Diabetes mellitus is correlated with modifications in bone micro architectural and mechanical strength, leading to increased bone fragility. The in cretin hormones, with a classical effect to increase insulin secretion following food ingestion, are now postulated to have important direct effects on bone. As such, glucose-dependent insulinotropic polypeptide (GIP) has dual actions on bone cells; enhancing boneforming activity of osteoblasts and suppressing bone resorption by osteoclasts. The sister incretin of GIP, glucagon-like peptide-l (GLP-I), is also suspected to directly influence bone health in a beneficial manner, although mechanism are less clear at present. The physiological actions of incretins are attenuated by dipeptidyl peptidase (DPP-4) activity and it is speculated that introduction of DPP-4 inhibitor may also positively affect quality of the skeleton. As such, this thesis evaluates the potential beneficial effects of a DPP-4 resistant GIP analogue, namely [D-Ala2]GIP, on osteoblastic-derived, SaOS-2 cells, and also preliminary in vivo studies on the impact of genetic deficiencies of GIPRs and GLP-IRs on bone mineral density and content. Further studies characterised the beneficial effects of incretin-based therapies on metabolic control, bone microstructure and bone mechanical integrity in animal models of pharmacologically-, genetically- and environmentally-induced diabetes. GIP and related stable analogue increased bone-forming biomarkers in SaOS-2 cells and importantly, [D-Ala2]GIP was shown to be more potent than native GIP. Knockout mouse studies revealed that both GIPR and GLP-IR signaling are important for optimum bone mass. All diabetic mouse models displayed reduced bone mass, altered bone micromorphology and impairment of bone mechanical strength, similar to the human situation, confirming their appropriateness. The incretin-based therapeutics, [D-Ala2]GIP and Liraglutide, in streptozotocin-diabetic significantly increased bone matrix properties, indicating recovery of bone strength at the tissue level. The beneficial effects of administration of [D-Ala2]GIPoxyntomodulin on bone health in db/db mice were more prominent as the Oxm analogue did not only improve bone strength at tissue level, but also at whole-bone level. These modifications were independent of metabolic status. Twice-daily Exendin-4 therapy improved glycaemic control and increased work required to resist bone fracture in high-fat fed mice. It was also established that Sitagliptin had neutral effects on bone microstructure and mechanical strength in high-fat mice.

Osteoclasts are large multinucleated cells responsible for the degradation of bone matrix. Osteoclast precursors (OCL-Ps) are of haematopoietic origin, being members of the mononuclear phagocyte system that circulate amongst the fraction of CD14+ cells in peripheral blood. OCL-Ps reach sites of osteoclast formation and remodelling via the vasculature and are therefore destined to encounter endothelium before migrating to the bone surface. The main aim of this study was to design a suitable model for the investigation of OCL-P-endothelial interactions. Adaptations of this culture system were also used to examine osteoclast formation in health and disease. Firstly, osteoclast formation from PBMCs was investigated using a soluble Receptor Activator of NF-kB Ligand (sRANKL)-based culture system. Osteoclasts could be consistently generated from the PBMCs of healthy volunteers. These cells were multinucleated, expressed TRAcP and VNR, formed F-Actin rings and were capable of forming extensive resorption lacunae on dentine slices. Osteoclast formation was most efficient in the presence of RANKL, MCSF, 1,25-VitD3, dexamethasone and PGE2, required 21 days of culture and was critically dependent on the presence of RANKL and MCSF. Cell selection studies revealed that the OCL-P resided solely within the CD14+ fraction and lymphocyte depletion had no significant effect on osteoclast formation. This culture system was subsequently utilised to examine and compare osteoclast formation in bone disease. No consistent differences in osteoclast formation or resorption were observed between patients with Paget's disease of bone (PDB) or osteoporosis compared to healthy volunteers. When osteoclast formation was compared between patients with Familial Expansile Osteolysis, a severe form of PDB and unaffected immediate relatives, no statistically significant differences were observed. However, osteoclast formation from patients with sclerotic bone disorder Camurati-Englemann disease (CED) was elevated 5-fold, and resorption 10-fold, when compared to both family-based and unrelated controls. CED is caused by mutations in the transforming growth factor-betal (TGF-1) gene, which leads to increased TGF-1 bioavailability.

