Efficacy of maggot debridement therapy on refractory leg ulcers of Behçet disease: an open-label study.

BACKGROUND: Cutaneous ulcers of Behçet disease (BD) are rare but have high morbidity and resistance to conventional therapies. An important and essential aspect of ulcer management is debridement. Regarding maggot therapy (MT), excretions of the green bottle fly, Lucilia sericata, have been shown to have the ability to remove necrotic debris and promote healing. AIM: To evaluate the efficacy of MT for cutaneous ulcers of BD. METHODS: In this open-label trial, patients with BD with refractory leg ulcers suitable for MT were enrolled. Maggot application was performed until complete debridement was achieved, and all patients were followed up for 12 months afterwards to assess the total healing of ulcers. RESULTS: In total, 24 patients with 32 ulcers were enrolled. Using MT, 91.6% of all ulcers were completely debrided. Mean time to debridement was 14.9 days and mean number of cycles required was 5.3. Mean ulcer size was decreased by 23% with treatment. Time to debridement was positively correlated with pretreatment ulcer size and ulcer duration (P = 0.01 and P < 0.01) but not with ulcer depth, comorbidities, smoking, age or sex (P > 0.05 for all). During follow-up, 79.1% of all ulcers healed completely. Mean time required for total healing was positively correlated with ulcer duration, pretreatment and post-treatment ulcer area, ulcer depth and mean time to total debridement (P < 0.03, P = 0.00, P = 0.04 and P < 0.01, respectively). CONCLUSIONS: To our knowledge, the findings presented in this first and unique study may provide key answers about factors affecting success rate of MT in BD cutaneous ulcers.

The impacts of larval density and protease inhibition on feeding in medicinal larvae of the greenbottle fly Lucilia sericata.

Larval therapy, the therapeutic use of blowfly larvae to treat chronic wounds, is primarily used in debridement. There are, however, gaps in current knowledge of the optimal clinical application of the therapy and mechanisms of action in the debridement process. Using an artificial assay, two studies were undertaken to investigate these aspects of larval debridement by Lucilia sericata Meigen (Diptera: Calliphoridae); the first studied the effects of the density of larvae on tissue digestion and larval mass, and the second considered the effects on the same parameters of incorporating protease inhibitors into the feeding substrate. The total mass of tissue digested increased with larval density until saturation was observed at 5.0-7.5 larvae/cm(2) . This range was considered optimal as lower doses resulted in the removal of less tissue and higher doses offered no additional tissue removal and appeared to exacerbate competition for feeding. In the second study, increased protease inhibitor concentration led to significant decreases in tissue digestion and larval mass, suggesting that serine proteases, particularly trypsin, may play major roles in larval digestion. Such information is important in elucidating the main constituents that make up larval digestive products and may be significant in the development of new therapies.

Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

Acetylcholinesterases of blood-feeding flies and ticks.

Acetylcholinesterase (AChE) is the biochemical target of organophosphate (OP) and carbamate pesticides for invertebrates, vertebrate nerve agents, and AChE inhibitors used to reduce effects of Alzheimer's disease. Organophosphate pesticides (OPs) are widely used to control blood-feeding arthropods, including biting flies and ticks. However, resistance to OPs in pests affecting animal and human health has compromised control efficacy. OP resistance often results from mutations producing an OP-insensitive AChE. Our studies have demonstrated production of OP-insensitive AChEs in biting flies and ticks. Complementary DNA (cDNA) sequences encoding AChEs were obtained for the horn fly, stable fly, sand fly, and the southern cattle tick. The availability of cDNA sequences enables the identification of mutations, expression and characterization of recombinant proteins, gene silencing for functional studies, as well as in vitro screening of novel inhibitors. The southern cattle tick expresses at least three different genes encoding AChE in their synganglion, i.e. brain. Gene amplification for each of the three known cattle tick AChE genes and expression of multiple alleles for each gene may reduce fitness cost associated with OP-resistance. AChE hydrolyzes the neurotransmitter, acetylcholine, but may have additional roles in physiology and development. The three cattle tick AChEs possess significantly different biochemical properties, and are expressed in neural and non-neural tissues, which suggest separation of structure and function. The remarkable complexity of AChEs in ticks suggested by combining genomic data from Ixodes scapularis with our genetic and biochemical data from Rhipicephalus microplus is suggestive of previously unknown gene duplication and diversification. Comparative studies between invertebrate and vertebrate AChEs could enhance our understanding of structure-activity relationships. Research with ticks as a model system offers the opportunity to elucidate structure-activity relationships for AChE that are important for advances in targeted pest control, as well as potential applications for medicine and biosecurity.

