The herpes simplex virus (HSV) and cytomegalovirus (CMV) are the prominent members of the Herpesviridae family collectively responsible for the majority of herpes-related morbidity and mortality in humans. These viruses are therefore the subjects of this study that was undertaken to improve our understanding of the nature of immune response and the mechanism of viral modulation leading to suppression of viral replication or evasion of the host immune response in vivo. The present study has examined the T helper responses to HSV-2 and murine CMV (MCMV) and provides new insights into the nature and the modulation of the T helper responses by these viruses in vivo. B7-1/B7-2 costimulation of T cells by antigen-presenting cells is essential for T cell activation by antigen. HSV-2 infection affects the expression of both B7-1 and B7-2 on monocytes, the key antigen presenting cells in vivo, potentially in two ways with opposing outcomes. It abrogates or diminishes the IFN-gamma-upregulation of B7-1 (in 8 out of 9 patients) and B7-2 (in 6 out of 9 patients) on monocytes. However, the infection also augments the expression of B7-1 and B7-2 on monocytes through an IFN-gamma-independent mechanism (in 9 out of 9 patients). Although the clinical significance of these opposing effects is presently unclear, these may be related to the immunological mechanism or strategy leading to recurrent disease in immunocompetent hosts. Like HSV-2, infections with MCMV in mice also led to a predominant Thl type immune response characterized by high levels of IFN-gamma and low IL-4 production. Studies with specific MCMV mutants, containing Tn3 transposon in the open reading frame of M25, M27, M43, or m09 gene, led to the identification of M43 gene that specifically suppresses IL-4 response. The Tn3 disruption of M43, but not M25, M27 or m09, gene of MCMV led to a strong IL-4 response (p = 0.0002) despite the presence of a dominant IFN-gamma response. These results provide insight into the possible role of a herpesvirus gene that can profoundly modulate the nature of T helper response in vivo. The presence of a homologous genetic element in other herpesvirus genomes may explain the dominant Th1 immune response triggered by HSV-2 and possibly other herpesviruses. The obvious importance of this finding, beyond herpesvirus immunopathogenesis, lies in the ability of M43 and homologous genes to globally modulate the nature of cytokine response in vivo and suppress Th2 cytokine-mediated diseases. (Abstract shortened by UMI.)

Corticotropin-releasing hormone (CRH) has been considered the quintessential 'stress' neuropeptide, as it mediates stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis and produces behavioural and autonomic responses analogous to natural stressors. However, the role of CRH in mediating the behavioural response to psychological stressors, also termed anxiety, is unclear due to inconsistent anxiolytic effects for CRH antagonists in processive stress models, and the central activation of CRH by non-anxiogenic stimuli. The first half of the current thesis examined the role of CRH, and amygdala CRH in particular, in the behavioural response to anxiety. The first experiment standardized the use of a neophobia-based feeding model for use as a behavioural index of anxiety. Using this model, the second experiment demonstrated that while exposure to an unfamiliar environment activates stress-relevant neural circuitry, activation of CRH receptors is not necessary for the expression of anxiety-like behavioral responses. In addition, amygdala CRH activation by novel stimuli is unaffected by changes in the organism's anxiety state. The second half of the current thesis focused on the role of CRH in neurotoxicity and behavioural deficits produced by global ischemic insults. It was demonstrated that global ischemic insults of 10--15 min duration produced short-term CRH increases in hypothalamic and amygdalar regions as well as the piriform cortex, perhaps mediating stress, inflammatory and/or epileptiform activity. This was followed by widespread CRH depletions throughout the brain at 24 h post-ischemia. However, by 72 h post-ischemia, just prior to the onset of cell death, CRH concentrations had normalized throughout the brain. Furthermore, there were no short or long term CRH changes at the CA1 hippocampal region, the area most vulnerable to cell death. Both selective and non-selective CRH antagonists failed to confer neuroprotection when administered before or shortly after an ischemic insult, and failed to protect against ischemia-induced spatial memory deficits. However, CRH1 antagonists did significantly attenuate ischemia-induced hyperactivity in the open field and in the elevated plus maze. Thus while ischemia-induced CRH activation does not appear to mediate subsequent neuronal degeneration, it may play a role in anxiety-related behavioural changes.

Type 1 diabetes (T1D) affects approximately 200,000 Canadians and is increasing at a rate of about 3.0% per year. The development of autoimmune diabetes requires genetic susceptibility and is influenced by environmental agents. Diets in which wheat protein is the sole amino acid source are associated with high diabetes frequency in diabetes-prone BioBreeding (BBdp) rats and non-obese diabetic (NOD) mice, two models of spontaneous autoimmune diabetes. We hypothesized that an abnormal immune response to wheat, as indicated by circulating IgG antibodies, could be used to identify T1D-related wheat proteins. Similar methods have been used to identify the diabetes-related autoantigens. A wheat cDNA expression library was immunoscreened with pooled IgG antibodies from wheat gluten-fed diabetic BBdp rats. Initially, three clones were isolated. When the proteins were screened with sera from individual rats at varying risk of disease, antibody reactivity to clone WP5212, a wheat storage globulin, was strongest in diabetic rats. This antibody reactivity was not only associated with disease but also closely correlated with islet damage. Additional studies revealed that WP5212 antibodies were linked to diabetes in NOD mice and, remarkably, in human T1D patients. Furthermore, patients with both T1D and the wheat sensitive enteropathy, celiac disease (CD), displayed high WP5212 antibody reactivity, possibly representing a marker of the predisposition of T1D patients to develop CD. WP5212 shared homology with a number of dietary proteins and allergens, suggesting that diabetes-promoting structures could exist in other food plants and that immunomodulatory molecules could interact with the immune system by a common mechanism. WP5212 also shared homology with self proteins, which could act as a means for the initiation or promotion of autoimmunity. WP5212 is the first candidate dietary wheat protein implicated in the development of diabetes. Thus, screening a cDNA expression library provides a novel approach that permits the identification of environmental antigens involved in autoimmune diseases. The evidence suggests that WP5212 is strongly antigenic in three separate species that develop spontaneous diabetes. The molecular identity of this immune-targeted dietary wheat protein will help elucidate the pathogenic mechanisms by which wheat-related diabetes occurs.

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