Endotoxin testing of a wound debridement device containing medicinal Lucilia sericata larvae

Alimentary products of medicinal Lucilia sericata larvae are studied to determine their mechanisms of action, particularly in the contexts of wound debridement and disinfection. Furthermore, the larvae can be applied to patients in contained devices, such as the BioBag (BioMonde). Here, we tested the materials and larval content of the most commonly used BioBag (the “BB-50”) to explore the possibility that endotoxins may be contributing to the bio-activity of the product, given that endotoxins are potent stimulants of cellular activation. Using standardised protocols to collect larval alimentary products (LAP), we proceeded to determine residual endotoxin levels in LAP derived from the BioBag, before and after the neutralisation of interfering enzymatic activity. The BB-50 device and its associated larval content was not a significant source of LPS activity. However, it is clear from these experiments that a failure to remove the confounding serine proteinase activity would have resulted in spuriously high and erroneous results. The residual LPS levels detected are unlikely to be active in wound healing assays, following cross-referencing to publications where LPS at much higher levels has been shown to have positive and negative effects on processes associated with wound repair and tissue regeneration

Quality control of a medicinal larval (Lucilia sericata) debridement device based on released gelatinase activity

Lucilia sericata Meigen (Diptera: Calliphoridae) larvae are manufactured worldwide for the treatment of chronic wounds. Published research has confirmed that the primary clinical effect of the product, debridement (the degradation of non-viable wound tissue), is accomplished by a range of enzymes released by the larvae during feeding. The quality assessment of larval activity is currently achieved during production using meat-based assays, which monitor insect growth and/or the reduction in substrate mass. To support this, we have developed a complementary radial-diffusion enzymatic assay (RDEA) to produce a visual and measureable indication of the activity of larval alimentary products (LAP) collected under standardised conditions, against a gelatin substrate. Using basic laboratory equipment and reagents, the assay is rapid and suited to high-throughput. Assay reproducibility is high (SD 0.06 - 0.27, CV 0.75 - 4.31%) and the LAP collection procedure does not adversely affect larval survival (mortality < 2%). As a cost- and time-effective method, it is suited to academic or industrial use, supporting good manufacturing (or laboratory) practice (GMP and GLP) as a quality control (QC) assay

Endotoxin testing of a wound debridement device containing medicinal Lucilia sericata larvae

Alimentary products of medicinal Lucilia sericata larvae are studied to determine their mechanisms of action, particularly in the contexts of wound debridement and disinfection. Furthermore, the larvae can be applied to patients in contained devices, such as the BioBag (BioMonde). Here, we tested the materials and larval content of the most commonly used BioBag (the “BB-50”) to explore the possibility that endotoxins may be contributing to the bio-activity of the product, given that endotoxins are potent stimulants of cellular activation. Using standardised protocols to collect larval alimentary products (LAP), we proceeded to determine residual endotoxin levels in LAP derived from the BioBag, before and after the neutralisation of interfering enzymatic activity. The BB-50 device and its associated larval content was not a significant source of LPS activity. However, it is clear from these experiments that a failure to remove the confounding serine proteinase activity would have resulted in spuriously high and erroneous results. The residual LPS levels detected are unlikely to be active in wound healing assays, following cross-referencing to publications where LPS at much higher levels has been shown to have positive and negative effects on processes associated with wound repair and tissue regeneration

TIME management by medicinal larvae

Wound bed preparation (WBP) is an integral part of the care programme for chronic wounds. The acronym TIME is used in the context of WBP and describes four barriers to healing in chronic wounds; namely, dead Tissue, Infection and inflammation, Moisture imbalance and a non-migrating Edge. Larval debridement therapy (LDT) stems from observations that larvae of the blowfly Lucilia sericata clean wounds of debris. Subsequent clinical studies have proven debriding efficacy, which is likely to occur as a result of enzymatically active alimentary products released by the insect. The antimicrobial, anti-inflammatory and wound healing activities of LDT have also been investigated, predominantly in a pre-clinical context. This review summarises the findings of investigations into the molecular mechanisms of LDT and places these in context with the clinical concept of WBP and TIME. It is clear from these findings that biotherapy with L. sericata conforms with TIME, through the enzymatic removal of dead tissue and its associated biofilm, coupled with the secretion of defined antimicrobial peptides. This biotherapeutic impact on the wound serves to reduce inflammation, with an associated capacity for an indirect effect on moisture imbalance. Furthermore, larval serine proteinases have the capacity to alter fibroblast behaviour in a manner conducive to the formation of granulation tissue

