Opportunistic mycoses continue to increase in frequency and importance. In 3 of the most common of these, candidiasis, aspergillosis, and mucormycosis, hyphae must be cleared from lesions, primarily by neutrophils (PMN), to prevent progressive disseminated infections. These studies focus upon a stepwise analysis of interactions between fungal hyphae and PMN that ultimately result in killing of the organisms. Initially, experiments will concentrate on interactions os PMN with Candida albicans, with additional studies using Aspergillus fumigatus and Rhizopus oryzae for specific aspects likely to differ, if time permits. Opsonized and unopsonized hyphae elicit different patterns of early PMN responses leading to activation of the PMN respiratory burst that is required for hyphal killing. Previous data provide initial clues to the identity of apparently separate PMN binding and activation sites as well as interacting ligands on opsonized and unopsonized hyphae. These will be further pursued using specific MAbs combined with hyphae selectively opsonized with individual serum factors reactive with PMN receptors. Activation of specific receptors will be linked to initiation of early PMN ion fluxes and biochemical events to determine events and pathways necessary and sufficient for activation of the respiratory burst and degranulation. Recent data imply that both are required for fungicidal effects. Identification of pathways for initial PMN activation will continue, including studies of temporal involvement of specific phospholipases, phospholipids, and possible endogenous mediators derived from arachidonic acid-containing or other lipids. Hyphal killing by PMN is a relatively slow process, not attributable to the almost immediate lethal effects of cell-free oxidants. To define the mechanisms and loci of lethal hyphal damage by PMN, a combined approach will employ both intact PMN and granule-depleted cytoplast with added granule constituents separately or in combination (e.g., myeloperoxidase, defensins, cathepsin G, lactoferrin, lysozyme and elastase). In addition to processes initiating PMN responses to hyphae, we will begin to identify and study factors modulating the sustained PMN activation response required for killing. To define effects of PMN on the target organisms, temporal biochemical and visual changes in multiple localized fluorescent markers loaded into live hyphae will be correlated with localized biochemical indicators of damage, PMN oxidant and granule release, and their coincidence with the onset of killing.
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