Idiopathic 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.

An imbalance of the T cell immune response is observed in inflammatory bowel disease. Intestinal microbes have been linked to the disease and the disease process leads to severe mucosal injury and systemic translocation of bacterial products. Aminosalicylates, corticosteroids and immunomodulators reduce these aggressive activities but are associated with potentially serious adverse events. The aim of this work was to investigate the effects of administration of prebiotics and probiotics that modulate the gut microflora and modulate the immune response, in ameliorating severity of colitis. The prebiotic, high amylose maize resistant starch was used at two different concentrations. A number of Bifidobacterium and Lactobacillus strains were used as probiotics. BALB/c mice were administered the prebiotics and probiotics and intrarectally infused with 2.5 mg trinitrobenzene sulfonic acid (TNBS) in 45% ethanol, thereby generating colitis. Mucosal cytokine responses, colonic microbial profiles and disease activity indices were monitored. The 5% concentration of high amylose maize resistant starch delayed progression of TNBS colitis as evidenced by reduced weight loss, lesser tissue damage, abrogation of the expression and synthesis of IFN-?? and upregulation of IL-4 and IL-10. The 30% concentration of high amylose maize resistant starch exacerbated the inflammatory response with an increase in acetic acid, coliforms and endopores in the colonic contents. Three strains of bifidobacteria and 3 strains of lactobacilli were individually screened for their activity against TNBS colitis. Each strain had a distinctive effect on the course of colon inflammation. Lactobacillus fermentum VRI 003 was selected for further study as it provided most protection. The ratio of immunosuppressive cytokines to pro-inflammatory cytokines was restored closer to the normal T cell cytokine levels. It also reduced the incidence of translocation of enteric bacteria into the spleens. Dosing a minimum daily dose of 6x109 CFU L. fermentum VRI-003 to ulcerative colitis patients in remission and maintained on standard therapy for 6 months prevented the exacerbation of symptoms, including diarrhea and abdominal pain, and improved the patient general well being. It also suppressed production of IFN-?? and sustained IL-10 levels. Moreover, absence of endospores and lower numbers of coliforms were detected in the faeces of UC patients during L. fermentum VRI-003 treatment. In summary, 5% high amylose maize resistant starch and L. fermentum VRI 003 prevented colon inflammation by changing the nature of the T cell immune response and modifying the colonic microflora in the murine model. The clinical evidence supported these findings.

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY), regulates anxiety and is a part of the stress response. The NPY system also modulates immune functions such as cytokine release, cell migration, and innate immune cell activity. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five receptors (Y1, Y2, Y4, Y5, and y6). Additionally, immune cells themselves release NPY following activation. Previous studies have shown that Y1 mediates NPY-immune effects and data presented here shows expression of Y1 on a wide range of immune cells. Results presented in this thesis, using Y1-deficient mice (Y1-/-), have uncovered a novel role for Y1 on immune cells. NPY acts endogenously to inhibit T cell activation whereas Y1-/- T cells are hyper-responsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signalling through the Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, in Y1-/- mice, T cell differentiation to Th1 T cells appears to be defective as these mice were resistant to T helper type 1 (Th1) cell???mediated inflammatory responses and showed reduced levels of the Th1 cell???promoting cytokine interleukin 12 and reduced interferon ?? production. This defect was due to functionally impaired antigen presenting cells (APCs). Y1-deficient APCs are defective in their ability to produce Th1-promoting cytokines and present antigens to T cells and consequently, Y1-/- mice had reduced numbers of effector T cells. Key reciprocal bone marrow chimera experiments indicated that this effect is intrinsic to immune cells and not driven by other Y1-expressing cell types. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. The findings presented in this thesis uncover a sophisticated molecular mechanism regulating immune cell functions and thus adds to a growing number of signalling pathways shared by the immune and nervous system.

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY), regulates anxiety and is a part of the stress response. The NPY system also modulates immune functions such as cytokine release, cell migration, and innate immune cell activity. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five receptors (Y1, Y2, Y4, Y5, and y6). Additionally, immune cells themselves release NPY following activation. Previous studies have shown that Y1 mediates NPY-immune effects and data presented here shows expression of Y1 on a wide range of immune cells. Results presented in this thesis, using Y1-deficient mice (Y1-/-), have uncovered a novel role for Y1 on immune cells. NPY acts endogenously to inhibit T cell activation whereas Y1-/- T cells are hyper-responsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signalling through the Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, in Y1-/- mice, T cell differentiation to Th1 T cells appears to be defective as these mice were resistant to T helper type 1 (Th1) cell???mediated inflammatory responses and showed reduced levels of the Th1 cell???promoting cytokine interleukin 12 and reduced interferon ?? production. This defect was due to functionally impaired antigen presenting cells (APCs). Y1-deficient APCs are defective in their ability to produce Th1-promoting cytokines and present antigens to T cells and consequently, Y1-/- mice had reduced numbers of effector T cells. Key reciprocal bone marrow chimera experiments indicated that this effect is intrinsic to immune cells and not driven by other Y1-expressing cell types. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. The findings presented in this thesis uncover a sophisticated molecular mechanism regulating immune cell functions and thus adds to a growing number of signalling pathways shared by the immune and nervous system.

