The long term objective of this research is to define in biochemical terms the microbicidal mechanisms of phagocytes in order to better understand the causes of opportunistic infection and to seek new means for its prevention and therapy. With their ability to ingest and kill potential pathogens, human neutrophils (PMN) are key components of host defenses against infection. We recently discovered that human PMN contain three antimicrobial peptides, named HNP-1, HNP-2 and HNP-3. The peptides, collectively referred to as """"""""defensins"""""""", were sequenced. They exhibit homology to a family of six antimicrobial peptides abundant in rabbit granulocytes and/or lung macrophages, the """"""""lysosomal cationic proteins"""""""" of Zeya and Spitznagel.
The specific aims are as follows: a. to determine the antimicrobial spectrum of the human defensins by studying their ability to kill or inactivate aerobic and anaerobic bacteria, yeast-phase fungi and selected viruses. b. to identify and analyse factors that modulate the antimicrobial activity of defensins. c. to examine the interactions of these peptides with other potentially antimicrobial constituents of human neutrophils, including lysozyme, BPI, myeloperoxidase and selected reactive oxygen intermediates. d. to establish the intracellular location of defensins in PMN and assess both their intracellular translocation to phagolysosomes and their extracellular release. e. to ascertain the mechanisms whereby defensins act on microorganisms. f. to develop and apply qualitative and quantitative procedures to measure the defensin content of normal PMN and determine whether PMN defensin deficiency underlies the occurrence of certain, presently unexplained infections in man. The methodology for performing these studies includes cellular fractionation, chromatographic purification and various electrophoretic, immunocytochemical and immunoquantitative (ELISA) techniques. Specific details and examples are provided in the body of the proposal.
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