The complement system has crucial functions in host defence. Novel data revealed a role for complement components in the pathology of osteoarthritis (OA). CD59a is a regulator of the terminal complement pathway in mice; the purpose of the study was to determine if CD59a-/- mice have an osteoarthritic bone phenotype. Osteoblast (OB) mineralisation, colony forming unit (CFU) and OCG assays were performed in vitro from bone marrow preparations of 8-20 week old mice. Decreased CFU differentiating towards osteoblasts and adipocytes (n=1 only), as well as an increased OCG, was revealed in male CD59a deficient (-/-) over wildtype (WT) mice. OCG in females were comparable. A human CD59 knockdown system utilising short hairpin (sh) ribonucleic acid (RNA) delivered by adenoviruses was established but did not differentiate into osteoclasts (OC). In vivo the bone phenotype of CD59a-/- mice was established for femora and vertebra L6 via X-ray, microcomputed tomography and histology. In male mice femoral length was increased in CD59a-/- versus WT mice at 8-10, 20 and 50 weeks. Cortical bone volume was increased whilst bone mineral density (BMD) was reduced in CD59a-/- versus WT mice at 8-10 and 20 weeks. Trabecular bone analysis of the distal femur (and spine) showed increased trabecular bone ratio, number, thickness, connectivity and total BMD in CD59a-/- over WT at 8-10 (and 20) weeks of age. In female mice there was no difference in femoral length and trabecular bone, but cortical BMD was raised at 50 weeks (CD59a-/- versus WT). Finally, histology revealed enhanced mineral apposition rate and OC surface as well as reduced osteoid surface in male CD59a-/- over WT mice at 8-10 weeks of age. Increased bone growth and turnover related to CD59a gene deletion were gender specific. These studies highlight CD59a as a potential target for OA treatment.

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease characterised by extensive synovitis resulting in cartilage and bone erosions. Both the innate and adaptive immune pathways contribute to the initiation and the maintenance of the disease. Understanding the role of these pathways is central to develop new therapeutics. We have developed a murine model of RA where ovalbumin (OVA) specific Th1 cells induced a breach of self-tolerance and a transient monoarthritis. This thesis aimed firstly to create a model of chronic autoimmune polyarthritis and then to investigate the contribution of B cells and innate inflammation to the induction of arthritis. Relapse of arthritis was associated with the nature of the antigen (OVA) employed and the route of administration. The analysis of collagen specific B cell response revealed that anti-type II collagen antibodies titres rise during the induction of the relapse of arthritis and that they were directed against the epitope U1. Although typical RA autoantibodies were detected in OVA-mediated arthritis, a mild arthritis could be elicited in absence of antigen presenting B cells and in complete absence of mature B cells. B cells were not necessary in the induction of pathology even though their presence was associated with a higher joint histology score. Finally, this thesis describes that an innate inflammatory stimulus, such as LPS, elicited joint pathology but was insufficient to breach B and T self-tolerance. On the contrary, antigen-specific T cell activation led to arthritis and the production of several autoantibodies typical of RA. The relapse and spread of arthritis developed in this thesis provides a useful tool to investigate the contribution of the innate and adaptive immune pathways in the development of autoreactive responses. A better understanding of these mechanisms will hopefully help to design new therapeutic intervention aiming to re-establish immunological tolerance.