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Introduction: Performing artistes have entertained audiences for thousands of years. Their repertoires require the integration of a well ‘tuned’ central nervous system and hypermobility. Hypermobility is a common phenomenon that is beneficial for some but not for others. This thesis discusses hypermobility associated with multisystemic symptoms referred to as Joint Hypermobility Syndrome (JHS). It is suggested that the multifactorial manifestations of the condition contribute to deconditioning thus impacting on the physical and mental well being of individuals with JHS. Purpose: To explore the multi-factorial manifestations of JHS including functional difficulties and their impact on quality of life. Methods: A two part study; part one, development of a questionnaire to assess for functional difficulties; part two, a mixed methods approach to explore aspects of JHS. Results: Principal Axis Factoring was employed to explore the structure of the 9-item Functional Difficulties Questionnaire (FDQ-9) to assess functional difficulties reported in childhood and adulthood. Internal consistency was high (0.81), correlations between items were > 0.5 and preliminary findings suggested satisfactory construct validity. Test-retest reliability was good (ICC 0.96 [95% CI 0.92 to 0.98]. Patients with JHS were 3 [95%CI 1.95 – 4.56] times more likely to report functional difficulties both as a child and as an adult than healthy volunteers. Patients with JHS were significantly more likely to report dislocations, autonomic nervous system, gastrointestinal and cardio-respiratory symptoms than healthy volunteers. Chronic widespread pain reported by 86% of patients with JHS was a significant predictor of quality of life using the SF-12. Patients with JHS had significantly lower physical component scores than healthy volunteers (29.2 [SD 10.6] and 54.5 [SD 5.7]) respectively. Conclusions The development of the FDQ-9 contributes to the understanding of the multi-factorial manifestations of JHS and their long term nature. These have important clinical implications as symptoms of JHS appear early in life. Management of this condition requires early recognition and an understanding of the multisystemic nature.

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This thesis is composed firstly of a literature review focusing on the attitudes of health professionals towards chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) and fibromyalgia. Secondly, a research paper explores the ways in which people living with CFS/ME have experienced psycho-emotional disablism. Finally, a critical appraisal discusses the process of conducting the research as well as its strengths and limitations. The literature review takes the form of a meta-synthesis regarding the attitudes of healthcare professionals towards CFS/ME and fibromyalgia. A meta-ethnographic approach was used with reciprocal translation producing the following themes: “Feeling hopeless and more hopeless”: psychological effects of lack of knowledge; “Your heart sinks when they come in the room”: stigma and stereotypes; and “I’m going to be with you through thick and thin”: management of the condition. The review highlights the difficulties faced by health professionals regarding the management and diagnosis of both conditions, as well as possible reasons for the negative attitudes held by some professionals. The research paper, which employed thematic analysis, explores the ways in which people living with CFS/ME have experienced psycho-emotional disablism. Three overarching themes were identified: “fighting to be heard”; “lack of legitimacy” and “feeling invisible”. Participants described the discrimination and stigma that they had encountered from many areas of society. Ideas for future research are proposed. The critical appraisal presents the author’s reflections on the research process as well as its strengths and limitations, and the five stages of the process are described as follows: choosing a thesis topic and designing the project; recruitment and research interviews; the interview process; analysis and writing up of the data; and the author’s reflections on the project.

An open label trial of B-lymphocyte depletion therapy (BLDT) based on rituximab in 5 patients with rheumatoid arthritis, initiated in 1998, had shown that this therapy was associated with major clinical improvement. B-lymphocyte repopulation had occurred before relapse in all patients and, interestingly, it had not been always associated with relapse. This thesis starts with an extension of the initial open label trial to 22 patients with results suggesting a dose response to rituximab. Further follow up of these and an additional 18 patients revealed that, although all patients eventually relapsed, as in the previous small study, relapse could occur either at the time of B-lymphocyte repopulation of the peripheral blood or at a variable time after this time point, up to almost 3 years. Re-treatment with BLDT was found to be well tolerated and effective. Upon re-treatment, patients usually repeated the same pattern of relapse. Disease-associated autoantibodies, rheumatoid factor of immunoglobulin A, G and M classes and antibodies to cyclic citrullinated peptides, fell in all patients following BLDT but only decreased significantly in those who responded to treatment. Comparison with changes observed in total immunoglobulin levels and anti-microbial antibodies, suggested a specific effect on autoantibodies. Relapse was almost always preceded or associated with a rise in autoantibody levels, particularly rheumatoid factor of immunoglobulin M isotype, and was more closely associated with a rise in autoantibody levels than with the presence of B lymphocytes per se. B-lymphocyte stimulator (BLyS) serum levels increased following BLDT. Levels decreased following B-lymphocyte repopulation but tended to be lower at the time of repopulation in the group of patients who relapsed at this time point. Immunophenotyping of peripheral blood B lymphocytes showed that depletion was major but not complete and that it involved all B-lymphocyte subpopulations. It suggested that a quantitative threshold of depletion needed to be reached for patients to respond to therapy. B-lymphocyte repopulation occurred mainly from naive cells with immature B lymphocytes present in increased numbers and frequency. Patients who relapsed at the time of B-lymphocyte repopulation tended to show increased frequency and increased numbers of circulating memory B lymphocytes at the time of repopulation. Immunophenotyping studies of bone marrow aspirates in 6 patients, 3 months after treatment with BLDT, showed that the frequency of cells of B-lymphocyte lineage at this time point varied between patients. The variable relationships between the frequencies of more immature and more mature B-lymphocyte precursors in the samples suggested different degrees of depletion. Samples from patients who relapsed the earliest showed a pattern suggesting less efficient depletion. In conclusion, the results presented here suggest that relapse following BLDT in patients with rheumatoid arthritis can be attributed to incomplete depletion of pathogenic B-lymphocyte clones, persistence of long-lived plasma cells producing pathogenic autoantibodies or to a combination of both. This will contribute to hypothesis-based development of B-lymphocyte targeting therapies.

