Rute Maria Gonçalves de Andrade

Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases

We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)‐dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD‐induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred. We show here that haemolysis of both PLD‐ and P1‐treated human erythrocytes was C dependent, but while PLD‐mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways. P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.

Sex-linked variation of Loxosceles intermedia spider venoms

In order to investigate intraspecific differences in Loxosceles intermedia spider venom we compared some biological properties of male and female venoms. Females produced higher amounts of venom than males. Furthermore, female venom presented more potent dermonecrotic and complement-dependent activities than male venom. Interestingly, the F35 toxin, a dermonecrotic and complement-dependent haemolytic factor, was also present in greater amounts in female venom, as demonstrated by ELISA. Therefore, the higher production and increased toxicity of venom in female specimens as compared to males may contribute to the variability observed in the severity of envenoming caused by L. intermedia spiders.

Ontogenetic development of Loxosceles intermedia spider venom

Envenomation by Loxosceles spider has become a public health problem in the South region of Brazil, mainly due to high levels of domiciliary infestation by Loxosceles intermedia spiders. The toxic effects of L. intermedia venom are mostly associated with a 35 kDa protein (F35) which presents complement-dependent haemolytic and dermonecrotic activities. The aim of this study was to detect, through biological and immunochemical assays, the appearance of the main toxic component, F35, during the ontogenetic development of L. intermedia spiders. The toxin appeared in its fully active form in venom of third instar spiderlings; from then on its activity increased throughout development until adulthood. On the other hand, F35 was not detected in extracts of either eggs or spiderlings of the first and second instars.

Loxosceles spider venom induces metalloproteinase mediated cleavage of MCP/CD46 and MHCI and induces protection against C-mediated lysis

We have recently shown that sphingomyelinase D toxins from the spider Loxosceles intermedia induce Complement (C) ‐dependent haemolysis of autologous erythrocytes by the induction of cleavage of cell‐surface glycophorins through activation of a membrane‐bound metalloproteinase. The aim of this study was to investigate the effects of these toxins on C‐regulator expression and the C‐resistance of nucleated cells. Cells were incubated with Loxosceles venom/toxins and the expression of C‐regulators was assessed by flow cytometry. A reduced expression of membrane co‐factor protein (MCP) was observed, while expression of decay‐accelerating factor (DAF) and CD59 was not affected. Analysis of other cell‐surface molecules showed a reduced expression of major histocompatibility complex I (MHCI). Western blotting showed that a truncated form of MCP was released into the supernatant. Release could be prevented by inhibitors of metalloproteinases of the adamalysin family but not by inhibitors specific for matrix metalloproteinases. Cleavage of MCP was induced close to or within the membrane as demonstrated by the cleavage of transmembrane chimeras of CD59 and MCP. Although the venom/toxins induced a release of MCP, the C‐susceptibility was decreased. The mechanism of this induction of resistance may involve a change in membrane fluidity induced by the sphingomyelinase activity of the toxin/venom and/or involvement of membrane‐bound proteases. The soluble forms of MCP found in tissues and body under pathological conditions like cancer and autoimmune diseases may be released by a similar mechanism. The identity of the metalloproteinase(s) activated by the spider venom and the role in pathology of Loxoscelism remains to be established.

Micropygomyia (Sauromyia) petari, a new species of Phlebotominae (Diptera, Psychodidae) from Vale do Ribeira, São Paulo State, Brazil

Micropygomyia (Sauromyia) petari sp. nov. (Diptera, Psychodidae, Phlebotominae) from speleological province of the Vale do Ribeira, Sao Paulo State, Brazil, is described and illustrated. This new taxon belongs to oswaldoi series.

Observações sobre oviposição, eclosão e tempo de vida de Triatoma matogrossensis Leite & Barbosa, 1953 (Hemiptera-Reduviidae) em função da alimentação em pombos e coelhos

A laboratory study was carried out concerning the influence of two kinds of blood-meal on egg laying, egg hatching and life span of Triatoma matogrossensis. 68 couples in 4 different groups with 20, 12, 20 and 16 individuals of each sex per group were formed. Maintained under laboratory conditions groups A1 and A2 were fed on pigeons and groups C1 and C2 were fed on rabbits. In relation to egg laying the best results were found in group A1. No differences on egg hatching were found between the groups fed on rabbits and those fed on pigeons. Concerning the life span, no differences between males and females in the 4 groups were observed but group A1 presented the longest life span and group C2 the shortest.

Microcirculation abnormalities provoked by Loxosceles spiders' envenomation.

Loxoscelism is caused by envenomation by spiders from Loxosceles genus. Clinical symptoms only appear a few hours after envenomation and can evolve in local reactions, such as dermonecrosis, and systemic reactions, including intravascular haemolysis, intravascular coagulation and renal failure. Considering that alterations in the microcirculatory network are involved in the pathogenesis of different diseases, including the inflammatory process, the aim of this study was to investigate the action of venoms of males and females of Loxosceles intermedia and Loxosceles laeta on the microcirculatory network and examine the systemic production of inflammatory mediators in a murine model of loxoscelism. We observed that during systemic envenomation, the alterations in the microcirculation include increase in the number of rolling cells, which was more intense in animals injected with female Loxosceles spider venoms. This positively correlated with increase in TNF-α and NO serum levels, induction of which was higher by female venoms when compared with male venoms. The increase of leukocytes rolling was not accompanied by increase of cell adhesion. The absence of leukocyte extravasation may explain why in mice, in contrast to humans, no cutaneous loxoscelism occurs. Thus, targeting the neutrophil adhesion and extravasation in Loxosceles envenomed patients may prevent cutaneous pathology.