Multiple sclerosis (MS) is a chronic disease of the human central nervous system (CNS) in which repeated episodes of inflammatory demyelination result in formation of persistently demyelinated plaques of gliotic scar tissue associated with varying degrees of axonal loss. MS is now considered a “complex trait” that is triggered in genetically susceptible individuals by environmental factors. The disease is also considered to contain an autoimmune component where both the adaptive and innate immune systems have been implicated in disease pathogenesis. There has been a steady accumulation of circumstantial evidence from both clinical and experimental studies that implicate a role for autoantibody dependent mechanisms. However this issue remains controversial in the absence of formal evidence that patients actually develop a pathogenic autoantibody response. The aim of this thesis was to resolve this question. To do this we developed an in vitro bioassay based on a dissociated myelinating culture system from embryonic rat spinal cord. We demonstrated that this in vitro system could reproduce many features of in vivo myelinated axons. To validate this model as a viable screening assay characterised complement mediated autoantibody responses using a series of monoclonal antibodies and anti-sera. Due to their significance in the literature we focussed in particular on the MOG specific and Nfasc specific responses and comprehensively demonstrated that our bioassay offered a robust screening strategy in which to detect pathogenic antibody responses in the presence and absence of exogenous complement. To determine whether we could use our model to detect pathogenic autoantibody responses in MS patients, we purified the IgG fraction from a cohort of MS patients (n=20), OND (n=10) and healthy controls (n=13). Using this patient purified IgG we demonstrated a MS specific demyelinating activity, which was present in ~50% of samples screened. However in 10% of patients demyelination occurred secondary to pronounced axonal injury. These effects were dependent on exogenous complement and were unique to the MS cohort. Pathogenic antibody responses tended to be most prevalent in those patients with an aggressive disease course. In addition to complement mediated CNS injury we also demonstrated that this pathogenic MS IgG could disrupt myelin formation in developing myelinating cultures. Attempts to define the specificity revealed that this was heterogeneous, however in one MS patient we discovered that Nfasc155 provided a dominant antigen for pathogenic autoantibody responses. Together these data provide formal demonstration that MS is associated with pathogenic autoantibody responses. This has significant long term consequences for the clinical management of the disease

Type 1 diabetes (T1D) is characterised by pancreatic cell autoimmunity and inflammation, resulting in cell islet destruction and insulin deficiency. Prospective studies from different continents have shown that insulin resistance is independently associated with risk for the development of T1D. We wanted to investigate the role of adiponectin in mediating this link. Adiponectin is a circulating adipokine whose anti-inflammatory and insulin sensitising actions appear to be mediated via two related receptors, AdipoR1 and AdipoR2. We began by characterising adiponectin receptor expression on PBMC by flow cytometry. We showed that monocytes express both receptors abundantly, that this expression correlates with insulin sensitivity in both health and diabetes. Furthermore, expression can be increased with lifestyle intervention. Adiponectin receptor expression on monocytes is reduced in T1D, and we demonstrate this leads to an apparent resistance in the ability of adiponectin to inhibit the stimulatory capacity of antigen presenting cells (APC). Specifically, we show that adiponectin inhibits the stimulatory capacity of APCs through down-regulation of CD86 expression, and that this effect is decreased in T1D. In this way, the release from the regulatory effects of adiponectin is one potential mechanism by which immune tolerance is lost in T1D.

Obesity is known to be associated with obstructive sleep apnoea (OSA) and Type 2 diabetes mellitus (T2DM). The effect of OSA in very severely obese individuals is not well documented. In this thesis, I compared the effect of OSA in South Asians and white Europeans, examined the effect of OSA on glycaemic control among T2DM, and explored the relationship between OSA and diabetic retinal and kidney diseases in a severely obese population. I also systematically reviewed the effect of OSA on diabetic kidney and retinal diseases. Findings from this thesis were 1) severely obese South Asians had greater severity of OSA compared to white Europeans and the mechanisms mediating this require further investigation, 2) a high OSA prevalence in T2DM individuals with a positive relationship between nocturnal hypoxia and glycaemic control, 3) severity of hypoxaemia during sleep may be an important factor in the development of diabetic retinal complications, 4) duration of hypoxaemia during sleep were inversely associated with renal function in T2DM and 5) from the systematic review, there is a need for future large cohort studies with long term follow-up data to examine the long-term effects of OSA and other sleep parameters on diabetic retinal and kidney diseases.