Title page, table of contents and summary only. The complete thesis in print form is available from the University of Adelaide Library. / Herpes simplex virus type I (HSV-I) produces acute muco-cutaneous infections, followed by spread to sensory nerve ganglia, and establishment of latency. In the peripheral nervous system, primary sensory neurons, which are found in dorsal root ganglia of the of the spinal nerves, are the target for HSV and they may undergo either productive or latent intection. Productive infection of sensory neurons generates the potential for lethal spread of virus through the nervous system but in immunocom petent hosts, viral replication is terminated by limely development of an adaptive immune response. The infection of dorsal root ganglia that follows cutaneous inoculation of the flanks of mice with HSV provides a well-characterized model of peripheral nervous system infection. The mechanisms responsible for clearance of HSV are complex. At mucosal and cutaneous sites, local innate immune mechanisms act to interrupt the initial spread of virus to the nervous system, while adaptive immunity is important in limiting replication in the ganglia and extension of the virus to adjacent dennatomes. Thus actions of both the innate and the adaptive immune systems are vital in defence against replicating HSV-1, while it is thought that latent infection in the ganglia is contained by the surveillance of the adaptive immune system. Natural killer T (NKT) cells are a conserved subpopulation of lymphocytes that recognize glycolipid antigens presented by the invariant MHC class I-like molecule CD1d. Upon activation through their semi-invariant T cell receptor, these cells rapidly release large amounts of immuno-modulating Th1 and Th2 cytokines. NKT cells have, therefore, been implicated in immune responses controlling various diseases, including infection, cancer, and autoimmunity, as well as having an involvement in allo-graft survival. Consideration of the important contributions of innate and adaptive immunity to clearance of HSV prompted this investigation of the role of CD1d and of CD1d-restricted NKT cells in the pathogenesis of HSV infection. The first part of this thesis (Chapter 3 and 4) describes investigations into the role of NKT cells in immunity to HSV-1, using a zosteriform model of infection and two gene knockout strains of C57BL/6 mice. CD1d GKO and Ja18 GKO mice, which are deficient in NKT cells, are compromised in controlling HSV-1 as evidenced by mortality, virus loads in skin and dorsal root ganglia, presence and size of skin lesions, persistence of HSV antigen, neuronal damage and extent of latency. Comparisons between wild type (NKT cell replete), Ja18 GKO (deficient in invariant Va14⁺ NKT cells) and CD1d GKO (deficient in all CD1d-dependant NKT cells) mice allowed assessment of CD1d-dependant NKT cell subsets in defence against the virus at various stages of infection. It was concluded that both subsets play important roles in controlling the virus and in preventing lethal neuro-invasive disease, that both are vital adjuncts to the adaptive immune response and that without them, low doses of neuropathogenic HSV-1 can establish quickly and cause fatal infections. The NKT-cell population appears to be quite dynamic in its response to a range of pathogens and other disease processes. The study described in Chapter 5 presents evidence suggesting that the response of NKT cells during HSV infection is no less dynamic. In the axillary lymph nodes, observations on numbers of cells expressing NK1.1 antigen and the invariant TCR suggest that NKT cells are activated in the regional lymph nodes draining the infection site. Observations on lymphocytes prepared from liver and spleen also suggested activation of NKT cells, indicating that NKT cells at these sites are also activated during the course of acute HSV infection. The role of NKT cells in the control of HSV infection was further examined by adoptive transfer studies, to investigate whether the defect in handling of HSV-1 by Ja18 GKO mice could be complemented by the adoptive transfer of lymphocytes from wt mice (Chapter 6). Finally, the relevance of activated NKT cells in the anti-HSV response was examined by observing the effects of a-GalactosylCeramide therapy on the severity of HSV-1 infection (Chapter 6). Activation of NKT cells by this compound delayed the onset of HSV disease, decreased prevalence and severity of zosteriform lesions and reduced viral titres in skin and ganglia. The beneficial effects of a-GalactosylCeramide on the outcome and severity of HSV infection in the skin were dose-dependent. Collectively, the studies described in this thesis provide insights into how NKT cells, normally a rare population of cells, has the ability to regulate the protective immune response to HSV-1. As more understanding is gained about how NKT cells become activated during HSV-1 infection, and how they mediate their antiviral effects, other ways may be developed to modulate and activate this interesting subset to the benefit of infected individuals. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1277283 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007

