Candida albicans is the most common fungal pathogen causing life- threatening disease in immunocompromised patients. Data suggest that the neutrophil (PMN) is the immune cell most important for defense against invasive candidiasis. The complex interactions between PMN and C. albicans that lead either to control of infection or development of invasive disease are incompletely understood. A Candida hyphal inhibitory product (CHIP) has been identified which may play an important role in host-pathogen interactions. Released by hyphal-phase C. albicans, CHIP profoundly inhibits PMN fungicidal responses, perhaps by selective blockade of PMN signal transduction pathways. This grant will focus on two goals that may help define the importance of CHIP in fungal pathogenesis: (1) The identity of CHIP will be explored. A systematic scheme of purification will be pursued, guided by previous data about the biochemical nature of CHIP. The semi-purified reagent currently available will be further analyzed, followed by the use of HPLC for significantly enhanced purification. Appropriate physicochemical studies such as mass spectrometry, amino acids analysis, and lipid/carbohydrate assays will guide purification. The success of each step in these efforts will be assessed by comparing the activity of new reagents with standard CHIP preparations. (2) The mechanisms by which CHIP inhibits fungicidal responses will be further analyzed. This broad goal has several objectives which, though related, will develop important data about different aspects of PMN-pathogen interaction. (a.) The effect of CHIP on PMN functional responses (chemotaxis, O-2 release, and degranulation) stimulated by potential immune modulators such as LTB-4, PAF, C5a, and immune complexes will be studied. These agents may be present at sites of infection and might modify PMN responses. (b.) To define site(s) of CHIP inhibition, analyses will be conducted of biochemical events that follow PMN activation by multiple stimuli, including Candida. CHIP effects on generation of lipid products (e.g. phosphoinositides, phospholipids, diacylglycerol, PAF, and arachidonate metabolites), protein kinase C activation, and cytosolic Ca++ will be studied. (c.) The complex interactions between fungus and phagocyte will be analyzed. Single cell digital imaging will allow direct observation of regional distribution of activation signals (e.g. Ca++, pH). Selective opsonization will be used to identify the receptor pathways blocked by CHIP. (d.) The ability of CHIP-treated PMNs to respond to selected cytokines (e.g. IFN-gamma, TNF, GM-CSF) will be explored.
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