Long term objectives. To define antimicrobial molecules involved in innate immunity, learn how they work, and use this knowledge to create novel ways to prevent and treat infections.
Specific aims. 1). To purify human hCAP-18 from leukocytes and secretions and examine its properties. 2) To learn how hCAP-18 and its murine homologue are processed by leukocytes. 3) To study the structures of human LL-37, murine CRAMP-38, and high molecular weight forms of hCAP-18 from plasma and secretions. 4) To define the minimal peptide structure required for LPS-binding and antimicrobial activity against P. aeruginosa. 5) To examine the effects of LPS and other stimuli on in vitro expression of hCAP-18 and examine in vivo synthesis of CRAMP in LPS-treated mice. 6) To measure hCAP-18 levels in secretions, and examine its interactions with other host defense peptides. Methods. These will include peptide synthesis, protein purification by preparative electrophoresis and chromatography (gel permeation, ion exchange, RP-HPLC and affinity), computer modeling, CD and FTIR measurements, antimicrobial testing and Northern and RT-PCR analyses. Health relatedness. The innate immune system is a key element of mucosal immunity that plays a major role in preventing infection. The proposal centers on two newly discovered, pro-antibiotics (""""""""cathelicidins""""""""): human hCAP-18 and its murine homologue, CRAMP. These peptides are expressed constitutively by leukocytes, are produced by epithelial cells, and are found in secretions such as human milk, tears and semen. Their C-terminal domains bind LPS avidly and have potent activity against P. aeruginosa and other pathogens, even under high salt condition. The experiments will expand our knowledge about these important peptides, and should provide the information needed to develop them as antibiotics for topical bronchopulmonary use in cystic fibrosis.
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