Title page, table of contents and summary only. The complete thesis in print form is available from the University of Adelaide Library. / Herpes simplex virus type I (HSV-I) produces acute muco-cutaneous infections, followed by spread to sensory nerve ganglia, and establishment of latency. In the peripheral nervous system, primary sensory neurons, which are found in dorsal root ganglia of the of the spinal nerves, are the target for HSV and they may undergo either productive or latent intection. Productive infection of sensory neurons generates the potential for lethal spread of virus through the nervous system but in immunocom petent hosts, viral replication is terminated by limely development of an adaptive immune response. The infection of dorsal root ganglia that follows cutaneous inoculation of the flanks of mice with HSV provides a well-characterized model of peripheral nervous system infection. The mechanisms responsible for clearance of HSV are complex. At mucosal and cutaneous sites, local innate immune mechanisms act to interrupt the initial spread of virus to the nervous system, while adaptive immunity is important in limiting replication in the ganglia and extension of the virus to adjacent dennatomes. Thus actions of both the innate and the adaptive immune systems are vital in defence against replicating HSV-1, while it is thought that latent infection in the ganglia is contained by the surveillance of the adaptive immune system. Natural killer T (NKT) cells are a conserved subpopulation of lymphocytes that recognize glycolipid antigens presented by the invariant MHC class I-like molecule CD1d. Upon activation through their semi-invariant T cell receptor, these cells rapidly release large amounts of immuno-modulating Th1 and Th2 cytokines. NKT cells have, therefore, been implicated in immune responses controlling various diseases, including infection, cancer, and autoimmunity, as well as having an involvement in allo-graft survival. Consideration of the important contributions of innate and adaptive immunity to clearance of HSV prompted this investigation of the role of CD1d and of CD1d-restricted NKT cells in the pathogenesis of HSV infection. The first part of this thesis (Chapter 3 and 4) describes investigations into the role of NKT cells in immunity to HSV-1, using a zosteriform model of infection and two gene knockout strains of C57BL/6 mice. CD1d GKO and Ja18 GKO mice, which are deficient in NKT cells, are compromised in controlling HSV-1 as evidenced by mortality, virus loads in skin and dorsal root ganglia, presence and size of skin lesions, persistence of HSV antigen, neuronal damage and extent of latency. Comparisons between wild type (NKT cell replete), Ja18 GKO (deficient in invariant Va14⁺ NKT cells) and CD1d GKO (deficient in all CD1d-dependant NKT cells) mice allowed assessment of CD1d-dependant NKT cell subsets in defence against the virus at various stages of infection. It was concluded that both subsets play important roles in controlling the virus and in preventing lethal neuro-invasive disease, that both are vital adjuncts to the adaptive immune response and that without them, low doses of neuropathogenic HSV-1 can establish quickly and cause fatal infections. The NKT-cell population appears to be quite dynamic in its response to a range of pathogens and other disease processes. The study described in Chapter 5 presents evidence suggesting that the response of NKT cells during HSV infection is no less dynamic. In the axillary lymph nodes, observations on numbers of cells expressing NK1.1 antigen and the invariant TCR suggest that NKT cells are activated in the regional lymph nodes draining the infection site. Observations on lymphocytes prepared from liver and spleen also suggested activation of NKT cells, indicating that NKT cells at these sites are also activated during the course of acute HSV infection. The role of NKT cells in the control of HSV infection was further examined by adoptive transfer studies, to investigate whether the defect in handling of HSV-1 by Ja18 GKO mice could be complemented by the adoptive transfer of lymphocytes from wt mice (Chapter 6). Finally, the relevance of activated NKT cells in the anti-HSV response was examined by observing the effects of a-GalactosylCeramide therapy on the severity of HSV-1 infection (Chapter 6). Activation of NKT cells by this compound delayed the onset of HSV disease, decreased prevalence and severity of zosteriform lesions and reduced viral titres in skin and ganglia. The beneficial effects of a-GalactosylCeramide on the outcome and severity of HSV infection in the skin were dose-dependent. Collectively, the studies described in this thesis provide insights into how NKT cells, normally a rare population of cells, has the ability to regulate the protective immune response to HSV-1. As more understanding is gained about how NKT cells become activated during HSV-1 infection, and how they mediate their antiviral effects, other ways may be developed to modulate and activate this interesting subset to the benefit of infected individuals. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1277283 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 194-230) Also available in print.

Thesis (Ph. D. in Microbiology and Immunology)--Vanderbilt University, May 2009. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D.)--University of Western Australia, 2007.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 117-147) Also available in print.