A novel approach of homozygous haplotype sharing identifies candidate genes in autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heritable disorder of complex and heterogeneous aetiology. It is primarily characterized by altered cognitive ability including impaired language and communication skills and fundamental deficits in social reciprocity. Despite some notable successes in neuropsychiatric genetics, overall, the high heritability of ASD (90%) remains poorly explained by common genetic risk variants. However, recent studies suggest that rare genomic variation, in particular copy number variation, may account for a significant proportion of the genetic basis of ASD. We present a large scale analysis to identify candidate genes which may contain low-frequency recessive variation contributing to ASD while taking into account the potential contribution of population differences to the genetic heterogeneity of ASD. Our strategy, homozygous haplotype (HH) mapping, aims to detect homozygous segments of identical haplotype structure that are shared at a higher frequency amongst ASD patients compared to parental controls. The analysis was performed on 1,402 Autism Genome Project trios genotyped for 1 million single nucleotide polymorphisms (SNPs). We identified 25 known and 1,218 novel ASD candidate genes in the discovery analysis including CADM2, ABHD14A, CHRFAM7A, GRIK2, GRM3, EPHA3, FGF10, KCND2, PDZK1, IMMP2L and FOXP2. Furthermore, 10 of the previously reported ASD genes and 300 of the novel candidates identified in the discovery analysis were replicated in an independent sample of 1,182 trios. Our results demonstrate that regions of HH are significantly enriched for previously reported ASD candidate genes and the observed association is independent of gene size (odds ratio 2.10). Our findings highlight the applicability of HH mapping in complex disorders such as ASD and offer an alternative approach to the analysis of genome-wide association data. © The Author(s) 2011

Gene-ontology enrichment analysis in two independent family-based samples highlights biologically plausible processes for autism spectrum disorders

Recent genome-wide association studies (GWAS) have implicated a range of genes from discrete biological pathways in the aetiology of autism. However, despite the strong influence of genetic factors, association studies have yet to identify statistically robust, replicated major effect genes or SNPs. We apply the principle of the SNP ratio test methodology described by O'Dushlaine et al to over 2100 families from the Autism Genome Project (AGP). Using a two-stage design we examine association enrichment in 5955 unique gene-ontology classifications across four groupings based on two phenotypic and two ancestral classifications. Based on estimates from simulation we identify excess of association enrichment across all analyses. We observe enrichment in association for sets of genes involved in diverse biological processes, including pyruvate metabolism, transcription factor activation, cell-signalling and cell-cycle regulation. Both genes and processes that show enrichment have previously been examined in autistic disorders and offer biologically plausibility to these findings

A genome-wide scan for common alleles affecting risk for autism

Although autism spectrum disorders (ASDs) have a substantial genetic basis, most of the known genetic risk has been traced to rare variants, principally copy number variants (CNVs). To identify common risk variation, the Autism Genome Project (AGP) Consortium genotyped 1558 rigorously defined ASD families for 1 million single-nucleotide polymorphisms (SNPs) and analyzed these SNP genotypes for association with ASD. In one of four primary association analyses, the association signal for marker rs4141463, located within MACROD2, crossed the genome-wide association significance threshold of P &amp;lt; 5 3 10-28. When a smaller replication sample was analyzed, the risk allele at rs4141463 was again over-transmitted; yet, consistent with the winner&apos;s curse, its effect size in the replication sample was much smaller; and, for the combined samples, the association signal barely fell below the P &amp;lt; 5 × 10-28 threshold. Exploratory analyses of phenotypic subtypes yielded no significant associations after correction for multiple testing. They did, however, yield strong signals within several genes, KIAA0564, PLD5, POU6F2, ST8SIA2 and TAF1C. © The Author 2010. Published by Oxford University Press. All rights reserved

Functional impact of global rare copy number variation in autism spectrum disorders

The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in reciprocal social interaction and communication, and the presence of restricted and repetitive behaviours 1. Individuals with an ASD vary greatly in cognitive development, which can range from above average to intellectual disability2. Although ASDs are known to be highly heritable ( ∼90%)3, the underlying genetic determinants are still largely unknown.Hereweanalysed the genome-wide characteristics of rare (&amp;lt;1%frequency) copy number variation in ASD using dense genotyping arrays. When comparing 996 ASD individuals of European ancestry to 1,287 matched controls, cases were found to carry a higher global burden of rare, genic copy number variants (CNVs) (1.19 fold, P=0.012), especially so for loci previously implicated in either ASDand/or intellectual disability (1.69 fold, P=3.4×310-4). Among the CNVs there were numerous de novo and inherited events, sometimes in combination in a given family, implicating many novel ASD genes such as SHANK2, SYNGAP1, DLGAP2 and the X-linked DDX53-PTCHD1 locus. We also discovered an enrichment of CNVs disrupting functional gene sets involved in cellular proliferation, projection and motility, and GTPase/Ras signalling. Our results reveal many new genetic and functional targets in ASD that may lead to final connected pathways. © 2010 Macmillan Publishers Limited. All rights reserved