Osteoarthritis (OA) is a common, multifactorial musculoskeletal disease that is characterised by joint pain and reduced joint function. It is a polygenic disease and progresses as a result of the focal loss of the articular cartilage of the synovial joint. rs143383 is a C to T transition single nucleotide polymorphism (SNP) located in the 5ʹ untranslated region (UTR) of the growth differentiation factor 5 gene GDF5. The T allele of the SNP is associated with an increased risk of OA and of a number of other common musculoskeletal diseases. This susceptibility is mediated by the T allele producing less GDF5 transcript relative to the C allele, a phenomenon known as differential allelic expression (DAE). The aim of my study was to identify trans-acting factors that bind to rs143383 and which regulate this GDF5 DAE. Protein binding to the gene was investigated by two experimental approaches: 1) competition and supershift electrophoretic mobility shift assays (EMSAs) and; 2) an oligonucleotide pull down assay followed by quantitative mass spectrometry. Binding was then confirmed in vivo by chromatin immunoprecipitation (ChIP), and the functional effects of candidate proteins investigated by RNA interference (RNAi) and over expression. Using these approaches the trans-acting factors Sp1, Sp3, P15 and DEAF-1 were identified as interacting with the GDF5 5ʹUTR. Knockdown and over expression of the factors demonstrated that they were repressors of GDF5 expression. Depletion of DEAF-1 modulated the DAE of GDF5 and this differential allelic effect was confirmed following over expression, with the rs143383 T allele being repressed to a significantly greater extent than the rs143383 C allele. In combination, Sp1 and DEAF-1 had the greatest repressive activity. The genotype of a second GDF5 5ʹUTR SNP, rs143384, which is located downstream of rs143383, has been previously found to impact upon the DAE at rs143383. Thus protein binding to rs143384 was also investigated using EMSAs. In conclusion, I identified four trans-acting factors that modulate the expression of GDF5 via the OA susceptibility locus rs143383.

Rheumatoid arthritis (RA) is a multifaceted inflammatory autoimmune disease characterised by the infiltration of leukocytes into synovial joints leading to the destruction of articular cartilage and bone. Autoreactive CD4+ T cells producing inflammatory cytokines are implicated in disease pathogenesis. The clinical efficacy of B cell-depletion therapy is not dependent on clearance of autoantibodies suggesting a critical role for B cells as antigen presenting cells (APC). To elucidate the role of B cells in activating CD4+ T cells in RA, this project examined B cell- mediated antigen presentation to CD4+ T cells isolated from T cell receptor (TCR)-transgenic mice specific for the major arthritogenic epitope of the candidate cartilage autoantigen, aggrecan. This system allows for direct comparison of aggrecan-specific B cells to dendritic cells (DC) and other APC in order to identify any unique consequences of B cell antigen presentation and modulation of CD4+ T cell effector cytokine production. The data presented here show the activation, proliferation and effector function of CD4 T cells co- cultured with different APC in the presence of aggrecan and demonstrate key differences in B cell and DC production of disease-relevant cytokines. Crucially, aggrecan-specific B cells induced greater CD4+ T cell production of the pathogenic cytokine IFN-γ than DC, despite equivalent IL-2 production. The role of aggrecan-mediated activation of several pattern recognition receptors in this process was excluded using an in vitro detection system. However, the identification of a differential requirement for CD80 and CD86 during antigen presentation by B cells and DC suggested a role for co-stimulatory molecules at the APC-T cell interface in mediating B cell induction in CD4 T cell IFN-γ production. In summary, this work highlights the role of the CD80/86-CD28 pathway in mediating the observed differential induction of CD4+ T cell IFN-γ production by antigen-specific B cells and DC following aggrecan presentation.

Aims: There has been very little lifecourse research looking at the risk of osteoarthritis (OA). A lifecourse analysis of risk factors for knee, hip and hand OA (defined using features on ultrasound) acting at different stages of life, including early life factors, was performed among members of the Newcastle Thousand Families birth cohort. Methods: Potential risk factors for OA (including birth weight, breast feeding data and socioeconomic status) have been collected prospectively in this birth cohort of subjects aged 63 (born in May-June 1947) and an a priori conceptual framework was developed. Subjects had both knees, hips and the dominant hand scanned with ultrasound. These data were analysed in relation to a range of factors from across the lifecourse using logistic and linear regression models. Results: Among 316 participants, duration of exclusive breast feeding showed a significant inverse association with knee osteophytes while BMI and total hip bone mineral density at age 50 increased the risk of knee osteophytes. The univariate effect of social class at birth on knee osteophytes was found to be mediated by its subsequent effect on breast feeding and total hip bone mineral density. The multivariate model for hip OA had three risk factors; BMI, physical activity and pack years of smoking at age 50. Smoking at age 50 and increased infections in childhood appeared to confer protection from hand OA. Conclusions: This is the first study to perform a lifecourse analysis of OA risk using prospectively collected data. The majority of the risk of OA at the three joint sites seemed to occur through factors acting in adulthood. However, breast feeding protected subjects from knee OA while infections in childhood decreased hand OA risk. These results suggest that modification of OA risk factors acting in adulthood would probably be more beneficial than intervening in early life.

