RESUMEN

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.

Asunto(s)

Hidrolasas Diéster Fosfóricas/toxicidad , Sustancias Protectoras/uso terapéutico , Insuficiencia Renal/inducido químicamente , Insuficiencia Renal/tratamiento farmacológico , Venenos de Araña/toxicidad , Tetraciclina/uso terapéutico , Animales , Caspasa 3/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Endogámicos BALB C , Sustancias Protectoras/farmacología , Proteinuria/inducido químicamente , Insuficiencia Renal/patología , Arañas , Tetraciclina/farmacología

RESUMEN

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.

Asunto(s)

Proteínas de Artrópodos/farmacología , Queratinocitos/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/farmacología , Venenos de Araña/farmacología , Arañas/enzimología , Animales , Antibacterianos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Queratinocitos/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Necrosis/prevención & control , Conejos , Piel/efectos de los fármacos , Piel/patología , Venenos de Araña/enzimología , Tetraciclina/farmacología , Factores de Tiempo

RESUMEN

BACKGROUND: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system. RESULTS: The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. CONCLUSION: These data show that a serine protease present in the Premolis semirufa's bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

Asunto(s)

Activación de Complemento/efectos de los fármacos , Proteínas de Insectos/farmacología , Mariposas Nocturnas/enzimología , Serina Proteasas/farmacología , Anafilatoxinas/química , Anafilatoxinas/aislamiento & purificación , Animales , Complejo de Ataque a Membrana del Sistema Complemento/química , Eritrocitos/efectos de los fármacos , Humanos , Proteínas de Insectos/aislamiento & purificación , Proteolisis , Serina Proteasas/aislamiento & purificación

RESUMEN

BACKGROUND: The spider family Sicariidae includes two genera, Sicarius and Loxosceles. Bites by Sicarius are uncommon in humans and, in Brazil, a single report is known of a 17-year old man bitten by a Sicarius species that developed a necrotic lesion similar to that caused by Loxosceles. Envenomation by Loxosceles spiders can result in dermonecrosis and severe ulceration. Sicarius and Loxosceles spider venoms share a common characteristic, i.e., the presence of Sphingomyelinases D (SMase D). We have previously shown that Loxosceles SMase D is the enzyme responsible for the main pathological effects of the venom. Recently, it was demonstrated that Sicarius species from Africa, like Loxosceles spiders from the Americas, present high venom SMase D activity. However, despite the presence of SMase D like proteins in venoms of several New World Sicarius species, they had reduced or no detectable SMase D activity. In order to contribute to a better understanding about the toxicity of New World Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from the Brazilian Sicarius ornatus spider and compare these with venoms from Loxosceles species of medical importance in Brazil. METHODOLOGY/PRINCIPAL FINDINGS: SDS-PAGE analysis showed variations in the composition of Loxosceles spp. and Sicarius ornatus venoms. Differences in the electrophoretic profiles of male and female venoms were also observed, indicating a possible intraspecific variation in the composition of the venom of Sicarius spider. The major component in all tested venoms had a Mr of 32-35 kDa, which was recognized by antiserum raised against Loxosceles SMases D. Moreover, male and female Sicarius ornatus spiders' venoms were able to hydrolyze sphingomyelin, thus showing an enzymatic activity similar to that determined for Loxosceles venoms. Sicarius ornatus venoms, as well as Loxosceles venoms, were able to render erythrocytes susceptible to lysis by autologous serum and to induce a significant loss of human keratinocyte cell viability; the female Sicarius ornatus venom was more efficient than male. CONCLUSION: We show here, for the first time, that the Brazilian Sicarius ornatus spider contains active Sphingomyelinase D and is able to cause haemolysis and keratinocyte cell death similar to the South American Loxosceles species, harmful effects that are associated with the presence of active SMases D. These results may suggest that envenomation by this Sicarius spider has the potential to cause similar pathological events as that caused by Loxosceles envenomation. Our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms composition may play a role in the toxic potential of the New World Sicarius venoms species.