Larvaterapia aplicada a heridas con poca carga de tejido necrótico y caracterización enzimática de la excreción, secreción y hemolinfa de larvas / Effect of maggot therapy on minimally necrotic tissues: characterization of larval enzymatic excretion/secretion

Introducción. Las úlceras crónicas son una afección con un impacto negativo importante en la calidad de vida de los pacientes y en el sistema de salud; la aparición de infecciones y su difícil manejo, así como la presencia de tejido necrótico, afectan el pronóstico de curación. La larvaterapia se presenta como una opción para el desbridamiento y el manejo de infecciones de úlceras crónicas. Objetivo. Evaluar la larvaterapia en heridas con poca carga de tejido necrótico y evaluar las excreciones, secreciones y la hemolinfa de las larvas, respecto a su contenido enzimático. Materiales y métodos. Se reporta una serie de tres casos clínicos con úlceras crónicas y poca carga de tejido necrótico, tratados con larvaterapia, y se evalúa su evolución por los índices PUSH (Pressure Ulcer Scale for Healing) y Wound Bed Score, así como el patrón electroforético y contenido enzimático por zimograma de las excreciones y secreciones, y de la hemolinfa de las larvas. Resultados. Con solo una aplicación de la larvaterapia se evidenció una mejoría del aspecto de la herida y en los puntajes evaluados; en el PUSH hubo una disminución de 2,3 puntos, en promedio, y con el Wound Bed Score, un incremento de 2,7, lo que demuestra una mejoría en ambas escalas. Conclusión. Se encontró una actividad enzimática diversa en su contenido de excreciones y secreciones, con predominio de actividad de la proteasa de tipo serina.

Introduction. Chronic leg ulcers are a burden for the health system and impact quality of life. The infections, the necrotic tissue and the difficult treatment affects the prognosis and healing time. Maggot therapy is presented as an acceptable alternative for the debridement and treatment of this pathology. Objective. The larval therapy was assessed on chronic leg ulcers with little necrotic tissue. Larval excretion and secretion (E/S) was characterized with respect to hemolymph (HL) enzymatic content. Materials and methods. Three patients with chronic leg ulcers and low necrotic tissue were treated with larval therapy and were assessed with the PUSH (pressure ulcer scale for healing) and Wound Bed Score. E/S and HL content was evaluated by SDS PAGE and zymogram. Results. The clinical aspect of the wounds showed improvement, and the scores demonstrated an average decrease of 2.3 for the PUSH and an average increase of 2.7 for the Wound Bed Score. A wide diversity of enzymatic activity in the E/S was demontrated with major activity belonging to serine protease family. Conclusions. Maggot therapy proved an effective treatment in cases with minimal tissue necrosis and can be considered a viable treatment option.

[Effect of maggot therapy on minimally necrotic tissues: characterization of larval enzymatic excretion/secretion]. / Larvaterapia aplicada a heridas con poca carga de tejido necrótico y caracterización enzimática de la excreción, secreción y hemolinfa de larvas.

INTRODUCTION: Chronic leg ulcers are a burden for the health system and impact quality of life. The infections, the necrotic tissue and the difficult treatment affects the prognosis and healing time. Maggot therapy is presented as an acceptable alternative for the debridement and treatment of this pathology. OBJECTIVE: The larval therapy was assessed on chronic leg ulcers with little necrotic tissue. Larval excretion and secretion (E/S) was characterized with respect to hemolymph (HL) enzymatic content. Materials and methods. Three patients with chronic leg ulcers and low necrotic tissue were treated with larval therapy and were assessed with the PUSH (pressure ulcer scale for healing) and Wound Bed Score. E/S and HL content was evaluated by SDS PAGE and zymogram. RESULTS: The clinical aspect of the wounds showed improvement, and the scores demonstrated an average decrease of 2.3 for the PUSH and an average increase of 2.7 for the Wound Bed Score. A wide diversity of enzymatic activity in the E/S was demonstrated with major activity belonging to serine protease family. CONCLUSIONS: Maggot therapy proved an effective treatment in cases with minimal tissue necrosis and can be considered a viable treatment option.

Kinetic studies on the glutathione peroxidase activity of selenium-containing glutathione transferase.

Selenium-containing glutathione transferase (seleno-GST) was generated by biologically incorporating selenocysteine into the active site of glutathione transferase (GST) from a blowfly Lucilia cuprina (Diptera: Calliphoridae). Seleno-GST mimicked the antioxidant enzyme glutathione peroxidase (GPx) and catalyzed the reduction of structurally different hydroperoxides by glutathione. Kinetic investigations reveal a ping-pong kinetic mechanism in analogy with that of the natural GPx cycle as opposed to the sequential one of the wild type GST. This difference of the mechanisms might result from the intrinsic chemical properties of the incorporated residue selenocysteine, and the selenium-dependent mechanism is suggested to contribute to enhancement of the enzymatic efficiency.

Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors.

Protease inhibitors have been proposed as potential defense molecules for increased insect resistance in crop plants. Compensatory over-production of insensitive proteases in the insect, however, has limited suitability of these proteins in plant protection, with very high levels of inhibitor required for increased plant resistance. In this study we have examined whether combined used of two inhibitors is effective to prevent this compensatory response. We show that leaf-specific over-expression of the potato PI-II and carboxypeptidase inhibitors (PCI) results in increased resistance to Heliothis obsoleta and Liriomyza trifolii larvae in homozygote tomato lines expressing high levels (>1% the total soluble proteins) of the transgenes. Leaf damage in hemizygous lines for these transformants was, however, more severe than in the controls, thus evidencing a compensation response of the larvae to the lower PI concentrations in these plants. Development of comparable adaptive responses in both insects suggests that insect adaptation does not entail specific recognition of the transgene, but rather represents a general adaptive mechanism triggered in response to the nutritional stress imposed by sub-lethal concentrations of the inhibitors. Combined expression of defense genes with different mechanisms of action rather than combinations of inhibitors may then offer a better strategy in pest management as it should be more effective in overcoming this general adaptive response in the insect.

Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans.

Numerous invertebrate species belonging to several phyla cannot synthesize sterols de novo and rely on a dietary source of the compound. SCPx (sterol carrier protein 2/3-oxoacyl-CoA thiolase) is a protein involved in the trafficking of sterols and oxidation of branched-chain fatty acids. We have isolated SCPx protein from Spodoptera littoralis (cotton leafworm) and have subjected it to limited amino acid sequencing. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.9 kb), which encodes a 57 kDa protein. Northern blotting detected two mRNA transcripts, one of 1.9 kb, encoding SCPx, and one of 0.95 kb, presumably encoding SCP2 (sterol carrier protein 2). The former mRNA was highly expressed in midgut and Malpighian tubules during the last larval instar. Furthermore, constitutive expression of the gene was detected in the prothoracic glands, which are the main tissue producing the insect moulting hormone. There was no significant change in the 1.9 kb mRNA in midgut throughout development, but slightly higher expression in the early stages. Conceptual translation of the cDNA and a database search revealed that the gene includes the SCP2 sequence and a putative peroxisomal targeting signal in the C-terminal region. Also a cysteine residue at the putative active site for the 3-oxoacyl-CoA thiolase is conserved. Southern blotting showed that SCPx is likely to be encoded by a single-copy gene. The mRNA expression pattern and the gene structure suggest that SCPx from S. littoralis (a lepidopteran) is evolutionarily closer to that of mammals than to that of dipterans.

Isolation and characterization of the Xanthine dehydrogenase gene of the Mediterranean fruit fly, Ceratitis capitata.

Xanthine dehydrogenase (XDH) is a member of the molybdenum hydroxylase family of enzymes catalyzing the oxidation of hypoxanthine and xanthine to uric acid. The enzyme is also required for the production of one of the major Drosophila eye pigments, drosopterin. The XDH gene has been isolated in many species representing a broad cross section of the major groups of living organisms, including the cDNA encoding XDH from the Mediterranean fruit fly Ceratitis capitata (CcXDH) described here. CcXDH is closely related to other insect XDHs and is able to rescue the phenotype of the Drosophila melanogaster XDH mutant, rosy, in germline transformation experiments. A previously identified medfly mutant, termed rosy, whose phenotype is suggestive of a disruption in XDH function, has been examined for possible mutations in the XDH gene. However, we find no direct evidence that a mutation in the CcXDH gene or that a reduction in the CcXDH enzyme activity is present in rosy medflies. Conclusive studies of the nature of the medfly rosy mutant will require rescue by germline transformation of mutant medflies.

Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata.

Prophenoloxidase (PPO) is a key enzyme associated with both melanin biosynthesis and sclerotization in insects. This enzyme is involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects. It was isolated from the larval hemolymph of Sarcophaga bullata and purified by employing ammonium sulfate precipitation, Phenyl Sepharose chromatography, DEAE-Sepharose chromatography, and Sephacryl S-200 column chromatography. The purified enzyme exhibited two closely moving bands on 7.5% SDS-PAGE under denaturing conditions. From the estimates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS-PAGE, which ranged from 90,000 to 100,000, it was inferred that the enzyme is made up of a single polypeptide chain. Activation of PPO (K(a)=40 microM) was achieved by the cationic detergent, cetyl pyridinium chloride below its critical micellar concentration (0.8 mM) indicating that the detergent molecules are binding specifically to the PPO and causing the activation. Neither anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The active enzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acid sequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encoded polypeptides of 685 and 691 amino acids. They contained two distinct copper binding regions and lacked the signal peptide sequence. They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sites and a conserved C-terminal region common to all known PPOs.