Background: Non-invasive cardiac imaging provides important diagnostic and prognostic information in cardiovascular disease. Assessment of ventricular function remains the fundamental imaging request in clinical practice. Cardiac magnetic resonance (CMR) is now the recognised reference standard for quantification of left and right ventricular systolic function, but not diastolic filling. Due to cost and limited availability of CMR, echocardiography remains the first line imaging modality for assessing ventricular function in most cases. Several echocardiographic methods are available for quantifying global ventricular function however despite significant advances in cardiac imaging techniques, visual assessment of ventricular systolic function remains the standard by which ventricular function is reported in many centres. This method is subjective and introduces inter-observer bias. In an era of multi-modal imaging, accurate, reproducible and widely available methods for quantifying ventricular function, which exhibit good inter-modal inter-technique concordance, are desirable. The overall aim of this thesis was to examine the accuracy and reproducibility of several new echocardiographic imaging techniques for quantifying left and right ventricular systolic function, indexed against CMR reference standards, and to examine a novel CMR technique for assessing diastolic function, indexed against current reference standards (invasive catheter recording of left ventricular end diastolic filling pressure (LVEDP)), in a heterogeneous cohort of patients as seen in clinical practice. Methods: All imaging modalities were performed within three hours of each other. Study 1 was designed to compare the accuracy of speckle tracking strain echocardiography for quantifying LV systolic function against biplane Simpson’s rule (SR) and 3D-echocardiography, using CMR LV ejection fraction (LVEF) as the reference standard. Study 2 was designed to investigate the accuracy of a novel modified regional wall motion scoring index (RWMSI) for calculating LVEF, and compare its accuracy against SR and CMR LVEF. Study 3 was designed to explore the clinical utility of velocity encoded (VEC) CMR for diagnosing LV diastolic dysfunction. VEC CMR E/Em velocity ratio was compared to LVEDP recorded during left heart catheterisation. Study 4 was a head-to-head comparison of 10 echocardiographic non-volumetric indices of right ventricular systolic function, based on current European Association of Echocardiography recommendations, indexed against CMR RVEF as the reference standard. Results: In study 1 we demonstrate that speckle tracking strain may be superior to SR for quantifying LV systolic function. In study 2 we suggest that, when specialist imaging software is unavailable, a modified RWMSI may be superior to SR for calculating LVEF. In study 3, we demonstrate a significant correlation between VEC-CMR E/Em ratios and LVEDP, and conclude that VEC-CMR may be a useful tool to diagnose diastolic dysfunction, especially in patients with preserved LVEF. In study 4, we demonstrate that RV free wall strain has a closer correlation to CMR-RVEF than nine alternative echocardiographic indices of RV function, and may be the method of choice for assessing RV systolic function by 2D-echocardiography in the future. Conclusions: This series of studies has confirmed that novel non-invasive cardiac imaging techniques may be used to accurately quantify cardiac ventricular function, and may confer significant advantage over current methods.

This thesis describes several interrelated studies concerning the effects of diphosphonates in the treatment of disorders of bone turnover. Diphosphonates have previously been shown to suppress bone turnover in Paget's disease when given orally for at least three months. However, five daily intravenous infusions of the diphosphonates, clodronate, etidronate and a newer agent, aminohexane diphosphonate (AHDP), induced marked and sustained biochemical and clinical changes similar to those achieved with long-term oral treatment. These new regimens may be useful in the management of Paget's disease. The assessment of biochemical response in Paget's disease, particularly in re-treated patients, is more complex than hitherto described. The pre-treatment serum alkaline phosphatase and, in re-treated patients, the extent of biochemical relapse are important determinants of response. Little was previously known of the effects of high doses of diphosphonates on mineral metabolism. Inhibition of mineralisation occurred following all 3 intravenous diphosphonates. However, this effect was partial and short-lived following clodronate or AHDP, but was complete and much more sustained following etidronate. All three diphosphonates induced hyperphosphataemia by increasing renal tubular reabsorption of phosphate. This latter effect was related, both temporally and quantitatively, to the effects of these agents on skeletal mineralisation suggesting a possible causal relationship. Hypocalcaemic responses were consistently observed in pagetic patients following intravenous clodronate or AHDP, but not following etidronate. Similarly, infusions of either clodronate or AHDP, but not etidronate, induced normalisation of mean serum calcium in patients with hypercalcaemia of malignancy. Furthermore, oral clodronate, but neither high-dose nor low-dose oral etidronate, induced significant hypocalcaemic responses in patients with primary hyperparathyroidism. The degree of inhibition of bone resorption was similar for each of the diphosphonates in all of the disorders studied, suggesting that the attenuated hypocalcaemic effects of etidronate resulted from the greater impairment of mineralisation that occurred following treatment with this agent.

