The ultimate aim of this project is to elucidate the mechanisms by which protein deficiency interferes with vaccine-induced resistance to pulmonary tuberculosis. New tuberculosis vaccines will be tested, ultimately, in malnourished humans. Thus, the focus of this project has never been a more urgent research priority. Previous work with a highly relevant guinea pig model, employing low-dose aerosol exposure to virulent Mycobacterium tuberculosis, has revealed diet-induced defects in trafficking, activation, and interactions of immune cells. The recent acquisition of cDNA clones for guinea pig chemokines (MCP-1, IL- 8, RANTES) and cytokines (IFNgamma, TNFalpha, TGFbeta, and IL- 1beta) provides a unique opportunity to apply molecular biological approaches to test four hypotheses: (a) protein deficiency affects trafficking of immune cells into inflammatory exudates by interfering with the production/function of chemokines; (b) abnormal granuloma formation in protein-deficient guinea pigs results from alterations in the production/function of TNFalpha; (c) failure of immune lymphocytes from protein- deficient guinea pigs to activate macrophages to suppress intracellular M. tuberculosis is due to decreased production/function of paracrine (IFNgamma) or autocrine (IL- 1beta, TNFalpha) cytokine signals; and (d) TGFbeta-mediated suppression of T lymphocytes and/or deactivation of macrophages leads to loss of control of intracellular mycobacteria in protein deficiency. Recombinant proteins and polyclonal antibodies will be produced for each of the chemokines and cytokines. Protein- deficient and well-nourished, BCG-vaccinated or nonvaccinated guinea pigs will be challenged by the pulmonary route and levels of these molecules assessed in freshly isolated or cultured cells by Northern blot and RT-PCR (for mRNA), or by bioassay (TNFalpha, TGFbeta) or ELISA. A tuberculous pleuritis model, previously established in the laboratory, and bronchoalveolar lavage will be used to assess the role of chemokines by instillation of recombinant chemokines or specific anti-chemokine antibodies. The effects of recombinant TNFalpha or anti-TNFalpha on granuloma formation will be determined. TGFbeta activity will be blocked in vivo by the injection of anti-TGFbeta antibodies or recombinant decorin. These experiments will provide important new insights into the contributions of these molecules to loss of tuberculosis vaccine efficacy observed in malnourished subjects.
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