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To examine the pathophysiological consequences of autoimmunity to TAG-1, CD4⁺ T cells and autoantibodies specific for TAG-1 were generated and their potential to induce EAE was investigated in a rat model. An adoptive transfer of T cells specific for the first two immunoglobulin domains of TAG-1 initiates an intense inflammatory response in both the cortex and spinal cord in the Dark Agouti and Lewis rats. This is particularly interesting as conventional models of EAE are of little help in investigating the pathomechanisms responsible for cortical tissue damage, as the cortex is only rarely targeted by myelin-specific autoimmune responses in this animal model of Multiple Sclerosis (MS). Moreover, when a demyelinating antibody like Z2 (anti-MOG) antibody was co-transferred with TAG-1-specific T cells it resulted in demyelination not only in the spinal cord white matter but also the grey matter and triggered demyelination coupled with macrophage infiltration in the perivascular areas of the cortex. This new model of cortical demyelination is exciting as it bears some resemblance with chronic progressive MS. However, two different monoclonal antibodies (4D7/TAG1) and 3.1C12) specific to TAG-1, when co-transferred with TAG-1 specific T cells, failed to mediate tissue damage in the CNS. Although the role of TAG-1-specific antibodies in EAE remained inconclusive from the preliminary experiments, this study clearly demonstrates TAG-1-specific T cells as a new tool to mediate EAE highlighting cortical pathology which can be exploited further to investigate the factors that control regional differences in the pathogenesis of inflammatory demyelinating lesions in the CNS. This new model might provide novel insights into the development of cortical pathology in MS patients and will ultimately guide to design therapeutic approaches in MS.

CD4+ T cells can be subdivided on the basis of their lymphokine repertoire produced on activation resulting in TH1 like and TH2 like populations. The purpose of this study was to measure the intraocular expression of cytokines as a means of defining the CD4+ lymphocyte subsets present during the development of uveoretinitis. Lewis rats were immunised with retinal antigen and pertussis toxin resulting in early signs of disease activity evident by day 9 with increasing severity evident by day 12. Extensive clinical and histological damage was observed by day 14 with a reduction in severity through to end stage disease at day 24. In this study, the failure to establish pure populations of retinal antigen specific T lymphocyte cell lines and the observation of the lack of sensitivity of Northern hybridization to signals expressed at low levels resulted in the more sensitive technique of RT-PCR being utilized. Both IL2 and IFN mRNA expression was detected at all stages of disease with highest levels being present early in uveitis. In contrast IL4 mRNA levels increased with disease progression. This study suggests a pathogenic role for TH1 like cells and a protective role for TH2 like cells in this autoimmune disease. In order to provide an insight into alternative treatment strategies of the disease, immunomodulation of EAU was carried out using the immunosuppressive drugs CsA, FK506 and rapamycin and the resultant cytokine mRNA profiles examined. The results indicated that CsA and FK506 downregulated the TH1 response having suppressive effects on the levels of IL2 and IFN mRNA respectively. In contrast rapamycin was found to modulate the TH2 response enhancing IL4 levels. From this data, a drug based strategy employing rapamycin appears to be the most favourable approach.

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The aims of this project were to investigate the binding characteristics of the cytosolic oestrogen receptor in the uterus, brain and thymus of immature Wistar rats. The specificities of the receptor in the uterus are well established. The specificities of the cytosolic receptor in the uterus and thymus of immature female Wistar rats were tested against a range of steroids and the values found for the thymus compared with those for the uterus. The concentrations and dissociation constant (Kd) of the cytosolic oestrogen receptor were determined in uterus, brain and thymus of male and female rats at 5,18 and 30 days of age. Clomiphene citrate (CC), an oestrogen antagonist/partial agonist, oestradiol (E2), CC+E2 or 4-hydroxyandrostenedione (4-OHA), an aromatase inhibitor, were administered to animals at 15 days in order to study the effects of these compounds on receptor binding characteristics at 30 days. Significant differences in specificity were found between the thymus and uterus, the cytosolic oestrogen receptor in the thymus exhibited significantly higher affinity for corticosterone than it did in the uterus. Male animals were heavier than females at 30 days. Uterus and thymus weights increased exponentially between 5 and 30 days. The tissue-to-body weight ratio increased in uterus between 18 and 30 days and increased in the thymus in both sexes between 5 and 18 days. In males at 30 days, the tissue-to-body weight ratio of the thymus was significantly lower than in females of the same age. Cytosolic oestrogen receptor concentrations in the uterus, brain and thymus differed between some age and/or sex groups. Cytosolic oestrogen receptor concentrations increased exponentially in the uterus between the different age groups. Cytosolic oestrogen receptor concentrations in both thymus and hypothalamus at 5 days were significantly higher in females than in males of the same age group. No differences in cytosolic oestrogen receptor concentrations were found between the sexes in the cortex at 5 and 18 days but at 30 days, receptors were not detectable in this brain area. The Kd for moxestrol, a synthetic oestrogen agonist that is not bound by alphafetoprotein present in the blood of immature rats, was similar in all tissues. E2 and CC+E'-' treatmentsr esulted in decreasedb ody and thymus weight in both sexes,i ncreased uterus weight and decreased thymus weight in both sexes but led to increased uterus weight. CC treatment decreased the concentration of the receptors in the female thymus only-, E2 and CC+E2 treatments decreased the concentratIon of the receptor to levels that were undetectable in hypothalamus and thymus in both sexes, 4-OHA treatment increased thymus weight and cytosolic receptor concentrations in the hypothalamus and thymus of males only. These results suggest that cytosolic oestrogen receptors in uterus, brain and thymus are similar and that sex differences in these tissues are mediated by differential exposure to oestradiol during the early postnatal period. The thymus is crucial to the development of the immune response. The finding that the cytosolic oestrogen receptor differed from the uterus receptor in its affinity for corticosterone and that sex differences in cytosolic oestrogen receptor concentrations were present in the thymus at 5 days could be relevant to the sex dimorphisms that exist in autoirnmune disease manifestation.