Coronary artery disease (CAD) is a disease of inflammatory aetiology, and remains the commonest cause of death globally despite therapeutic advances. Monocytes are implicated in the pathogenesis of CAD, but also in reparative mechanisms after myocardial infarction (MI) due to subset heterogeneity. The aim of this thesis was to provide a detailed phenotypic comparison of differences between the three human monocyte subsets in CAD and after MI, with particular emphasis on CD16+ monocytes which have previously been analysed as a single population rather than two distinct subsets. Longitudinal changes were analysed following MI and relationships explored with plasma cytokines. Multiple significant novel changes in monocyte phenotype attributable to specific subsets were identified, particularly related to the CD14++CD16+CCR2+ ‘Mon2’/‘Intermediate’ subset which increased in number on day 1 after MI and appeared highly functionally active. There were significant changes in expression of a range of receptors associated with inflammation, migration and reparative processes. Significant relations to plasma cytokines and the degree of myocardial damage were observed. Most monocyte parameters predictive of left ventricular ejection fraction six weeks after MI were related to the Mon2 subset. This suggests an important role for this subset in the acute phase of MI.

No description available.

Abstract Background: Pulmonary hypertension (PH) is a rare condition that can occur as a primary disease process, Idiopathic Pulmonary Hypertension (IPH) or secondary to other disorders. In Familial IPH mutations have been identified in the bone morphogenetic protein receptor II gene (BMPRII) (chromosome 2q32-31) a member of the Transforming Growth Factor  (TGF) (Lane et al, 2000). Despite the mutation being present in all cells, vascular wall remodelling is only seen in the pulmonary circulation with marked thickening of the intima and neointimal formation, muscularisation of small-generation resistance vessels and thickening of the adventitial layer together with increased ECM deposition. Similar appearances are noted in the pulmonary circulation’s response to hypoxia. for this projectProlonged exposure of the pulmonary circulation to hypoxia results in vasoconstriction and subsequent vascular wall remodelling. The hypothesis of this work is that the pulmonary circulation’s response to hypoxia may be partially explained by the existence of differences exist in cell signalling pathways in between adventitial fibroblasts from pulmonary and systemic arteries in HPAF. Studies from the Scottish Pulmonary Vascular (SPVU) Laboratory have shown that pulmonary arterial fibroblasts (PAFB) in bovine and rat models of acute hypoxic exposure preferentially proliferate to hypoxia, whereas systemic arterial fibroblasts (SAFB) do not , that the stress mitogen activated protein kinase p38 MAPK is consistently activated in PAFB exposed to acute hypoxia, and is constitutively upregulated in PAFB cultured from rats exposed to chronic hypoxia (Welsh et al, 1998; Welsh et al; 2001). This response to hypoxic exposure has been shown to be dependent on p38 MAPK activity, as use of SB203580 can block the hypoxia-mediated proliferative response to acute hypoxia (Scott et al, 1998; Welsh et al, 2001). Aims and methods: We wished to establish whether the pro-proliferative response of PAFB to acute hypoxic exposure previously noted in bovine and rat models could also be demonstrated in a human model. We wished to establish a role for both classic MAPK and stress MAPKs in hypoxia-mediated PAFB proliferation. We also wished to examine the role of hypoxia inducible factor 1 (HIF1) in human arterial fibroblast responses to acute hypoxia. There is a body of literature that documents cross talk between p38 MAPK and the Bone Morphogenetic Protein (BMPR) signalling pathways. We wished to establish whether Smad proteins (involved in the downstream signalling cascade from BMPR) might play a role in human pulmonary and systemic arterial fibroblast proliferation to acute hypoxia. Following approval from the local Ethics Committee, PAFB were harvested from patients undergoing lobectomy for the treatment of lung cancer. Left internal mammary arteries (SAFB) were harvested from patients undergoing coronary artery bypass grafting. Cells from systemic and pulmonary arterial fibroblasts were grown in conditions of normoxia or acute hypoxia (PO2 35 mmHg ~ 5% O2). Cellular proliferation was assessed using [3H]Thymidine uptake as a surrogate. p38, p44/p42 - ERK1/2 and JNK MAPKs and Smad protein activity was assessed using Western Blotting Techniques with the use of appropriate primary and secondary antibodies and Chemiluminescence to detect the presence of protein. p38 MAPK isoform activity was assessed using Catch and Release® immunophoresis techniques. Findings and conclusions: We demonstrated that acute hypoxic exposure results in human PAFB proliferation, associated with increased p44/p42 – ERK 1/2 MAPK activity, but dependent on p38 MAPK  activity. We also found that the p38 MAPK  isoform was expressed in human PAFB following hypoxic exposure but this did not appear to be involved in the hypoxia-mediated proliferative response. p38 MAPK  activity appeared to occur in a bi-phasic pattern with peaks of activity at t = 6 and 16 hours, the second peak was found to be responsible for the hypoxia-mediated proliferation seen in these cells in agreement with previous work from the SPVU laboratory (Scott et al, 1998; Welsh et al., 2001). The second peak in p38 MAPK  activity was synchronous with peak HIF1 activity (between t = 8 –16 hours). We demonstrated that HIF1 activity can be abrogated by pre-incubation of human PAFB with SB203580 suggesting a mechanistic link between p38 MAPK  activation and HIF1 in a human model of acute hypoxic exposure. We have also demonstrated that that BMPR2-associated Smad 1, 5 and 8 activation is increased in hypoxic human SAFB, suggestive of the activation of an anti-proliferative pathway in these cells that is not associated with p38 MAPK activity. To our knowledge this is the first demonstration of an active response in SAFB to acute hypoxic exposure that involves the active upregulation of an anti-proliferative pathway in these cells. In addition we have demonstrated that in hypoxic pulmonary arterial fibroblasts phospho Smad 1, 5 and 8 expression is reduced (suggestive of the down-regulation of an anti-proliferative pathway) and can be further abrogated by pre-incubation with SB203580. This suggests that in SAFB Smad 1, 5 and 8 activation occurs independent of p38 MAPK activation while in PAFB, p38 MAPK activity augments Smad 1, 5 and 8 activation.

