS in Portuguese patients; 2- to investigate MHC and non-MHC gene associations with susceptibility and phenotypic features of AS and; 3- to identify candidate genes associated with AS by means of whole-genome microarray. Material and Methods: AS was defined in accordance to the modified New York criteria and AS cases were recruited from hospital outcares patient clinics. Demographic and clinical data were recorded and blood samples collected. A random group of HLA-B27 positive patients and controls were selected and typed for HLA class I and II by PCR-rSSOP. The extended HLA haplotypes were estimated by Expectation Maximization Algorithm using Arlequin v3.11 software. Genotyping of IL23R, ERAP1 and ANKH allelic variants was carried out with TaqMan allelic discrimination assays. Association analysis was performed using the Cochrane-Armitage and linear regression tests as implemented in PLINK, for dichotomous and quantitative variables, respectively. Gene expression profile was carried out using Illumina HT-12 Whole-Genome Expression BeadChips and candidate genes were validated using qPCR-based TaqMan Low Density Arrays (TLDAs). Results: A total of 369 patients (62.3% male; mean age 45.4±13.2 years; mean disease duration 11.4±10.5 years), were included. Regarding clinical disease pattern, at the time of assessment, 49.9% had axial disease, 2.4% peripheral disease, 40.9% mixed disease and 7.1% isolated enthesopathic disease. Acute anterior uveitis (33.6%) was the most common extra-articular manifestation. 80.3% of AS patients were HLA-B27 positive. The haplotype A*02/B*27/Cw*02/DRB1*01/DQB1*05 seems to confer susceptibility to AS, whereas A*02/B*27/Cw*01/DRB1*08/DQB1*04 seems to provide protection in terms of disease activity, functional and radiological repercussion. Three markers (two for IL23R and one for ERAP1) showed significant single-locus disease associations. Association of these genes with AS in the Portuguese population was confirmed, whereas ANKH markers studied did not show an association with AS. No association was seen between non-MHC genes and clinical manifestations of AS. A gene expression signature for AS was established; among the fourteen validated genes, a number of them have a well-documented inflammatory role or in modulation of cartilage and bone metabolism. Conclusions: A demographic and clinical profile of patients with AS in Portugal was established. Identification of genetic variants of target genes as well as gene expression signatures could provide a better understanding of AS pathophysiology and could be useful to establish models with relevance in terms of susceptibility, prognosis, and potential therapeutic guidance.

Mechanisms of tolerance by which the human body distinguishes self from non-self serve to protect us from pathogens, and are central to the understanding of immunology. Breach of self- tolerance can result in inappropriate immune responses targeting one's own proteins, cells 0 tissues and lead to autoimmune disease. Ankylosing Spondylitis (AS) is a systemic, rheumatic autoimmune disease, which affects up to 0. 1% of the population. Despite a strong association with the MHC class I allele, HLA-B27, the aetiology of AS is unknown. The work in this thesis describes the use of existing and the development of new proteomic methods to characterize and quantify differences in protein expression in both cellular an humoral components of the immune system in patients with AS. Monocytes from AS patients have been characterized by 2D-gel electrophoresis and label-free quantitative nano-UPLC-MSE mass spectrometry analysis, which has revealed differential expression in proteins from the Ubiquitin Proteasome Pathway (UPP) as well as an up-regulation of 13 proteins that are induced by Tumour Necrosis Factor. An in vitro proteasomal digest assay demonstrated that one protein upregulated in the UPP, the proteasomal activator PA2S, increase the generation ofHLA•B27•restricted epitopes. In parallel, novel methods for the MALDI-TOF analysis of endogenous and tryptic plasma peptides were developed with the aim of identifying diagnostic biomarkers in AS plasma. The depletion of abundant plasma proteins followed by plasma fractionalion based on isoelectric focusing or nano-UPLC•MSE mass spectrometry analysis was also employed to this end Preliminary plasma cytokine profiling showed a specific upregulation of interleukin-27 in AS patients. Levels of AS plasma matrix-metallo proteins (MMPs) analysed demonstrated a~ upregulation of two collagenases, however this find ing was not supported by plasma MM enzymatic activity assays. Finally, in collaboration with the Harvard Institute of Proteomics, the novel finding 0 autoantibodies in AS patients was characterized and quantified by the use of Nucleic Acid Programmable Protein Arrays. 44% of AS patients studied demonstrated a broad, non-specific autoantibody response and multiple AS patients showed responses to common putative autoantigens. The AS cohort autoantibody response was more specific compared to that from the RA cohort and autoantibodies from AS patients showed a bias towards antigens involved i6 skeletal and connective tissue disorders. Taken together, evidence for differential expression of proteins in the Ubiquitin Proteasome Pathway and a broad, non-specific autoantibody response in AS patients support the hypothesis ~f a role in antigen presentation for HLA-B27 in Ankylosing Spondylitis. The work in this thesis demonstrates that clinical proteomics is a viable method for the investigation of au toimmuI1f diseases, provides entry points to target cellular and molecular mechanisms for further investigation of disease pathogenesis, disease diagnosis and therapeutic benefit.