Asunto(s)

Arácnidos , Hidrolasas Diéster Fosfóricas/toxicidad , Picaduras de Arañas/patología , Ponzoñas/enzimología , Animales , Brasil , Electroforesis en Gel de Poliacrilamida , Eritrocitos/efectos de los fármacos , Femenino , Hemólisis , Humanos , Masculino , Peso Molecular , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/aislamiento & purificación , Esfingomielinas/metabolismo , Ponzoñas/química

RESUMEN

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, such as intravascular haemolysis, intravascular coagulation and renal failure. Current therapies are not effective, often not based in scientific research and can be even detrimental. A lack of understanding of the mechanism of action of the venom of the Loxosceles spider had thus far prevented development of effective therapies. In this review we aim to give an overview of our contributions to the understanding of the mechanism of action of the Loxosceles venom and propose targets and therapeutics for medical intervention.

Asunto(s)

Picaduras de Arañas/etiología , Venenos de Araña/toxicidad , Animales , Antivenenos/química , Antivenenos/uso terapéutico , Humanos , Modelos Animales , Conejos , Picaduras de Arañas/tratamiento farmacológico , Picaduras de Arañas/patología , Arañas/anatomía & histología , Arañas/química , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

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 asdermonecrosis, and systemic reactions, such as intravascular haemolysis, intravascular coagulation and renal failure. Current therapies are not effective, often not based in scientific research and can be even detrimental. A lack of understanding of the mechanism of action of the venom of the Loxosceles spider had thus far prevented development of effective therapies. In this review we aim to give an overview of our contributions to the understanding of the mechanism of action of the Loxosceles venom and propose targets and therapeutics for medical intervention.

Asunto(s)

Humanos , Arácnidos/clasificación , Venenos de Araña/efectos adversos , Venenos de Araña/inmunología , Venenos de Araña/uso terapéutico , Hemólisis , Metaloproteasas , Tetraciclinas , Venenos de Araña/química

RESUMEN

Sphingomyelinase D (SMase D) present in the venoms of Loxosceles spiders is the principal component responsible for local and systemic effects observed in the loxoscelism. By using "expressed sequencing tag", it was possible to identify, in a L. laeta venom gland library, clones containing inserts coding for proteins with similarity to SMase D. One of these clones was expressed and the recombinant protein compared with the previously characterized SMase I from L. laeta, in terms of their biological, biochemical and structural properties. The new recombinant protein, SMase II, possesses all the biological properties ascribed to the whole venom and SMase I. SMase II shares 40% and 77% sequence similarity with SMase I and Lb3, respectively; the latter, a SMase D isoform from L. boneti, catalytically inactive. Molecular modeling and molecular dynamics simulations were employed to understand the structural basis, especially the presence of an additional disulfide bridge, in an attempt to account for the observed differences in SMases D activity.

Asunto(s)

Glándulas Exocrinas/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Venenos de Araña/enzimología , Secuencia de Aminoácidos , Western Blotting , Tampones (Química) , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Eritrocitos/efectos de los fármacos , Citometría de Flujo , Hemólisis/efectos de los fármacos , Humanos , Indicadores y Reactivos , Modelos Moleculares , Datos de Secuencia Molecular , Necrosis/inducido químicamente , Necrosis/patología , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Proteínas Recombinantes/química , Piel/patología

RESUMEN

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.

Asunto(s)

Venenos de Araña/toxicidad , Arañas/química , Animales , Western Blotting , Edema/inducido químicamente , Electroforesis en Gel de Poliacrilamida , Femenino , Hiperalgesia/inducido químicamente , Masculino , Ratas , Ratas Wistar , Venenos de Araña/enzimología

RESUMEN

BACKGROUND: 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.

