Cell mediated immunity (CMI) appears to be crucial in the host response to the dimorphic fungus, Blastomyces dermatitidis. However, patients with defects in CMI do not experience more frequent or serious infection with this organism. Likewise, patients with neuropenia are not at increased risk for developing rapidly progressive, difficult to treat blastomycosis. Because the signal transduction mechanisms culminating in human monocyte/macrophage antifungal activity have yet to be studied, these cells are found in blastomycotic lesions, and have been shown to kill B. dermatitidis conidia and yeasts, we propose to study the biochemical pathways triggered by incubation of human monocytes and monocyte derived macrophages (Mphi) with B. dermatitidis conidia and yeasts.
Our Specific Aims are to: (1) Define specific effects of duration of growth (differentiation) and culture conditions (e.g., presence or absence of cytokines) on killing of B. dermatitidis conidia and yeasts. (2) Define fungal and effector cell surface factors required for mediating (a) attachment of conidia and yeasts and (b) fungicidal or fungistatic activity. These factors include: (i) identification of relative proportions of specific complement components (C3b, C3bi) present after serum opsonization of the conidia and yeasts in the presence and absence of antibodies and their relative importance for generation of fungicidal activity and (ii) identification of alternative surface receptors (e.g., Fc, CR1, CR3, lectins) that may be important for generation of monocyte/Mphi antifungal activity following binding to fungal ligands. (3) Correlate antifungal activity itself and the major cellular processes mediating it (oxidant generation and degranulation) with specific early signal transduction responses (e.g., ion fluxes [Na+, K+, H+, Ca+2]); activity of the cytoskeleton (e.g., actin polymerization); alteration in membrane composition (e.g., membrane fluidity, phospholipid metabolism, including phosphoinositide turnover, specific arachidonic acid products, generation of platelet activating factor or other lipid mediators, diacylglycerol generation, etc.); protein phosphorylation activity (e.g., kinase C activation), so as to determine the separate patterns or pathways of effector cell signal-transduction responses triggering killing of conidia and yeasts. The data obtained during the course of these studies will enhance our understanding of biologically relevant responses of human monocytes/macrophages to an important fungal pathogen and will provide pertinent useful information concerning the pathogenesis of blastomycosis.
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