Ankylosing Spondylitis (AS), or Bechterew’s disease, is an inflammatory rheumaticdisease that through the formation of additional bone tissue in the spine eventuallyleads to the complete fusion of the vertebrae, in effect turning the spine into one longbone. Due to the reduced flexibility of the spine with the long lever arms, spinalfractures in AS-patients are relatively common even after minor trauma. The aim of this thesis was to use an existing finite element model of a healthy spineand adapt it to the conditions of AS, thus gaining some insight into the effects ofsurgical stabilization of cervical fractures, using posterior screws and rods. Althoughthis type of surgery is often performed, it has not been previously investigated in abiomechanical model. This thesis should be considered as a starting point for how afinite element model of the spine could be used to investigate the effect of spinalimplants in the case of a fracture in the ankylosed spine. An existing FE-model was modified to some of the conditions of AS: The vertebraewere fused by adding ossifications at the intervertebral discs (with the Head-C1 andC1-C2 joints left mobile). A fracture was simulated at the C6C7 disc level. Fourdifferent implant configurations were tested: Short instrumentation C6C7, mediuminstrumentation C5toT1, long instrumentation C3toT3, and a long instrumentationC3C6C7T3 with skipped intermediate levels. Three loads (1.5g, 3.0g, 4.5g) wereapplied according to a specific load curve. Kinematic data such as the gap distance inthe fracture site were obtained. Furthermore the stresses in the ossified parts of thediscs were evaluated. It was shown that the chosen methods of adapting the model to the AS conditions, andmodeling the fracture and implant, changed the kinematics so that less movementoccurred between the vertebra, which is typical for AS. Measured as fracture gap, alltested implant configurations were equally good at stabilizing the fracture, althoughthey all allowed more movement than the non-fractured AS-model did. All implantconfigurations were also able to stabilize the fracture in terms of the horizontal translation in the fracture. The disc ossifications were somewhat shielded from stress for those ossifications that were within the range of the implant. This was so for all implant configurations. No increased stress was observed in the ossifications immediately outside the range for the implants, relative the non-fractured AS-model. For the C6C7 and C5toT1 implant configurations as well as the non-fractured ASmodel,the stresses were highest at the T1T2 level. Stresses in the ossifications in the thoracic spine were generally low, apart from the T1T2 level. The results show that the chosen AS-adaptations and the modeled implant seem reasonable for testing some of the considerations of cervical fractures in the ankylosed spine as well as for some implant configurations. The results also make it possible to speculate about the optimal type of implant. The effects of screw placement and anchoring, osteoporosis, muscle activation and possible spinal deformity on the implant stability were not investigated, and should be a matter for further studies.
