Improving The Sensitivity And Predictability Of Testing

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We have been evaluating the effect of immune modulation, accomplished through administration of monoclonal antibodies or genetic manipulation, on allergic responses and susceptibility to infection or tumors. To date we have characterized differential responses to allergen challenge and influenza infection in COX deficient mice and examined the role of depletion of specific T cell populations in pulmonary responses to influenza, tumor cell challenge and parasitic infection. In B6C3F1 mice depletion of CD4 or CD8 positive peripheral blood lymphocytes via the administration of monoclonal antibodies had little effect on the clinical outcome of influenza infection. However, depletion of circulating CD8 positive cells prior to infection significantly ameliorated the inflammatory response to the virus in the lung, inhibiting the recruitment of macrophages and PMN and reducing the release of Lactate dehyrogenase., Cell numbers in bronchioalveolar lavage fluid and measures of pulmonary inflammation were unchanged in mice depleted of CD4 positive cells. Evaluation of cytokine levels in the lavage fluid is ongoing. A similar lack of effects on the clinical outcome of infection was observed following challenge with syngenic tumors or malarial parasites. In the confirmed absence of circulating and splenic T lymphocytes these results were somewhat surprising. To evaluate whether monoclonal antibody administration altered systemic immune responses, the antibody response against sheep erythrocytes was evaluated one day following CD4 or CD8 depletion. Depletion of CD4 positive cells suppressed the antigen-specific IgM antibody forming response by more than 75%, levels comparable to responses following treatment with the potent immunosuppressive agent cyclophosphamide. In contrast, depletion of CD8 positive cells slightly enhanced the antigen-specific antibody response. Additional studies examining systemic responses, depletion of cells responsible for innate immune responses and mechanistic studies to compare responses in animals depleted of specific cell populations and naive animals are in progress. As part of continued efforts to evaluate the potential for exogenous factors to target the immune system, we have been examinig the utility of gene expression profiling as a method to screen for immunomodulation. Exposure to environmental agents can affect a number of adverse immunological outcomes, including changes in the incidence of infectious disease. Diethystilbesterol (DES), Dexamethasone (DEX), Cyclophosphamide (CPS), and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) are immunosuppressive chemicals that can induce similar pathophysiological endpoints in the thymus, however the mechanism of toxicity is different for each compound. We have examined differential gene expression in the spleen and thymus following chemical exposure and correlated these changes with alterations in functional immune endpoints and our knowledge of the known mechanisms of action. RNA from the spleen and thymus has been analyzed using Illumina Sentrix arrays and BeadStudio software. Preliminary data suggest that DES induced the greatest number of gene changes in the spleen, while DEX induced the most changes in the thymus. Many of the differentially expressed genes are known to play a role in apoptosis, host defense, cell growth and differentiation. The majority of gene alterations were unique to a single compound, however a number of genes were similarly altered across compounds. In the spleen, changes common to multiple chemicals include upregulation of IL-18, lymphotoxin B receptor, and colony stimulating factor receptor, and downregulation of RANTES and histocompatibility antigens. In the thymus gene changes common to all compounds included downregulation of the interferon gamma receptor and upregulation of caspase 1 and ApoE. Genomic analysis revealed gene expression changes that may be commonly associated with xenobiotic-induced immune system perturbations. However, distinct gene profiles were also found and may specifically relate to cellular targets and mode of action.