Rho-associated kinase 1 (ROCK1) is a serine/threonine kinase important for the regulation of the cellular cytoskeleton through the induction of actin stress fibres and acto-myosin contractility. The cleavage and subsequent activation of ROCK1 by caspase 3 during apoptosis is believed to cause many morphological phenomena associated with programmed cell death such as dynamic membrane blebbing. I now formally prove the necessity of ROCK1 cleavage for apoptotic blebbing by knocking-in a caspase cleavage resistant mutant of ROCK1 in a genetically modified model. In addition, animals homozygous for non-cleavable ROCK1 demonstrate a phenotype consistent with auto-immune disease suggesting that apoptotic blebbing is important to mediate rapid efferocytosis, which is a rapid phagocytic clearance of the cellular corpse, and thus maintain self-tolerance. Furthermore, apoptotic blebbing is important for the clearance of apoptotic cells and I demonstrate a novel mechanism for ROCK to mediate the release of factors participating in macrophage migration to dying cells. ROCK induced apoptotic blebs and bodies lose membrane integrity prior to secondary necrosis and leak intracellular material. Using quantitative mass spectrometry I identified numerous proteins that were previously unrecognized to be released during apoptosis. The release of protein was found to be impaired following ROCK antagonism with Y27632 which underscores the importance of ROCK activity in apoptotic protein release. One of these proteins, gelsolin, was released following caspase cleavage and encourages macrophage motility towards apoptotic cells. Finally, I now demonstrate that the three nonsynonymous somatic mutations in the ROCK1 gene identified in the Cancer Genome Project lead to elevated kinase activity and drive actin cytoskeleton rearrangements that promote increased motility and decreased adhesion, characteristics of cancer progression. Mapping of the kinase-interacting regions of the carboxy-terminus combined with structural modeling provides insight into how these mutations likely affect the regulation of ROCK1. Consistent with the frequency of ROCK1 mutations in human cancer, these results support the conclusion that there is selective pressure for the ROCK1 gene to acquire ‘driver’ mutations that result in kinase activation.

