Beta-defensin 7 antimicrobial peptide, partial

Sepsis is a common and serious complication of major burn injury and accounts for over 54% of deaths in burn patients. Burns are associated with high levels of circulating pro-inflammatory cytokines and immunosuppression, promoting systemic inflammatory response syndrome (SIRS) and sepsis, for which no effective treatment is currently available. Defensins, a family of cationic, naturally occurring, antimicrobial peptides are important components of the innate immune system, playing a major role in the body's defence by inhibiting activities of bacteria, fungi and enveloped viruses. These natural antimicrobials also chemoattract immature dendritic cells, some types of T and B-lymphocytes, neutrophils and macrophages, and act as an adjuvant, enhancing adaptive immunity. Our prior studies suggested a decreased expression of human beta defensin 2 (HBD2) in burn wounds. Here we have identified HBD2 protein in skin samples of partial and full thickness burns and in normal skin using fluorescence deconvolution microscopy. Images showed that in normal skin the majority of HBD2 is located in the Malpighian layer and, in smaller amounts, in the more superficial layers, a pattern that is absent in burned skin in which the epidermis is destroyed or damaged. However, surviving dermal and subcutaneous layers revealed the presence of HBD2 in a number of other cell types and structures, such as hair follicles and sweat gland acini, but not in vascular endothelium and fat cells. The results of these studies further contribute to an understanding of the role of antimicrobial peptides in the pathophysiology of burn injury, associated immunosuppression and sepsis and the possibility of using these other sites of HBD2 deposition for upregulation of antimicrobial synthesis in the treatment of burns.

Binding of melanocortin peptide agonists to the melanocortin-1 receptor of melanocytes results in eumelanin production, whereas binding of the agouti signalling protein inverse agonist results in pheomelanin synthesis. Recently, a novel melanocortin-1 receptor ligand was reported. A β-defensin gene mutation was found to be responsible for black coat colour in domestic dogs. Notably, the human equivalent, β-defensin 3, was found to bind with high affinity to the melanocortin-1 receptor; however, the action of β-defensin as an agonist or antagonist was unknown. Here, we use in vitro assays to show that β-defensin 3 is able to act as a weak partial agonist for cAMP signalling in human embryonic kidney (HEK) cells expressing human melanocortin-1 receptor. β-defensin 3 is also able to activate MAPK signalling in HEK cells stably expressing either wild type or variant melanocortin-1 receptors. We suggest that β-defensin 3 may be a novel melanocortin-1 receptor agonist involved in regulating melanocyte responses in humans.

Antimicrobial midgut proteins and peptides that result from blood digestion in feeding American dog ticks Dermacentor variabilis (Say) were identified. Midgut extracts from these ticks showed antimicrobial activity against Micrococcus luteus, regardless of whether they were challenged with peptidoglycan, blood meal components, rabbit blood, Bacillus subtilis, Escherischia coli or Borrelia burgdorferi. However, no peptide band co-migrating with defensin was found in midgut extracts from the challenged ticks. Partial purification of the midgut extracts using C(18) Sep Paks and gel electrophoresis showed the presence of 4 distinct bands with rMW 4.1, 5.3, 5.7 and 8.0 kDa identified by tryptic digestion-mass fingerprinting as digestive fragments of rabbit alpha-, beta-, gamma-chain hemoglobin, and rabbit ubiquitin. No evidence of varisin, a defensin previously identified in the hemolymph of D. variabilis, was found in the tryptic digest, although varisin was found in a hemocyte lysate using the same methods. However, varisin transcript was detected in midgut cell lysates. Also present in all midgut samples was a cluster of 3 overlapping bands with rMW 13.0, 14.1 and 14.7 kDa which were identified by tryptic-digestion LC-MS and MALDI-TOF as rabbit alpha- and beta-chain hemoglobin (undigested) and transtherytin. Lysozyme transcript was detected in midgut cell extracts but the peptide was not. Studies done on other tick species demonstrated that hemoglobin digestion resulted in antimicrobial fragments. Antimicrobial hemoglobin fragments (including fragments larger than any reported previously) also were found in D. variabilis, as well as ubiquitin, a peptide known to occur as part of an antimicrobial complex in vertebrate leukocytes. In addition, we noted that Borrelia burgdorferi spirochetes were not lysed in the midgut lumen, which would be expected if defensin and lysozyme were active in this location. In this respect, the midgut's response to microbial challenge differs from that of the hemolymph. In summary, the midgut's antimicrobial activity appears to be primarily a byproduct of hemoglobin digestion rather than expression of immune peptides and proteins.