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(2007)ThesisIdiopathic Membranous nephropathy (MN) is a common cause of nephrotic syndrome in humans, and many patients progress to end-stage kidney disease. The best available animal model of MN is active Heymann nephritis (HN) in which rats are immunized with renal tubular antigen (RTA) in complete Freund's adjuvant (CFA). Rats develop heavy proteinuria, a key measure of glomerular damage, and the disease is histologically identical to human MN. It has been thought that HN is mediated by antibody-based mechanisms. More recent evidence demonstrates a critical role for cytotoxic T cells. This thesis aims to further examine the role of T cell responses in active HN. First, the effect of the anti-CD3 monocIonal antibody (mAb) G4.18 was investigated. Anti-CD3 given 4 weeks after immunization prevented the development of proteinuria, delayed anti-RTA antibody responses, and reduced glomerular infiltration of CD8+ T cells and macrophages, but did not affect glomerular deposition of IgG or complement. Increased mRNA expression of the Th2 cytokines IL-4 and IL-5 was detected in draining lymph nodes. These findings suggest that immune deviation to a Th2 response reduces glomerular injury in HN. Second, the role of CD4+ T cells in immune tolerance was examined. Rats were given RTA in incomplete Freund's adjnvant (lFA) to induce tolerance to RTA, and three weeks later were immunized with RTA in CFA. Anti-CD4 mAb therapy at the time of RTA1IFA treatment had no effect on subsequent proteinuria or anti-RTA autibodies. Third, the role of IL-4 in this model of immune tolerance was examined. Anti-IL-4 mAb therapy blocked the induction of tolerance, and led to the development of proteinuria. Finally, the effect of treatment with IL-4 and IL-5 was examined. Treatment with these cytokines separately or together after immunization blocked the development of proteinuria, without a consistent effect on anti-RTA antibodies. These results demonstrate a central role for T cell regulation in HN, and show that immune deviation to a Th2 response is protective against glomerular injury. The findings may have implications in the future for focused therapeutic intervention in human idiopathic MN.
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(2008)ThesisThe complement system is one of the most ancient immune defense mechanisms, providing rapid protection against invading micro-organisms. It is essential for the complement cascade to be under tight regulation in order to prevent excessive production of complement proteins. C5a is the most potent anaphylotoxin produced by the complement system, it binds C5a receptor (C5aR, CD88) and C5L2 (GPR77). C5a binding to C5aR induces leukocyte chemotaxis and release of inflammatory mediators. Over-production of C5a is known to be involved in many inflammatory and pathological conditions such as RA, I1R injury and sepsis, making it an attractive therapeutic target. Human and mouse C5aR share low homology and blocking C5a/C5aR signaling with small molecules has been challenging. We generated human C5aR knockout/knockin (hC5aR KI) mice in which the mouse C5aR coding region was replaced with that of human C5aR to utilize them for the development of human therapeutics targeting C5aR. hC5aR KI mice showed normal development, and leukocytes from hC5aR KI mice responded well to mouse C5a. We used two approaches to generate monoclonal antibodies (mAbs) against hC5aR. We used a mouse cell line transfected with hC5aR or neutrophils from hC5aR KI mice to immunize wild-type mice and generated high-affinity antagonistic mAbs which are specific to human C5aR. Anti-hC5aR mAb 7F3 blocked C5a-induced signaling completely without agonistic activity in vitro. In the animal model of K/BxN inflammatory arthritis, 7F3 both prevented and reversed inflammation. Currently, the function of the second C5a receptor, C5L2, remains controversial. There are contradicting reports from C5L2 KO mice that were generated by independent groups. We assessed the function of human C5L2 using an antagonistic mAb that specifically blocks C5L2 function and not C5aR. In vitro analysis using the C5L2-blocking mAb showed that C5a does not signal via C5L2 to affect chemotaxis or phagocytosis by neutrophils, indicating that C5L2 is not a signaling receptor for C5a, at least in these cellular functions.
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(2008)ThesisScleroderma is a chronic autoimmune connective tissue disease of unknown etiology characterized by excessive fibrosis and is broadly divided into two clinical entities: localized scleroderma (LSc) and systemic sclerosis (SSc). SSc is a multi-system disease resulting in both skin and visceral organ fibrosis. The more benign disorder, LSc is for the most part self-limited with the disease pathology being confined to the skin and subcutaneous tissues. Proposed factors involved in the pathogenesis of these disorders include endothelial cell injury and dysfunction, immunological alterations and inflammatory activation, and abnormal ECM production by activated fibroblasts. However, the initiating mechanisms that leads to these changes remains largely unknown. This thesis examines the hypothesis that the transcriptional expression at the edge and centre of expanding LSc plaques could represent the metabolic changes involved in the different stages of disease. The major finding of this thesis was the identification of two panels of genes that showed significant changes in expression between LSc patients and healthy controls irrespective of whether the sample was taken from a diseased or clinically unaffected area of the patient. The first panel consisted of inflammatory genes including those genes characteristic of the Thl response and those induced by NF-KB. The Thl response was supported by an increased infiltration of CD4+ T cells in the LSc patients. The second panel consisted of a subset of array identified genes (scleroderma Signature) in SSc patients. Of interest, WIF1 was down regulated in both disorders and showed a gradual decrease in expression across the clinically different areas of the LSc patients. Both panels of genes showed the biggest changes of expression at the edge of the plaque suggesting their involvement in the initiating events of the disease. These results suggest that, like SSc, the underlying pathology of LSc is related to systemic changes in genes controlling amongst others, immunological and inflammatory responses. This information not only sheds light on the mechanisms involved in the initiation and progression of scleroderma, but could also contribute to the creation of a diagnostic test for the early detection of sufferers of this rare, but important disease.
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(2008)ThesisType 2 diabetes accounts for 85-90% of all people with diabetes and is currently estimated to affect more than 180 million people worldwide, a figure estimated to double by the year 2030. Thus understanding the basic biology of glucose homeostasis and how it is altered during disease progression is crucial to the development of safe and effective treatment regimes. The link between high dietary fat and the development of type Il diabetes is well established. Chronic treatment of pancreatic islets with the lipid palmitate induces defects in glucose stimulated insulin secretion (GSIS) akin to those seen in the development of type Il diabetes. Previous studies from our group have identified the lipid-activated kinase protein kinase C epsilon (PKCε) as a potential mediator of some of these effects. Deletion of PKCε in mice results in complete protection from high-fat diet induced glucose intolerance. This protection is associated with enhanced circulating insulin suggesting that PKCε may be involved in the regulation of insulin release from the pancreatic β-Cell. The data presented here suggests that PKCs plays an important role in the regulation of insulin secretion under both physiological and pathophysiological conditions. We demonstrate that PKCε can be activated by chronic lipid treatment and acute cholinergic stimulation. Under these conditions insulin secretion is enhanced by PKCε deletion or inhibition suggesting that PKCε is a negative regulator of insulin secretion. Mechanistically the PKCs mediated inhibition of insulin release by acute or chronic PKCε activation appears to be distinct. The effect of PKCε induced by palmitate pre-treatment appears to be distal to calcium influx. The pool of pre-docked vesicles is enhanced in palmitate pre-treated β-cells lacking PKCε suggesting that PKCε may be involved in the regulation of vesicle dynamics. In contrast, calcium dynamics induced by cholinergic stimulation are altered by PKCε deletion, suggesting an effect on either the calcium channels themselves or on the upstream signalling. Given the ability of PKCε to inhibit insulin secretion, inhibition of PKCε in the β-cells of people suffering from insulin resistance and (or) type II diabetes represents a novel target for the treatment of type II diabetes.
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