Laminopathies are a heterogeneous group of diseases associated with mutations in A-type lamins, which together with B-type lamins, form the nuclear lamina: a proteinaceous network underlying the nuclear membrane. A-type lamins are encoded by the LA1NA gene, and more than 300 mutations have been described, associated with more than 16 phenotypes. The majority of mutations affect striated muscle to cause Emery-Dreifuss muscular dystrophy (EDMD) or cardiomyopathy, while others result in lipodystrophy, neuropathy or premature ageing syndromes. However, clear genotype-phenotype correlations are not established, the pathogenic mechanisms are little understood and therapies are lacking. -- This thesis first explores genotype-phenotype for LMZV/4 mutations and makes correlations by characterising both physical-chemical properties of the amino-acid change, and position in the 3D structure of lamin A. LMNsl mutations associated with muscular dystrophies, premature ageing disorders and lipodystrophies clustered in the Ig-fold domain of lamin A and resulted in a similar change in charge, suggesting that modification of specific protein-protein interactions contribute to different phenotypes. -- Next, I investigated the effects of four pathogenic EDMD mutations on nuclear morphology, nuclear protein distribution and myogenic cell function. I found that some mutations led to severe nuclear deformations and mislocalisation of lamin B, while others caused accumulation of lamin A-positive nuclear foci. Myogenic differentiation was mildly affected by some mutant lamin A species. -- Finally, I describe a series of proof-of-principle experiments investigating a potential therapeutic intervention for laminopathies. A lamin A variant with a deletion corresponding to regions encoded by exon 5, removing 42 amino acids in the central rod domain, localised correctly.

Rheumatoid arthritis (RA) is a chronic inflammatory immune disease affecting the joints. CD4+CD25+ regulatory T cells (Tregs) are abundantly present in the inflamed joints of patients with RA, but inflammation still persists. The notion that Tregs are terminally differentiated suppressor cells has recently been disputed by studies showing that Tregs can display a significant degree of plasticity and even convert into IL-17-producing cells under inflammatory conditions. Therefore, the overall hypothesis of this thesis was that the pro-inflammatory environment impairs Treg function in RA by converting them into IL-17-producing cells. We show in this thesis that frequencies of CD25+CD127low Tregs with a CD45RO+ memory phenotype were increased at the site of inflammation in RA. These Tregs displayed a regulatory phenotype, with increased expression of the Th17 marker CD161. Furthermore, CD14+ monocytes with an activated phenotype were found in high numbers in the RA joint. Monocytes and Tregs could be found in close proximity in human tissue suggesting that they interact in vivo. We next studied the effects of in vitro-activated monocytes on Treg phenotype and function. Activated monocytes increased the percentages of IL-17+, IFNγ+, TNF-α+, and IL-10+ memory (CD45RA-) Tregs. Tregs from these co-cultures showed no loss in Treg markers or suppressive capacity, and were rather enhanced in their suppressor functions. Finally, Tregs from the peripheral blood of patients with RA showed a similar phenotype and cytokine expression profile compared to Tregs from healthy controls. RA Tregs were capable of suppressing autologous effector T cell proliferation and cytokine secretion. However, Tregs from some patients showed a hampered ability to suppress monocyte-derived chemokines and cytokines. Together, these data suggest that Treg function is not globally impaired in patients with RA and that Tregs exposed to a pro-inflammatory environment, such as occurs in the inflamed rheumatic joint, do not lose their regulatory function.

