Biochemical and Antibacterial Analysis of Human Wound and Blister Fluid

Abstract

Fluid from a post-operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous acetonitrile containing 1 % trifluoroacetic acid. The pooled supernatants were 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, inhibition zone assay on plates with Bacillus megaterium (anti-Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1 -11,l-15 and 1 -16, intact thymosin p-4, defensins HNPl, 2 and 3, lysozyme and the peptide antibiotic FALL-39 and its precursor(s). The HPLC-separated blister fluid was extremely rich in anti-Bm activity (mainly defensins) and lysozyme. It may also contttin factors not identified before. The plate assays scored 50-fold differences in anti-Bm activities and more than 10-fold differences in lysozyme, factors which together with thymosin could be active in wound healing. It is concluded that analysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further.

The Expression of the Gene Coding for the Antibacterial Peptide LL-37 Is Induced in Human Keratinocytes during Inflammatory Disorders

“…The gene is expressed in bone marrow and testis, and the peptide and its proform have been localized in granulocytes (8,11). In addition, we have detected LL-37 together with several other antibacterial peptides in human wound and blister fluids (12).…”

Transient cutaneous adenoviral gene therapy with human host defense peptide hCAP-18/LL-37 is effective for the treatment of burn wound infections

“…[10][11][12] Extracellular cleavage into a cathelinlike domain of 14.3 kDa and the 37-amino-acid-long C-terminus (LL-37) activates the pro peptide, which by its own has been identified in plasma, airway surface fluid and in wound secretions. 10,11,13 The peptide is induced by pro-inflammatory infectious stimuli and displays direct antimicrobial activity by interaction with the cell membranes of microorganisms. 14 Primarily understood as endogenous antibiotics, HDP obtain increasing importance as multifunctional mediators in the complex responses of innate immunity.…”

Porcine antimicrobial peptides: New prospects for ancient molecules of host defense

“…The broad antimicrobial spectrum and strategic locations of AMPs in leukocytic phagocytes or mucosal epithelial cells provide the most obvious indirect argument for their participation in inflammation and infection. Several AMPs, particularly cathelicidins and defensins, have been found in blisters and wound fluid, some at antimicrobial concentrations [29,101]. Increased levels of AMPs also are associated with animal or human infections [2,133].…”