BIOCHEMICAL AND ANTIBACTERIAL ANALYSIS OF HUMAN WOUND AND BLISTER FLUID

Fluid from a post-operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immuno logical techniques. The results were compared with those from sera. Al l samples were lyophilized and extracted twice with 60% aqueous aceton itrile containing 1% trifluoroacetic acid. The pooled supernatants wer e lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the blister exudate only with three): reverse-phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, in hibition zone assay on plates with Bacillus megaterium (anti-Bm activi ty) and zone clearing on plates with cell walls from Micrococcus luteu s (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1-11, 1-1 5 and 1-16, intact thymosin beta-4, defensins HNP1, 2 and 3, lysozyme and the peptide antibiotic FALL-39 and its precursor(s). The HPLC-sepa rated blister fluid was extremely rich in anti-Bm activity (mainly def ensins) and lysozyme. It may also contain factors not identified befor e. The plate assays scored 50-fold differences in anti-Bm activities a nd more than 10-fold differences in lysozyme, factors which together w ith thymosin could be active in wound healing. It is concluded that an alysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that ca n be useful to understand molecular mechanisms of wound healing furthe r.