Ankyloserande Spondylit (AS), eller Bechterew’s sjukdom, är en inflammatorisk, reumatisk sjukdom som innebär att ny benvävnad formas i ryggraden. Detta leder till att kotorna slutligen smälter samma. I praktiken leder detta till att ryggraden fogas samman till ett enda långt ben. Vid en reducerad flexibilitet med långa hävstänger är frakturer i kotpelaren relativt vanliga för patienter med AS även vid mindre trauma. Syftet med denna uppsats var att använda en existerande FE-modell av en frisk rygg och nacke, och anpassa den till det tillstånd som är typiskt för AS. Vidare var syftet att erhålla insikt om hur kirurgisk stabilisering med skruvar och stänger av en fraktur i nacken beter sig. Trots att denna typ av operation utförs förhållandevis ofta, så har detta inte tidigare undersökts i någon biomekanisk modell. Denna uppsats kan ses som en utgångspunkt för hur en FE-modell av ryggraden kan användas för att undersöka effekten av stabiliserande implantat av en fraktur vid AS. En existerande FE-modell modifierades för att överensstämma med några av de villkor som gäller vid AS: Kotorna fogades samman via förbeningar i diskarna. (Huvud-C1 och C1-C2-lederna lämnades rörliga). En fraktur simulerades i C6C7- nivån. Fyra olika konfigurationer av implantat provades: Kort implantat C6C7, mellanlångt implantat: C5tillT1, långt implantat C3tillT3 och ett långt implantat C3C6C7T3 där mellanliggande kotor hoppades över. Tre lastfall (1.5g, 3.0g, 4.5g) applicerades enligt en specifik lastkurva. Kinematisk data såsom “fracture gap distance” i frakturen registrerades. Vidare registrerades också spänningarna i de förbenade delarna av diskarna. Det var möjligt att, genom att anpassa modellen till AS och modellera fraktur och implantat, påverka nackens kinematik så att mindre rörelse skedde mellan kotorna, vilket är typiskt för AS. Med utgångspunkt i mätningarna av “fracture gap distance”, var det möjligt för alla konfigurationer av implantat att stabilisera frakturen, även om implantaten tillät mer rörelse än AS-modellen som saknade fraktur. Detta gällde även för horisontell translation i frakturen. Vad gällde spänningarna i förbeningarna så avlastades dessa för de förbeningar som var belägna inom implantatets räckvidd. Detta gällde för alla implantatkonfigurationer. Inga spänningskoncentrationer observerades i förbeningarna precis utanför implantatens räckvidd i förhållande till AS-modellen utan fraktur. Vad gällde C6C7 och C5toT1 implantaten, liksom ASmodellen utan fraktur, så registrerades de högsta spänningarna i T1T2-nivån. Spänningarna i förbeningarna i bröstryggen var generellt låga, bortsett från T1T2- nivån. Resultaten visade att de valda anpassningarna till AS och sättet att modellera implantaten verkade vettiga för att undersöka några av de överväganden som uppstår vid nackfrakturer i en ankyloserad kotpelare, samt ett par implantatvarianter.  Resultaten möjliggör även spekulationer kring vilken typ av implantat som är optimal. Huruvida implantatstabiliteten påverkades av placeringen av skruvar, infästningen i benvävnaden, osteoporos, muskelaktivering och möjlig missbildning av ryggen undersöktes inte. Detta kan undersökas i framtida studier.

