Project description:ObjectivesCommercially produced sterile green bottle fly Lucilia sericata maggots are successfully employed by practitioners worldwide to clean a multitude of chronic necrotic wounds and reduce wound bacterial burdens during maggot debridement therapy (MDT). Secretions from the maggots exhibit antimicrobial activity along with other activities beneficial for wound healing. With the rise of multidrug-resistant bacteria, new approaches to identifying the active compounds responsible for the antimicrobial activity within this treatment are imperative. Therefore, the aim of this study was to use a novel approach to investigate the output of secreted proteins from the maggots under conditions mimicking clinical treatments.MethodscDNA libraries constructed from microdissected salivary glands and whole maggots, respectively, were treated with transposon-assisted signal trapping (TAST), a technique selecting for the identification of secreted proteins. Several putative secreted components of insect immunity were identified, including a defensin named lucifensin, which was produced recombinantly as a Trx-fusion protein in Escherichia coli, purified using immobilized metal affinity chromatography and reverse-phase HPLC, and tested in vitro against Gram-positive and Gram-negative bacterial strains.ResultsLucifensin was active against Staphylococcus carnosus, Streptococcus pyogenes and Streptococcus pneumoniae (MIC 2 mg/L), as well as Staphylococcus aureus (MIC 16 mg/L). The peptide did not show antimicrobial activity towards Gram-negative bacteria. The MIC of lucifensin for the methicillin-resistant S. aureus and glycopeptide-intermediate S. aureus isolates tested ranged from 8 to >128 mg/L.ConclusionsThe TAST results did not reveal any highly secreted compounds with putative antimicrobial activity, implying an alternative antimicrobial activity of MDT. Lucifensin showed antimicrobial activities comparable to other defensins and could have potential as a future drug candidate scaffold, for redesign for other applications besides the topical treatment of infected wounds.

Project description:BackgroundDiabetes and its concurrent complications impact a significant proportion of the population of the US and create a large financial burden on the American health care system. FDA-approved maggot debridement therapy (MDT), the application of sterile laboratory-reared Lucilia sericata (green bottle fly) larvae to wounds, is a cost-effective and successful treatment for diabetic foot ulcers and other medical conditions. Human platelet derived growth factor-BB (PDGF-BB) is a secreted dimeric peptide growth factor that binds the PDGF receptor. PDGF-BB stimulates cell proliferation and survival, promotes wound healing, and has been investigated as a possible topical treatment for non-healing wounds. Genetic engineering has allowed for expression and secretion of human growth factors and other proteins in transgenic insects. Here, we present a novel concept in MDT technology that combines the established benefits of MDT with the power of genetic engineering to promote healing. The focus of this study is to create and characterize strains of transgenic L. sericata that express and secrete PDGF-BB at detectable levels in adult hemolymph, whole larval lysate, and maggot excretions/ secretions (ES), with potential for clinical utility in wound healing.ResultsWe have engineered and confirmed transgene insertion in several strains of L. sericata that express human PDGF-BB. Using a heat-inducible promoter to control the pdgf-b gene, pdgf-b mRNA was detected via semi-quantitative PCR upon heat shock. PDGF-BB protein was also detectable in larval lysates and adult hemolymph but not larval ES. An alternative, tetracycline-repressible pdgf-b system mediated expression of pdgf-b mRNA when maggots were raised on diet that lacked tetracycline. Further, PDGF-BB protein was readily detected in whole larval lysate as well as larval ES.ConclusionsHere we show robust, inducible expression and production of human PDGF-BB protein from two conditional expression systems in transgenic L. sericata larvae. The tetracycline-repressible system appears to be the most promising as PDGF-BB protein was detectable in larval ES following induction. Our system could potentially be used to deliver a variety of growth factors and anti-microbial peptides to the wound environment with the aim of enhancing wound healing, thereby improving patient outcome in a cost-effective manner.

Project description:Enzymatic debridement (ED) has become a reliable tool for eschar removal. Although ED application is simple, wound bed evaluation and therapy decision post-intervention are prone to subjectivity and failure. Experience in ED might be the key, but this has not been proven yet. The aim of this study was to assess interrater reliability (IR) in post-intervention wound bed evaluation and therapy decision as well as the impact of experience. In addition, the authors introduce video assessment as a valuable tool for post-ED decision-making and education. A video-based survey was conducted among physicians with various experiences in ED. The survey involved multiple-choice and 5-point Likert scale questions about professional status, experience in ED, confidence in post-ED wound bed evaluation, and therapy decision. Subsequently, videos of 15 mixed pattern to full-thickness burns immediately after removal of the enzyme complex were demonstrated. Participants were asked for evaluation of each burn wound, including bleeding pattern and consequent therapy decision. IR ≥ 80% was considered as a consensus. Responses were stratified according to participants' experience in applying ED (<10, 10-19, 20-49, and ≥50 applications). IR was assessed by chi-square test (raw agreement [RA]; ≥80% was considered as a consensus) and by calculation of Krippendorff's alpha. In addition, expert consensus for therapy decision was compared with the actual clinical course of each shown patient. Last, participants were asked for their opinion on video as an assessment tool for post-ED wound bed evaluation, decision-making, and training. Thirty-one physicians from 11 burn centers participated in the survey. The overall consensus (raw agreement [RA] ≥ 80%) in post-ED wound bed evaluation and therapy decision was achieved in 20 and 40%, respectively. Krippendorff's alpha is given by 0.32 (95% confidence interval: 0.15, 0.49) and 0.31 (95% confidence interval: 0.16, 0.47), respectively. Subgroup analysis revealed that physicians with high experience in ED achieved significantly more consensus in post-intervention wound bed evaluation and therapy decision compared with physicians with moderate experience (60 vs 13.3%; P = .02 and 86.7 vs 33.3%; P = .04, respectively). Video analysis was considered a feasible (90.3%) and beneficial (93.5%) tool for post-intervention wound bed evaluation and therapy decision as well as useful for training purposes (100%). Reliability of wound bed evaluation and therapy decision after ED depends on the experience of the rating physician. Video analysis is deemed to be a valuable tool for ED evaluation, decision-making, and user training.

Dataset Information

Maggot therapy for calciphylaxis wound debridement complicated by bleeding.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets