Cinthya K. Okamoto

C5a receptor is cleaved by metalloproteases induced by sphingomyelinase D from Loxosceles spider venom.

Neutrophils are involved in numerous pathologies and are considered to be major contributors to the establishment of cutaneous loxoscelism after envenomation by the Loxosceles spider. Neutrophils are attracted to the site of envenomation by locally generated C5a and contribute to the tissue destruction. We have investigated the effects of this spider venom on the receptor for C5a: C5aR/CD88, a seven transmembrane G-protein coupled receptor. We show here that the Loxosceles venom induces the cleavage of the C5aR at two sites, resulting in the release of the extracellular N-terminus, while retaining part of the transmembrane regions. Using specific inhibitors, it was shown that the cleavage was induced by activation of an endogenous metalloprotease of the adamalysin (ADAM) family, which was activated by the sphingomyelinase D in the venom. Although it resulted in the near complete loss of the N-terminus, C5a was still able to induce a small increase in intracellular calcium and increase secretion of IL-8. The cleavage of the C5aR may well be a protective response after envenomation, rather than contributing to the pathology of Loxosceles envenomation and may represent a general mechanism for how the body protects itself against excess C5a generation in pathological circumstances such as sepsis.

Characterization of anti-crotalic antibodies

Crotalus durissus terrificus, C. d. collilineatus, C. d. cascavella and C. d. marajoensis are responsible minor but severe snake bites in Brazil. The venoms of these snakes share the presence of crotoxin, a neurotoxin comprising of two associated components, crotapotin and phospholipase A2 (PLA2). Treatment of the victims with specific antiserum is the unique effective therapeutic measure. The ability of anti-Crotalus antisera produced by the routine using crude venom to immunize horses or purified crotoxin and PLA2 as individual immunogens was compared. Antisera obtained from horses immunized with C. durissus terrificus crude venom were able to recognize and neutralize not only the toxins presents in C. durissus terrificus, but also the ones present in the venoms from C. d. collilineatus, C. d. cascavella and C. d. marajoensis. Antisera from horses immunized with individual crotoxin or PLA2, although in lesser titers, were also able of recognizing the toxins in all four Crotalus species and neutralize the lethality of the C. d. terrificus venom.

Ctenus medius and Phoneutria nigriventer spiders venoms share noxious proinflammatory activities.

ABSTRACT Ctenus medius Keyserling, 1891 (Araneae: Ctenidae) co-occurs in various microhabitats of the Brazilian Atlantic Forest and can be easily misidentified as the medically important spider Phoneutria nigriventer Keyserling, 1981 (Ctenidae). Despite being phylogenetically close to Phoneutria, no data are available about the toxic potential of Ctenus medius venom. Here we show that, although presenting different profile of protein composition, C. medius venom displays some of the toxic properties exhibited by P. nigriventer venom, including proteolytic, hyaluronidasic and phospholipasic activities, as well as the ability of causing hyperalgesia and edema. Moreover, C. medius venom interferes in the activation of the complement system in concentrations that P. nigriventer venom is inactive. Thus, these data show that venoms of spiders from Ctenidae family share important proinflammatory properties and suggest that the C. medius bite may have an important noxious effect in human accidents.

Sphingomyelinase D from Loxosceles laeta Venom Induces the Expression of MMP7 in Human Keratinocytes: Contribution to Dermonecrosis

Envenomation by Loxosceles spider is characterized by the development of dermonecrosis. In previous studies, we have demonstrated that increased expression/secretion of matrix metalloproteinases 2 and 9, induced by Loxosceles intermedia venom Class 2 SMases D (the main toxin in the spider venom), contribute to the development of cutaneous loxoscelism. In the present study we show that the more potent venom containing the Class 1 SMase D from Loxosceles laeta, in addition to increasing the expression/secretion of MMP2 and MMP9, also stimulates the expression of MMP7 (Matrilysin-1), which was associated with keratinocyte cell death. Tetracycline, a matrix metalloproteinase inhibitor, prevented cell death and reduced MMPs expression. Considering that L. laeta venom is more potent at inducing dermonecrosis than L. intermedia venom, our results suggest that MMP7 may play an important role in the severity of dermonecrosis induced by L. laeta spider venom SMase D. In addition, the inhibition of MMPs by e.g. tetracyclines may be considered for the treatment of the cutaneous loxoscelism.

Tetracycline Reduces Kidney Damage Induced by Loxosceles Spider Venom

Envenomation by Loxosceles spider can result in two clinical manifestations: cutaneous and systemic loxoscelism, the latter of which includes renal failure. Although incidence of renal failure is low, it is the main cause of death, occurring mainly in children. The sphingomyelinase D (SMase D) is the main component in Loxosceles spider venom responsible for local and systemic manifestations. This study aimed to investigate the toxicity of L. intermedia venom and SMase D on kidney cells, using both In vitro and in vivo models, and the possible involvement of endogenous metalloproteinases (MMP). Results demonstrated that venom and SMase D are able to cause death of human kidney cells by apoptosis, concomitant with activation and secretion of extracellular matrix metalloproteases, MMP-2 and MMP-9. Furthermore, cell death and MMP synthesis and secretion can be prevented by tetracycline. In a mouse model of systemic loxoscelism, Loxosceles venom-induced kidney failure was observed, which was abrogated by administration of tetracycline. These results indicate that MMPs may play an important role in Loxosceles venom-induced kidney injury and that tetracycline administration may be useful in the treatment of human systemic loxoscelism.

Molecular Cloning, Expression, Function, Structure and Immunoreactivities of a Sphingomyelinase D from Loxosceles adelaida, a Brazilian Brown Spider from Karstic Areas

Loxosceles is the most poisonous spider in Brazil and, at least, three different species of medical importance are known in Brazil (L. intermedia, L. gaucho, L. laeta), with more than 5000 cases of envenomation reported each year. In South Africa, L. parrami and L. spinulosa are responsible for cutaneous loxoscelism (Newlands et al., 1982). In Australia, a cosmopolitan species, L. rufescens, is capable of causing ulceration in humans. In the USA, at least five Loxosceles species, including L. reclusa (brown recluse), L. apachea, L. arizonica, L. unicolor and L. deserta are known to cause numerous incidents (Ginsburg W Gendron, 1990; Bey et al., 1997; Desai et al., 2000). Several studies have indicated that sphingomyelinase D (SMase D) present in the venoms of Loxosceles spiders is the main component responsible for the local and systemic effects observed in loxoscelism (Forrester et al., 1978; Kurpiewski et al., 1981; Tambourgi et al., 1998, 2000, 2002, 2004, 2005, 2007; van den Berg et al., 2002, 2007; Fernandes Pedrosa et al., 2002; Paixao Cavalcanti et al., 2006, Tambourgi et al., 2010). SMases D hydrolyze sphingomyelin resulting in the formation of ceramide-1-phosphate and choline (Forrester et al., 1978; Kurpiewski et al., 1981; Tambourgi et al., 1998) and, in the presence of Mg2+, are able to catalyze the release of choline from lysophosphatidylcholine (van Meeteren et al., 2004). All spider venom SMases D sequenced to date display a significant level of sequence similarity and thus likely possess the same (┙/┚)8 or TIM barrel fold (Murakami et al., 2005, 2006). Based on sequence identity, biochemical activity and molecular modelling, a scheme for classification of spider venom SMases D was proposed (Murakami et al., 2006). The class 1 enzymes include SMase I and H13, SMases D from L. laeta, which possess a single disulphide bridge and contain an extended hydrophobic loop or variable loop (Murakami et