Background: Patient uncertainty is considered to be an inherent part of the illness experience, and particularly relevant in unpredictable conditions; however, it has not been thoroughly investigated in systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) and no appropriate instrument is available for its quantification. This thesis presents mixed-method studies aiming to address this gap in the literature. Phase-1: Qualitative interviews with 32 patients and 8 health care professionals were conducted in order to conceptualise patient uncertainty in SLE and RA. These findings were used to develop a new self-report instrument for patient uncertainty. Items of the new instrument were qualitatively tested through cognitive debriefing interviews. Phase-2: A field test was set up to evaluate and revise the newly developed instrument psychometrically, using the modern technique of Rasch analysis in a sample of 388 patients. The instrument was subsequently evaluated using traditional psychometrics tests. Phase-3 (part-1): A second field test was set up to evaluate the psychometric properties of the second draft of the new instrument using a combination of modern and traditional psychometric techniques in an independent sample of 279 patients. The final draft of the instrument consisted of five scales; symptoms and flares, medication, trust in doctor, self-management and impact. Phase-3 (part-2): The construct validity of the new instrument, as well as the contribution of the five patient uncertainty scales to SLE and RA patient outcomes, including treatment adherence, mood and health related quality of life, were explored. Statistical tests, including correlational analyses and multiple linear regressions, were used for this exploration. Conclusions: This thesis offers a conceptual framework and a self-report instrument for the assessment of patient uncertainty in SLE and RA. The findings offer implications for the role of patient uncertainty in these conditions and demonstrate the importance of comprehensive methodology in assessing such constructs.

Dystonia is a disabling condition characterised by involuntary muscle spasms and abnormal postures. Its pathophysiology is incompletely understood but most lines of evidence point to an underlying defect of basal ganglia function leading to abnormal corticomotor output. Various abnormalities have been shown, including abnormal neuronal activity in basal ganglia output nuclei, defective neural inhibition at the spinal, brainstem, cortical level and sensorimotor misprocessing. More recently, increased neural plasticity has been found in dystonia patients in response to transcranial magnetic stimulation (TMS) protocols which induce motor cortex plasticity. Excessive plasticity might contribute to dystonia by promoting or reinforcing abnormal patterns of connectivity. The most significant advance in the treatment of generalised dystonia has been the development of globus pallidus internus (GPi) deep brain stimulation (DBS). Interestingly its beneficial effects are progressive over weeks to months rather than immediate. A plasticity effect has been implicated but physiological evidence has been lacking. Furthermore it is unknown what impact GPi DBS has on the underlying pathophysiology such as defective inhibition or excessive plasticity. The aim of the present work was to examine the impact of GPi DBS on underlying pathophysiological features such as disinhibition and abnormal motor cortical plasticity. In this thesis, studies in a consecutive series of dystonia patients, mainly those with primary generalised dystonia, who underwent bilateral GPi DBS, are presented. Patients were studied in a prospective, longitudinal manner with both clinical assessment of dystonia using a validated rating scale and electrophysiological studies including blink reflex excitability and forearm H-reflex reciprocal inhibition. In addition, once stable improvement had been achieved, the impact of GPi DBS on motor cortex plasticity was studied using TMS paired associative stimulation (PAS). The clinical study of these patients confirmed the therapeutic efficacy of GPi DBS and provided direct evidence of the superiority of the posteroventral globus pallidus as the optimal target. The longitudinal studies of blink and H-reflex, showed progressive normalisation of brainstem and spinal excitability, which correlated with the time-course of clinical improvement. These data provide the first evidence of reversal of underlying dystonia pathophysiology by GPi DBS and are compatible with progressive long-term neural reorganisation (plasticity) playing a role in the mechanism of action of GPi DBS. Furthermore, the result of TMS PAS experiments demonstrated that GPi DBS reduces the short-term plasticity of the motor cortex, the magnitude of this effect also correlated with therapeutic effect. This result is compatible with the concept that excessive plasticity promotes dystonia and reversal of these abnormalities may be another mechanism by which GPi DBS acts. In conclusion, work presented in this thesis provides the first electrophysiological correlates of clinical improvement in dystonia after GPi DBS, which collectively supports the notion that both long and short-term plasticity within the central nervous system are involved in the mechanism of GPi DBS action.

This review examines the role of fear and avoidance in chronic pain, focusing in particular on their role in activity limitation. It examines the existing fear-avoidance models, in particular Vlaeyen and Linton's (2000) model, it reviews the evidence for this model, and considers it in the context of models of fear and avoidance in other psychological disorders. Evidence is reviewed for other factors which contribute to activity limitation in chronic pain. In the context of this evidence, it is concluded that whilst there is much support for Vlaeyen and Linton's model, it may be useful to consider alternative models describing the process of activity limitation, and future research is proposed to support this.