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.

Does maggot therapy promote wound healing? The clinical and cellular evidence.

The larvae of Lucillia sericata, or maggots of the green-bottle fly, are used worldwide to help debride chronic, necrotic and infected wounds. Whilst there is abundant clinical and scientific evidence to support the role of maggots for debriding and disinfecting wounds, not so much emphasis has been placed on their role in stimulating wound healing. However, there is accumulating evidence to suggest that maggots and their externalized secretions may also promote wound healing in stubborn, recalcitrant chronic ulcers. There are a growing number of clinical reports which support the observation that wounds which have been exposed to a course of maggot debridement therapy also show earlier healing and closure end-points. In addition, recent pre-clinical laboratory studies also indicate that maggot secretions can promote important cellular processes which explain this increased healing activity. Such processes include activation of fibroblast migration, angiogenesis (the formation of new blood vessels from pre-existing vessels) within the wound bed, and an enhanced production of growth factors within the wound environment. Thus, in this review, we summarize the clinical evidence which links maggots and improved wound healing, and we précis recent scientific studies which examine and identify the role of maggots, particularly individual components of maggot secretions, on specific cellular aspects of wound healing.

Maximising the secondary beneficial effects of larval debridement therapy.

Laboratory-based clinical investigations have shown that maggots and their secretions promote, among other activities, fibroblast motogenesis and angiogenesis. These events would contribute to re-granulation if translated to the wound environment. Maggot secretions also have ascribed antibacterial actions and may exhibit anti-inflammatory effects. Many of these biological events would be lost in the presence of necrotic tissue, making debridement a prerequisite for the release of larval-secreted secondary beneficial effects on the wound. We argue that Larval Debridement Therapy (LDT) should be considered as a primary and secondary treatment in wound management, with the primary application designed to debride the wound, and with subsequent applications to the debrided wound targeted to cellular events that promote healing. This review lends support to a re-evaluation of larval application protocols, in order to optimally harness the potential secondary beneficial clinical effects of larval therapy.

The role of oxygen-associated therapies for the healing of chronic wounds, particularly in patients with diabetes.

This paper discusses the role of molecular oxygen as an aid to wound healing, and the potential value of the three major therapies which allow the delivery of oxygen to the wound site: Hyperbaric Oxygen Therapy (HBOT), Topical Oxygen Therapy (TOT) and a new sterile wound dressing, Oxyzyme™. We summarize studies which have been undertaken using these interventions, and discuss their reported effect on chronic, non-healing wounds, in particular, on ulcers associated with Diabetes. The main conclusions drawn from the studies reviewed indicate that therapeutic oxygen can be used as an aid to the healing of chronic wounds; and benefits are clearly evident with the use of both HBOT and TOT. There is also potential for the use of a new, portable, topical oxygen delivery system, oxyzyme. However its use is still embryonic and studies on its effectiveness are limited. More robust measures of its efficacy are urgently needed.

Amino acid derivatives from Lucilia sericata excretions/secretions may contribute to the beneficial effects of maggot therapy via increased angiogenesis.

BACKGROUND: Maggot therapy, utilizing the larvae of Lucilia sericata, has been reported to reduce the bacterial load within wounds and also to enhance wound healing. Maggot excretions/secretions (ES) have been shown to have a role in the success of maggot therapy. While the protein content of ES has been investigated, to date little research has focused on the small metabolites present in ES and their potential contribution to the therapy. Study of the molecular composition of the secretions and the potential bioactivities present will allow for a more detailed evaluation of the efficacy of maggot therapy. OBJECTIVES: We studied the amino acid-like compounds present in ES of L. sericata larvae in order to determine the compounds present and their potential role in the wound healing process. METHODS: These included thin-layer chromatography/mass spectrometric analysis of ES to identify amino acid-like components, a turbidometric assay to investigate their potential antibacterial activity and cell proliferation studies to investigate their potential mitogenic ability. RESULTS: Three prominent compounds were detected and identified as histidine, valinol and 3-guanidinopropionic acid. While these amino acids were not shown to exhibit antibacterial activity, a proliferative effect on the growth of human endothelial cells, but not fibroblasts, was noted. CONCLUSIONS: The demonstrated proliferative effect, selectively on endothelial cells, suggests that the amino acid-like compounds present in maggot ES may have a role in wound healing, by stimulating angiogenesis.

A preliminary investigation into bacterial contamination of Welsh emergency ambulances.