Osteoporosis (OP) is a common disease, especially among postmenopausal women. OP is regarded as the main cause of fragility fractures. Bisphosphonate (BP) medications, approved by the FDA in 1995, have been adopted as the most common and frontline treatment to OP. The most obvious positive effect of BP is their ability to improve bone mineral density (BMD). However, lots of concerns have been raised after BP medications coming into market for about ten years. A new type of hip fractures (atypical fractures) has been reported and linked with long-term BP treatment. It is still debatable on the effect of BP on bone health. Previous studies have evaluated BP’s effect either by measuring BMD quantitatively or following up fracture cases. There are very limit studies from an engineering background investigating BP’s effect on bone strength and microstructure, especially on those bones sustaining fractures despite BP treatment. The femoral heads from trauma patients’ hip replacement operations were collected and worked as a BP study group (10 femoral heads) and fracture control group (13 femoral heads) depending on whether or not the patient had been treated with BP before fractures. Cadaver samples were collected from an elderly control group (5 femoral heads). Five cylindrical sub-samples were cored from the same location of each femoral head. All the five cylinder sub-samples were micro-CT scanned for microstructure measurements. Two of the five cylinder sub-samples were selected randomly and compressively mechanically tested for apparent strength. Another two sub-samples were further synchrotron radiation scanned for a sub-micro features study, especially focusing on the trabecular microcracks and fully broken trabeculae. The apparent strength for BP treated samples is 29% and 48% lower than that of non-treated fracture controls and elderly controls, respectively. The density and microstructure parameters for the BP study group are slightly higher than or at a similar level as those of non-treated fracture controls. However, there are 24% and 55% more microcracks existing in BP treated samples than that of non-treated fracture controls and elderly controls, respectively. There are a sub-group of patients with whom BP treatment does not work very well, as they still fractured even with BP treatment for years. The bone mass density and trabecular microstructure may not be the cause of lower apparent strength. Microcracks and fully broken trabeculae can partly explain the lower strength, but further studies at the sub-micro and fibrillar levels are highly suggested.

Disorders of the circulation of the leg are a problem in both the developed and developing world, and are a major cause of morbidity and mortality. The incidence of diabetes is increasing, the population is ageing, and resources to treat vascular disease are finite. Exercise can augment the circulation, and has many beneficial effects on the cardiovascular system as a whole. Electrical stimulation of the muscles of the leg may work in a similar fashion to both voluntary contraction and external muscle compression, in that venous return is increased, and symptoms of vascular disease are reduced. This body of work aims to delineate the clinical problems to which this solution should be applied, carry out preliminary testing and pilot trials to assess feasibility and effect size of electrical therapy, and to design a device which best addresses the needs of the identified population. Hypothesis Neuromuscular electrical stimulation (NMES) of the leg increases the circulatory parameters of the leg, and treatment with NMES over time will alleviate clinical symptoms of vascular disease and improve quality of life. Methods The incidence of medical morbidity related to vascular disease was explored, and target populations requiring development of current inadequate management strategies were identified. Neuromuscular electrical stimulation has been defined, and the history of the use of electrical therapies has been reviewed. Bench testing and small trials were conducted to evaluate the physiological impact of neuromuscular electrical stimulation device usage, and how they compare to current standards of non-pharmaceutical and non-operative medical care (e.g. graduated compression, intermittent pneumatic compression). Clinical trials in patient groups of interest were instituted, looking specifically at chronic venous disease, peripheral arterial disease, and diabetic vascular complications. Results Flow rates and speed of blood in the femoral vessels increased in both healthy subjects and those with vascular disease. Those patients with severe diabetic foot complications were more resistant to flow changes. Fluximetry measurements taken from the foot increased with NMES device use. Improvements in functional outcomes were shown, particularly in the management of claudication and wound healing in the diabetic foot. Quality of life measures improved but on the whole were not statistically significant. Conclusions The judicious use of NMES in patients with vascular disease could prove useful, especially in the setting of multiple comorbidities and polypharmacy. The risk profile is very low, especially when compared to anticoagulants or surgery. The health economic benefits are not known, but future studies could use the data in this thesis to power future trials. The biological effects of device usage, both long and short term have not been explored, and may be amenable to testing in further trials in humans.