Natural killer cells (NKC) of a variety of host species appear to eliminate eukaryotic cells, including fungi such as Cryptococcus neoformans (Cn) and Candida albicans (Ca), by both direct cytotoxic effects and/or by indirect immunoregulatory effects on the functional responses of other immune effector cells. However, an understanding of the mechanisms these NKC responses is very incomplete, particularly as it pertains to antifungal effects of human NKC. Because elucidation of such mechanisms may be important in determining the potential relevance of NKC in host defense against opportunistic fungi, the major focus of our efforts will be to determine the direct and/or indirect effects of human NKC against both Cn and Ca and then to determine the mechanisms by which such effects occur. Direct effects against Cn and Ca will be assessed by determining the in vitro growth inhibitory/killing activity of normal and cytokine (IFN-g, IL- 2, TNF, GM-CSF)-activated human peripheral blood NKC against the fungi. Parallel assessments will also be made of the requirement for antibody (by examination of whole nonimmune and immune sera and by specific antibody fractions) in mediating NKC antifungal activity for Cn and Ca. Indirect effects against the fungi will be determined by coincubation of NKC with the critical relevant immune effector known to mediate specific resistance (i.e. monocyte/macrophage for Cn, PMN for Ca) for assessment of additive, synergistic or antagonistic effects. Studies will also be performed to determine whether soluble factors produced in culture supernatants from incubations of NKC and the fungi have effects on these phagocytes as well. Lastly, we shall determine the factors mediating direct and/or indirect NKC antifungal activity by determining (a) mechanisms of target cell interactions (directed degranulation/secretory response; participation of granules/membranes proteins; oxidative metabolism); (b) the requirement for expression of specific NKC markers and receptors in triggering antifungal effects and (c) the biochemical definition of specific patterns of intracellular responses triggered by stimulation of specific events related to changes: in membrane lipid metabolism (arachidonate release, PAF, phospholipase C and/or second messengers); in ion (Na+/K+, H+, Ca++) fluxes and concentrations in individual NKC and distributed in NKC subpopulation; in release of other mediators such as histamine/leucine; in cytoskeletal rearrangements; and in RNA transcription and protein synthesis.
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