Kinetic microplate-based assays for inhibitors of mitochondrial NADH:ubiquinone oxidoreductase (complex I) and succinate:cytochrome c oxidoreductase.

Kinetic microplate-based assays for both mitochondrial NADH:ubiquinone oxidoreductase (complex I) and succinate:cytochrome c oxidoreductase using insect submitochondrial particles as the source of the enzyme activities have been developed. These assays have been used to design high-throughput screens for inhibitors of these mitochondrial electron transfer activities to assess their intrinsic in vitro efficacies as potential pesticides. These methods can be used to test up to 60 compounds per day without the use of automated sample handling and diluting technology. The accuracy, specificity, and reproducibility of the microplate methods compared well with conventional spectrophotometer-based assays.

Comparative studies of suidatrestin, a specific inhibitor of trehalases.

Suidatrestin, isolated from a Streptomyces strain, was characterized as a new trehalase inhibitor. Its inhibitory potential was 7 to 50-fold higher than that of validamycin when tested against insect, fungal and mammalian trehalases. The kinetic properties of suidatrestin were studied in vitro with trehalases from flight muscle mitochondria of the fly, Protophormia terraenovae, from larval midgut of the moth, Spodoptera littoralis, and from porcine kidney, as well as with maltase from yeast. Suidatrestin was inactive on maltase but inhibited all trehalases with IC50 values of 0.08-0.1 microM; Ki values ranged from 0.02 to 0.05 microM. The very low Ki/K(m) ratios (3.9 x 10(-6) -4.9 x 10(-6)) indicated excellent in vitro inhibitory action of suidatrestin. When injected into larvae of S. littoralis, suidatrestin required high and repetitive doses which lead to reversible inhibition of larval growth only. Consecutive omission of the inhibitor even stimulated weight increase above that of controls. Significant mortality was achieved at a rather high dose only. Injection of a growth-inhibiting dose of suidatrestin did not change hemolymph osmolality as a measure of sugar concentration. The discrepancy between in vitro and in vivo potency of suidatrestin may be understood once its chemical structure is fully known.

Cloning and functional expression of poly(ADP-ribose) polymerase cDNA from Sarcophaga peregrina.

A cDNA spanning the entire coding region for poly(ADP-ribose) polymerase (PARP) of Sarcophaga peregrina was isolated and the nucleotide sequence was determined. The longest open reading frame encodes a polypeptide of 996 amino acid residues with a molecular mass of 113,033 Da. The similarities to the human PARP in amino acid sequence were relatively low in the DNA-binding and auto-modification domains, but very high in the C-terminal catalytic domain: identity of amino acids is 34% in the N-terminal DNA-binding domain (residues 1-369), 27% in the auto-modification domain (residues 370-507), and 56% in the C-terminal NAD-binding domain (residues 508-996). Two zinc-fingers (C-X2-C-X28-H-X2-C and C-X2-C-X31-H-X2-C)2 and a basic region in the N-terminal DNA-binding domain recognized in other PARP are conserved. Downstream of the basic region, another cysteine-rich motif (C-X2-C-X13-C-X9-C), a putative zinc-finger, was found to be well conserved in the PARP of Sarcophaga, Drosophila and human. A leucine-zipper motif (L-X6-L-X6-L-X6-L) which was found in the auto-modification domain of Drosophila PARP, is disrupted in the Sarcophaga enzyme: the second leucine is replaced by proline, and the third leucine by valine. Full-length cDNA for Sarcophaga PARP was cloned into an expression plasmid and expressed in Escherichia coli. A lysate of E. coli cells containing expressed protein reacted with antibody against Sarcophaga PARP, and PARP activity was detected. Thus, we conclude that isolated cDNA encodes a functional Sarcophaga PARP cDNA.

Studies on catalase in ageing Zaprionus paravittiger (Diptera) with special reference to an antioxidant feeding.

Zaprionus paravittiger fed with an antioxidant (sodium hypophosphite, 1 X 10(3) microM) supplemented diet exhibited adaptive compensatory responses in catalase activity (quantitative as well as qualitative). The longevity and catalase activity were found to be positively linked. The study denotes that free radical formation and antioxygenic defenses are closely associated and are the possible determinants of life span and ageing.