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.

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.

The aim of the project was to develop a PCR method to detect HLA-B27 at the Immunology Department of St. James hospital in Dublin. The HLA-B27 gene is common among patients with ankylosing spondylitis (AS). Ninety percent of patients with AS have the HLA-B27 gene and it is therefore counted as a risk factor and could be used as part of the diagnosis. Twenty-two frozen blood samples from patients with AS or suspected AS were donated from the rheumatology department at St. James hospital. PCR is a well known and common technique, many hospital laboratories have a PCR machine and therefore PCR is a good choice for detection of the HLA-B27 gene. A multiplex PCR was developed where a PCR control, primers to the β-globin gene, was used in the same tube as the HLA-B27 primers, to secure that the PCR worked in every tube. Finally a blind test was performed to test the specificity of the PCR. The result shows that the specificity was 100%. Of all patient samples, sixteen was HLA-B27 positive and six were HLA-B27 negative. In addition, optimal conditions for the PCR and the way to extract DNA from frozen blood were successfully established. For future diagnosis, the described PCR can be used to detect the HLA-B27 gene in patients and it can be considered as a start for further development of a real-time PCR for detection of the HLA-B27.

The unfolded protein response (UPR) detects the presence of misfolded proteins in the endoplasmic reticulum (ER) and subsequently relieves ER stress by increasing the folding capacity of the ER. The secretory pathway substrate HLA-B27 is highly associated with the chronic inflammatory disease ankylosing spondylitis (AS) and has a tendency to misfold in the ER. Here, we show that overexpression of HLA-B27 and non-disease associated HLA-B7 in immortalised cell lines leads to heavy chain misoxidation, which is accompanied by upregulation of BiP and splicing of XBP1, a key step in the IRE1 pathway of the UPR which is increasingly being linked with intestinal inflammation. We also demonstrate that different cell lines respond to different ER stress stimuli in distinct ways. We establish that HT1080 cells inefficiently induce a UPR in response to tunicamycin and that this has consequences for cell survival. However, inefficient activation of the UPR in HT1080 cells can be overcome by secondary signals, since co-administration of the tyrosine kinase inhibitor genistein leads to activation of XBP1. Furthermore, we show that genistein can inhibit UPR induction of BiP in response to a range of ER stresses indicating that the cancer drug genistein can inhibit or activate the UPR depending on the environment and cell type. This has implications for inflammatory disease since regulation of the UPR is important in determining a cell’s tendency towards apoptosis.

Possession of the Major Histocompatibility Complex (MHC) allele, HLA-B27 (B27), strongly predisposes to the development of spondyloarthritis. Furthermore, B27 exists as polymorphic variants, with some subtypes (such as B*2705) being more strongly associated with disease than others (B*2709). The immunological function of MHC molecules is to present peptides in a heterotrimeric complex with beta-2-microglobulin (β2m); however, B27 has also been observed to form non-classical (β₂m –free) homodimers at the cell surface. It has been suggested that there may be a pathogenic role for cell surface B27 homodimer interactions with Natural Killer (NK) cell receptors, such as Leukocyte Immunoglobulin like Receptors (LILRs). In this thesis I characterise these interactions and investigate molecular differences between two B27 subtypes. Here I show that the B*2705 subtype forms homodimers more readily than the B*2709 subtype, but once formed, B27 homodimers of the 2 different subtypes exhibit comparable binding specificities and affinities to the NK receptors. On the other hand, I show significant differences in the binding specificities and affinities of these receptors to B27 homodimers and heterotrimers. LILRB1 does not bind B27 homodimers, but does bind B27 heterotrimers. LILRB2 binds B27 heterotrimers with a KD of 22μM, whereas LILRB2 binds B27 homodimers more strongly with a KD of 2.5μM. In addition to these main findings, I have characterised the specificity and affinity of candidate B27 homodimer-specific antibodies. I have performed epitope-mapping experiments and developed a model for binding to the B27 homodimer. Finally, I have identified crystallisation conditions for the B27 homodimer in complex with a Fab, allowing for X-ray crystallography studies. In this thesis, I have characterised for the first time the molecular interactions of the B27 homodimer with NK cell ligands and show that they are different from those with the B27 heterotrimer. This work supports a hypothesis of B27 homodimer induced pathology involving NK receptors.

Physiotherapy lacks valid and useful measures to assess outcome of treatment interventions. Body charts are used widely by physiotherapists for the assessment of pain in patients. The aim of this thesis was to validate the use of a scored body chart as an outcome measure in Ankylosing Spondylitis (AS). 69 AS patients (Modified New York criteria) were assessed three monthly by a single observer for one year. Mean age was 42.4 years (range 21-74, M:F 4:1), median duration of disease, 5 years, and symptoms, 15 years. Patients shaded a body chart to show their areas of pain, and scored each individual area of pain as; 1=mild, 2=moderate, 3=severe or 4=very severe. The body chart score is the sum of the individual pain intensity scores. Disability questions and visual analogue scales (VAS) for pain and stiffness were completed. Objective measures of tenderness (enthesitis) and movement were recorded. Results were analysed using the Number Cruncher Statistical System. The median body chart score at visit one was 12 (range 0-63, 25%tile=8, 75%tile=21). At the first visit, statistically significant correlations (Spearman's) were found between the body chart and VAS present pain (r=0.318, p<0.008), VAS stiffness (r=0.535, p