Infections due to the encapsulated fungus Cryptococcus neoformans are significant causes of morbidity and mortality in patients with impaired host defenses. Phagocytes, especially macrophages, presumably play important roles in containing this ubiquitous yeast in both the early and late stages of infection. However, the condition under which phagocytes bind, phagocytose, and eventually inhibit or kill C. neoformans are incompletely understood. This grant proposes to focus on several specific aspects of the host phagocyte response to C. neoformans: (1) Opsonic receptors on phagocytic cells that mediate binding of C. neoformans will be characterized by quantitating binding of organisms selectively opsonized with complement or immunoglobulins under conditions known to disable specific phagocytic receptors (e.g., treatment with anti-receptor monoclonal antibodies, proteases or divalent cation chelators). Moreover, the modulation of receptor-ligand binding by selected cytokines (e.g., TNF, IFN-gamma, GM-CSF) and extracellular matrix proteins (e.g., fibronectin, collagen) will be explored. (2) The type and sequence of biochemical events that follow specific receptor-ligand interactions will be studied. Early events to be measured include generation of specific bioactive phospholipid products (such as phosphoinositides, diacylglycerol, platelet activating factor, and arachidonate metabolites including prostaglandins and leukotrienes), kinase activation (protein kinase C translocation) and cytosolic calcium fluxes. Later events to be studied include generation of respiratory burst products (e.g. superoxide anion and H2O2), and lysosomal enzyme release. Heterogeneity of response to many of these events among individual phagocytes and phagocyte subpopulations will be determined using FACS and image analysis techniques. (3) Triggering of these biochemical events will be correlated with the ability of the phagocyte to contain the organism by mounting functional responses such as phagocytosis, fungistasis and fungicidal activity. The role cryptococcal capsule plays as a virulence factor will be explored by comparison of encapsulated strains with mutant strains that lack capsule. Emphasis in these studies will be on human macrophage populations (both culture-derived and bronchoalveolar), however, monocytes and neutrophils will also be studied.
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