Characterization of the gene encoding component C3 of the complement system from the spider Loxosceles laeta venom glands: Phylogenetic implications.

A transcriptome analysis of the venom glands of the spider Loxosceles laeta, performed by our group, in a previous study (Fernandes-Pedrosa et al., 2008), revealed a transcript with a sequence similar to the human complement component C3. Here we present the analysis of this transcript. cDNA fragments encoding the C3 homologue (Lox-C3) were amplified from total RNA isolated from the venom glands of L. laeta by RACE-PCR. Lox-C3 is a 5178 bps cDNA sequence encoding a 190kDa protein, with a domain configuration similar to human C3. Multiple alignments of C3-like proteins revealed two processing sites, suggesting that Lox-C3 is composed of three chains. Furthermore, the amino acids consensus sequences for the thioester was found, in addition to putative sequences responsible for FB binding. The phylogenetic analysis showed that Lox-C3 belongs to the same group as two C3 isoforms from the spider Hasarius adansoni (Family Salcitidae), showing 53% homology with these. This is the first characterization of a Loxosceles cDNA sequence encoding a human C3 homologue, and this finding, together with our previous finding of the expression of a FB-like molecule, suggests that this spider species also has a complement system. This work will help to improve our understanding of the innate immune system in these spiders and the ancestral structure of C3.

A new anti-loxoscelic serum produced against recombinant sphingomyelinase D Results of preclinical trials

Envenomation by Loxosceles species (brown spider) can lead to local dermonecrosis and to serious systemic effects. The main toxic component in the venom of these spiders is sphingomyelinase D (SMase D) and various isoforms of this toxin are present in Loxosceles venoms. We have produced a new anti-loxoscelic serum by immunizing horses with recombinant SMase D. In the present study, we compared the neutralization efficacy of the new anti-loxoscelic serum and anti-arachnidic serum (the latter serum is used for therapy for loxoscelism in Brazil) against the toxic effects of venoms from spiders of the genus Loxosceles. Neutralization tests showed that anti-SMase D serum has a higher activity against toxic effects of L. intermedia and L. laeta venoms and similar or slightly weaker activity against toxic effects of L. gaucho than that of Arachnidic serum. These results demonstrate that recombinant SMase D can replace venom for anti-venom production and therapy.

Transcriptome analysis of Loxosceles laeta (Araneae, Sicariidae) spider venomous gland using expressed sequence tags

The bite of spiders belonging to the genus Loxosceles can induce a variety of clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis, and persistent inflammation. In order to examine the transcripts expressed in venom gland of Loxosceles laeta spider and to unveil the potential of its products on cellular structure and functional aspects, we generated 3,008 expressed sequence tags (ESTs) from a cDNA library. Results: All ESTs were clustered into 1,357 clusters, of which 16.4% of the total ESTs belong to recognized toxin-coding sequences, being the Sphingomyelinases D the most abundant transcript; 14.5% include "possible toxins", whose transcripts correspond to metalloproteinases, serinoproteinases, hyaluronidases, lipases, C-lectins, cystein peptidases and inhibitors. Thirty three percent of the ESTs are similar to cellular transcripts, being the major part represented by molecules involved in gene and protein expression, reflecting the specialization of this tissue for protein synthesis. In addition, a considerable number of sequences, 25%, has no significant similarity to any known sequence. Conclusion: This study provides a first global view of the gene expression scenario of the venom gland of L. laeta described so far, indicating the molecular bases of its venom composition.

Loxosceles spider venom induces the release of thrombomodulin and endothelial protein C receptor: implications for the pathogenesis of intravascular coagulation as observed in loxoscelism.

BACKGROUND: The venom of the spider Loxosceles can cause both local and systemic effects including disseminated intravascular coagulation. AIM: The aim of this study was to investigate the effects of the venom of Loxosceles intermedia (L. intermedia) and the purified Sphingomyelinase D (SMaseD) toxin upon the Protein C (PC) natural anticoagulant pathway. RESULTS: Both the venom and e purified SMaseD reduced the cell surface expression of thrombomodulin (TM) and Endothelial PC Receptor on endothelial cells in culture. The reduction of cell surface expression was caused by cleavage from the cell surface mediated by activation of an endogenous metalloproteinase. Reduction of TM and Endothelial PC Receptor on the surface of these cells resulted in an impaired ability of the cells to assist in the thrombin-induced activation of PC. CONCLUSION: This novel observation gives further insight into the mechanisms of the pathology induced by venom from Loxosceles spiders and may aid the development of a suitable therapy.

Sphingomyelinases D induce direct association of C1q to the erythrocyte membrane causing complement mediated autologous haemolysis.

Bites by Loxosceles spiders can induce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis and persistent inflammation. The causative toxin is a sphingomyelinase D (SMase D) that cleaves sphingomyelin into choline and ceramide-1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. We have previously found that SMase D toxins led to an increased susceptibility of human erythrocytes (E) to activation of complement (C) via the classical pathway (CP) in the absence of antibodies. In the present study we have investigated the CP initiating components involved in the haemolysis induced by SMases from Corynebacterium pseudotuberculosis (PLD) and from Loxosceles intermedia venom (P1). When P1 or PLD treated E were incubated with C8-depleted human serum, an increase in C1q, serum amyloid protein (SAP) and C-reactive protein (CRP) binding was observed. While purified C1q, SAP and CRP were found to bind to P1 or PLD treated E, depletion of SAP or CRP from human serum did not prevent C-mediated lysis, suggesting that pentraxins are not involved in the initiation of C-activation. However depletion of C1 lead to a greatly reduced haemolysis, demonstrating that the activation of the CP is caused by direct binding of C1q to the SMase treated cells. Binding of fluid phase C-regulators C4b-binding protein and factor H was also observed, however these C-regulators in conjunction with the membrane bound C-regulators were unable to prevent haemolysis, demonstrating the potency of SMase D facilitated binding of C1 and activation of C.

[Presence of Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae) in Serra da Bodoquena, State of Mato Grosso do Sul, Brazil]. / Presença de Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae) na Serra da Bodoquena, Estado de Mato Grosso do Sul, Brasil.

The venom of Loxosceles spiders causes dermonecrotic lesion and induces complement-dependent intravascular haemolysis that characterizes a severe systemic effect. In Brazil, L. gaucho, L. intermedia and L. laeta, present in the anthropic environment, have been pointed out as the most important agents of the loxoscelism. Besides these species there are others that, by predominating in the natural environment, have not been evaluated regarding human health risk, as in the case of Loxosceles similis. The development of a research project in Bodoquena Range, for ecological observation and identification of insects of medical interest, enabled the capture of Loxosceles similis specimens in the "Pitangueiras" cave and "Lago Azul" cave, in Bodoquena Range, municipality of Bonito, State of Mato Grosso do Sul, Brazil. The objectives of this study were to define the parameters for identification, environmental features of the habitat of this species, as well as an update of its geographical distribution.

Presença de Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae) na Serra da Bodoquena, Estado de Mato Grosso do Sul, Brasil / Presence of Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae) in Serra da Bodoquena, State of Mato Grosso do Sul, Brazil

The venom of Loxosceles spiders causes dermonecrotic lesion and induces complement-dependent intravascular haemolysis that characterizes a severe systemic effect. In Brazil, L. gaucho, L. intermedia and L. laeta, present in the anthropic environment, have been pointed out as the most important agents of the loxoscelism. Besides these species there are others that, by predominating in the natural environment, have not been evaluated regarding human health risk, as in the case of Loxosceles similis. The development of a research project in Bodoquena Range, for ecological observation and identification of insects of medical interest, enabled the capture of Loxosceles similis specimens in the [quot ]Pitangueiras[quot ] cave and [quot ]Lago Azul[quot ] cave, in Bodoquena Range, municipality of Bonito, State of Mato Grosso do Sul, Brazil. The objectives of this study were to define the parameters for identification, environmental features of the habitat of this species, as well as an update of its geographical distribution.

O veneno das aranhas do gênero Loxosceles causa lesão dermonecrótica e induz hemólise intravascular dependente de complemento, configurando um quadro clínico de intensa gravidade. No Brasil, as espécies L. gaucho L. intermedia e L. laeta, presentes no ambiente antrópico, têm sido apontadas como principais agentes do loxoscelismo. Além destas, existem outras espécies, que por predominarem no ambiente natural, não têm sido avaliadas quanto ao risco à saúde do homem, como é o caso de Loxosceles similis. O desenvolvimento de projeto de pesquisa, na Serra da Bodoquena, para observações ecológicas e identificação de insetos de interesse médico, possibilitou a captura de espécimens de Loxosceles similis na Serra da Bodoquena, Município de Bonito, Estado do Mato Grosso do Sul, Brasil, nas grutas Pitangueiras e do Lago Azul. Os parâmetros para identificação, características ambientais do habitat da espécie, bem como atualização de sua distribuição geográfica são objetos deste trabalho.

Loxosceles intermedia spider envenomation induces activation of an endogenous metalloproteinase, resulting in cleavage of glycophorins from the erythrocyte surface and facilitating complement-mediated lysis.

Loxosceles is the most venomous spider in Brazil, and envenomation causes dermonecrosis and complement (C)-dependent intravascular hemolysis. The authors studied the mechanism of induction of C-induced hemolysis. Purified Loxosceles toxins rendered human erythrocytes susceptible to lysis by human C but did not have an effect on the E-bound C-regulators DAF, CR1, or CD59. However, incubation with venom toxins caused cleavage of glycophorin from the erythrocyte (E) surface, facilitating C activation and hemolysis. The results suggest that glycophorin is an important factor in the protection of E against homologous C. Cleavage of glycophorin (GP) A, GPB, and GPC occurred at sites close to the membrane but could not be accomplished using purified GPA and purified toxins, demonstrating that cleavage was not an effect of a direct proteolytic action of the Loxosceles toxins on the glycophorins. Inhibition of the cleavage of glycophorins induced by Loxosceles venom was achieved with 1,10-phenanthroline. The authors propose that the sphingomyelinase activity of the toxins induces activation of an endogenous metalloproteinase, which then cleaves glycophorins. They observed the transfer of C-dependent hemolysis to other cells, suggesting that the Loxosceles toxins can act on multiple cells. This observation can explain the extent of hemolysis observed in patients after envenomation. Identification of the mechanism of induction of susceptibility to C-mediated lysis after Loxosceles envenomation opens up the possibility of the development of an effective therapeutic strategy. (Blood. 2000;95:683-691)

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.

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.

[Observations on oviposition, hatching and the life span of Triatoma matogrossensis Leite & Barbosa, 1953 (Hemiptera, Reduviidae) as a function of their feeding on pigeons and rabbits]. / 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.