Systemic lupus erythematosus (SLE) is a chronic, multi-organ autoimmune disease characterized by the production of antibodies against self nuclear antigens. Genetics play a dominant role in disease pathogenesis and functional examination of spontaneously-arising lupus-prone animal models has provided key insights into understanding the genetic complexity of the disease. The overarching goal of the work presented here is to identify the underlying immunologic abnormalities, together with lupus susceptibility loci that produce them, that promote the development of autoimmunity in the lupus-prone New Zealand Black (NZB) background. Chapter 2 identifies the critical role of CD40-CD40L interactions in the pathogenesis of disease in NZB mice. We showed that this interaction is required for the production of class-switched IgG autoantibodies and development of hemolytic anemia and kidney disease. Polyclonal B cell activation, expansion of plasmacytoid dendritic cells (pDC), and elevated gene expression of baff were maintained in CD40L-deficient NZB mice, despite the lack of IgG immune complexes. Chapter 3 utilizes bicongenic mice carrying both NZB chromosomes 1 and 13 to investigate the genetic complexity in disease pathogenesis. In addition to identifying new phenotypes, examination of bicongenic mice showed that chronic stimulation of pDC due to the persistence of nuclear antigens leads to a refractory state with a failure to produce more IFN-α upon subsequent stimulation. Chapter 4 identifies a novel lupus susceptibility locus on NZB chromosome 13 associated with impaired clearance of apoptotic debris, a key initiating step in the development of autoimmunity. Using subcongenic mice, this locus was localized and examined its impact on immune function. Work from this thesis will contribute to understanding the complex immunogenetic mechanisms that lead to development of SLE.

Systemic lupus erythematosus (SLE) is a chronic, multi-organ autoimmune disease characterized by the production of antibodies against self nuclear antigens. Genetics play a dominant role in disease pathogenesis and functional examination of spontaneously-arising lupus-prone animal models has provided key insights into understanding the genetic complexity of the disease. The overarching goal of the work presented here is to identify the underlying immunologic abnormalities, together with lupus susceptibility loci that produce them, that promote the development of autoimmunity in the lupus-prone New Zealand Black (NZB) background. Chapter 2 identifies the critical role of CD40-CD40L interactions in the pathogenesis of disease in NZB mice. We showed that this interaction is required for the production of class-switched IgG autoantibodies and development of hemolytic anemia and kidney disease. Polyclonal B cell activation, expansion of plasmacytoid dendritic cells (pDC), and elevated gene expression of baff were maintained in CD40L-deficient NZB mice, despite the lack of IgG immune complexes. Chapter 3 utilizes bicongenic mice carrying both NZB chromosomes 1 and 13 to investigate the genetic complexity in disease pathogenesis. In addition to identifying new phenotypes, examination of bicongenic mice showed that chronic stimulation of pDC due to the persistence of nuclear antigens leads to a refractory state with a failure to produce more IFN-α upon subsequent stimulation. Chapter 4 identifies a novel lupus susceptibility locus on NZB chromosome 13 associated with impaired clearance of apoptotic debris, a key initiating step in the development of autoimmunity. Using subcongenic mice, this locus was localized and examined its impact on immune function. Work from this thesis will contribute to understanding the complex immunogenetic mechanisms that lead to development of SLE.