• Gastro-oesophageal reflux disease (GORD) is the commonest chronic disease in Western countries. Symptomatic GORD is the strongest risk factor for the development of oesophageal adenocarcinoma with obesity and male sex also linked to the development of neoplasia at this site. Recent decades have seen a significant increase in the incidence of this highly lethal cancer among Western populations with Scotland having the highest recorded incidence worldwide. • Human saliva has a high nitrite content derived from the entero-salivary recirculation of nitrate in our diet which has resulted from the increased use of nitrogenous fertilisers over the past 50-60 years. • The luminal chemistry produced at the gastro-oesophageal junction (GOJ) when swallowed salivary nitrite reacts with gastric acid, and most notably the production of nitric oxide (NO), may explain most of the physiological abnormalities that contribute to the pathogenesis of GORD. NO has been shown to reduce lower oesophageal sphincter (LOS) pressure, impair oesophageal clearance, delay gastric emptying and may be the final mediator of transient lower oesophageal sphincter relaxations (TLOSRs). Previous studies to investigate the role of this luminal chemistry in the pathogenesis of GORD show conflicting results. • In addition to the distal oesophageal acidification produced by traditional trans-sphincteric reflux, previous studies suggest ‘splaying open’ of the distal lower oesophageal sphincter following a meal may expose the gastric cardia and the most distal oesophageal squamous mucosa to the noxious effects of gastric acid. • Although the gastric cardia is an important site of pathology in the upper gastrointestinal tract, it is a complex and poorly understood area. It has been proposed, from autopsy studies, that cardia mucosa itself may be pathological and in fact an ‘acquired cardia’ due to metaplasia of the most distal oesophageal squamous mucosa. • A series of studies were designed to examine the effect of salivary nitrite on post-prandial GORD, gastro-oesophageal function and GOJ morphology in 20 healthy, asymptomatic adult volunteers using high-resolution pH manometry, an isotope gastric emptying breath testing and X-ray localisation of the squamo-columnar junction (SCJ). • Despite an excellent range of salivary nitrite concentrations extending over and above the normal physiological range no effect of salivary nitrite on gastro-oesophageal reflux, function or morphology was demonstrated. However, the studies did confirm, for the first time using high-resolution manometry, that distal opening of the LOS occurs after a meal. • The relationship of age and obesity to the SCJ position relative to the proximal border of the gastro-oesophageal high pressure zone (HPZ) was examined in 15 Helicobacter Pylori negative healthy volunteers. Strong negative correlations were seen between SCJ position relative to the proximal HPZ and increasing age, body mass index (BMI) and waist circumference (WC) respectively. These correlations were stronger in the male sub-group. • In 25 healthy volunteers, parietal cell density was measured from endoscopic biopsies taken from the macroscopic SCJ, 1cm distal to the SCJ, the gastric body and the gastric antrum. Again, a strong negative correlation was seen between increasing age and parietal cell density at the SCJ. This effect was localised to the SCJ and not seen at the other biopsy sites. • Our findings suggest that salivary nitrite does not alter gastro-oesophageal function, the integrity of the gastro-oesophageal barrier or gastro-oesophageal reflux in healthy volunteers. They confirm distal opening of the LOS after meals. The strong negative correlations between age and both SCJ position relative to the proximal HPZ and parietal cell density support the hypothesis of an ‘acquired’ cardia. The development of cardia mucosa may also be linked to obesity, visceral obesity and male sex. • Future work could examine the carcinogenic effect of salivary nitrite and its luminal chemistry but this would require large scale epidemiological research. Further, larger clinical studies are needed to investigate the role of distal opening of the LOS after meals and to improve our understanding of the gastric cardia. Such studies should focus on the role of obesity and posture.