OBJECTIVES: The objective of this regional study across Wales was to perform a preliminary examination of Welsh emergency vehicles to determine levels of bacterial contamination. METHODS: Specific sites within emergency vehicles were swabbed, before and after vehicle cleaning, over a 12 month period, on a monthly basis. All swabs were sent to pathology laboratories for culturing and analysis. RESULTS: Results showed that most sites within emergency vehicles across Wales were contaminated with a range of bacterial species before vehicle cleaning. After vehicle cleaning, many sites in vehicles were still contaminated, and some sites that were previously uncontaminated, became freshly contaminated as a result of cleaning methods used. CONCLUSIONS: The authors conclude that the Welsh emergency vehicles examined exhibited an unacceptable level of bacterial contamination. This finding should be carefully considered and all attempts must be made to tackle the problem of vehicle cleanliness and infection control.

Role of superoxide and reactive nitrogen intermediates in Rhodnius prolixus (Reduviidae)/Trypanosoma rangeli interactions.

This study compares aspects of the superoxide, nitric oxide and prophenoloxidase pathways in Rhodnius prolixus hemolymph, measured in parallel, in response to Trypanosoma rangeli inoculation. Responses to two strains of T. rangeli, and two developmental forms, were studied, and the results obtained were correlated with the ability of the parasites to survive, multiply, and complete their life cycles in the hemolymph of the host. T. rangeli H14 strain parasites, which fail to complete their life cycle in Rhodnius by invading the salivary glands, stimulated high levels of superoxide and prophenoloxidase activity, which peaked 24 h after inoculation. Simultaneously, the concentration of hemolymph nitrites and nitrates increased, indicative of nitric oxide activity, but parasite numbers remained low. T. rangeli Choachi strain parasite inoculation also stimulated superoxide and prophenoloxidase activity, which, though significantly lower than the equivalent responses to the H14 strain, also peaked at 24 h. However, nitrate and nitrite levels in Choachi strain-inoculated hemolymph remained low, and this parasite strain multiplied rapidly, especially following peak superoxide activity, and eventually invaded the salivary glands for transmission to a vertebrate host. In both strains, short form epimastigotes stimulated greater superoxide and prophenoloxidase responses than long form epimastigotes. Injection of the NADPH oxidase inhibitor N-ethylmaleimide or the inducible nitric oxide synthase inhibitor S-methyl isothiourea sulfate caused significantly higher insect mortalities in groups of R. prolixus inoculated with either parasite strain compared with those of uninfected control insects. This indicates that both NADPH oxidase and nitric oxide synthase activity may be involved in the immune response of R. prolixus to infection by T. rangeli. Finally, Western blotting of R. prolixus hemocyte lysates revealed the presence of a protein immunologically related to the human NADPH oxidase complex, the initiator enzyme of the respiratory burst.

Studies on a haemolymph lectin isolated from Rhodnius prolixus and its interaction with Trypanosoma rangeli.

We demonstrated that in Rhodnius prolixus haemocyte monolayers, both Trypanosoma cruzi and Trypanosoma rangeli are capable of inducing haemocyte/parasite clump formation. We also purified, by one-step affinity chromatography, a haemolymph galactoside-binding lectin from R. prolixus which we believe could play an important role in the development of T. rangeli in the haemocoel of the insect vector. This lectin markedly enhanced the activation of clump formation by T. rangeli in R. prolixus haemocyte monolayers, with an increase in clump size and haemocyte aggregation. The haemolymph lectin also significantly affected the motilitity and survival of T. rangeli culture short forms, but not the long forms, when they were incubated in vitro. This molecule is also one of the few described in insects with agglutination activity independent of calcium ions. The partial N-terminal amino acid sequence of this lectin demonstrated similarity to a bacterial xylulose kinase and in preliminary experiments the purified haemolymph lectin phosphorylated a tyrosine kinase substrate in a dose-dependent manner. The possible role of this haemolymph lectin in the life cycle of T. rangeli is discussed.

Trypanosoma cruzi and erythrocyte agglutinins: a comparative study of occurrence and properties in the gut and hemolymph of Rhodnius prolixus.

The activity of agglutinins found in the gut tissues and hemolymph of Rhodnius prolixus was tested using rabbit erythrocytes, Trypanosoma cruzi, or Trypanosoma rangeli as test particles. In addition, investigations were made of the influence of parasitic infection and insect diet on the agglutination titers. A range of physicochemical tests and carbohydrate-binding studies were performed and inhibitors were subsequently found for both the crop agglutinin (p-nitrophenol-derived sugars) and the hemolymph agglutinin (galactose-type sugars). As a result, affinity chromatography was utilized for an attempted purification of these agglutinins, and a one-step purification protocol for the R. prolixus hemolymph agglutinin has been developed. Preliminary results of some biological and physicochemical characteristics of this pure agglutinin are described. These results represent a starting point for future studies of lectin/parasite interaction in this reduviid-trypanosome model.