Asunto(s)

Arañas/genética , Secuencia de Aminoácidos , Animales , Etiquetas de Secuencia Expresada , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Biblioteca de Genes , Humanos , Hialuronoglucosaminidasa/genética , Datos de Secuencia Molecular , Hidrolasas Diéster Fosfóricas/genética , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Picaduras de Arañas , Venenos de Araña/genética , Arañas/patogenicidad , Arañas/fisiología

RESUMEN

Envenomation by spiders belonging to the Loxosceles genus (brown spider) often results in local dermonecrotic lesions. We have previously shown that Loxosceles sphingomyelinase D (SMase D), the venom component responsible for all the pathological effects, induced the expression of matrix metalloproteinases (MMPs) in rabbits and in human keratinocytic cells. We also showed that the SMase D-induced apoptosis and MMP expression of keratinocytes was inhibited by tetracyclines. We have further investigated the ability of tetracyclines to inhibit or prevent the dermonecrotic lesion induced by Loxosceles venom in vivo and in vitro models. Primary cultures of rabbit fibroblasts incubated with increasing concentrations of venom or SMase D showed a decrease in cell viability, which was prevented by tetracyclines. In vivo experiments showed that topical treatments with tetracycline of rabbits, inoculated with crude Loxosceles intermedia venom or recombinant SMase D, significantly reduced the progression of the dermonecrotic lesion. Furthermore, tetracyclines also reduced the expression of MMP-2 and prevented the induction of MMP-9. Our results suggest that tetracycline may be an effective therapeutic agent for the treatment of cutaneous loxoscelism.

Asunto(s)

Hidrolasas Diéster Fosfóricas/toxicidad , Enfermedades de la Piel/tratamiento farmacológico , Enfermedades de la Piel/etiología , Venenos de Araña/toxicidad , Tetraciclinas/farmacología , Animales , Mordeduras y Picaduras , Línea Celular , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Necrosis , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Conejos , Enfermedades de la Piel/patología , Esfingomielina Fosfodiesterasa/toxicidad , Arañas

RESUMEN

Envenomation by the spider Loxosceles (brown spider) can result in dermonecrosis and severe ulceration. We have previously shown that Loxosceles sphingomyelinase D (SMaseD), the enzyme responsible for these pathological effects, induced expression of matrix metalloproteinase-9 (MMP-9), which is possibly one of the main factors involved in the pathogenesis of the cutaneous loxoscelism. The aim of this study was to further investigate the molecular mechanisms triggered by Loxosceles SMaseD involved in the initiation of the dermonecrotic lesion, using HaCaT cultures, a human keratinocyte cell line, as an in vitro model for cutaneous loxoscelism. We show here that SMaseD from Loxosceles spider venom induces apoptosis in human keratinocytes, which is associated with an increased expression of metalloproteinase-2 and -9, and that the use of metalloproteinase inhibitors, such as tetracycline, may prevent cell death and potentially may prevent tissue destruction after envenomation.

Asunto(s)

Apoptosis , Queratinocitos/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Hidrolasas Diéster Fosfóricas/farmacología , Venenos de Araña/enzimología , Anexina A5/metabolismo , Biomarcadores/análisis , Línea Celular Transformada , Humanos , Queratinocitos/enzimología , Propidio/metabolismo

RESUMEN

Envenomation by the spider Loxosceles can result in dermonecrosis and severe ulceration. Our aim was to investigate the role of the complement system and of the endogenous metalloproteinases in the initiation of the pathology of dermonecrosis. Histological analysis of skin of rabbits injected with Loxosceles intermedia venom and purified or recombinant sphingomyelinases showed a large influx of neutrophils, concomitant with dissociation of the collagenous fibers in the dermis. Decomplementation, using cobra venom factor, largely prevented the influx of neutrophils, while influx of neutrophils was also reduced in genetically C6-deficient rabbits, suggesting roles for both C5a and the membrane attack complex in the induction of dermonecrosis. However, C-depletion and C6 deficiency did not prevent the haemorrhage and the collagen injury. Zymography analysis of skin extracts showed the induction of expression of the endogenous gelatinase MMP-9 in the skin of envenomated animals. Rabbit neutrophils contained high levels of MMP-9, expression of which was further increased after incubation with venom, suggesting that these cells may be a source of the MMP-9 found in the skin of envenomated animals. Furthermore, skin fibroblasts also secreted MMP-9 and MMP-2 upon incubation with venom, suggesting that locally produced MMPs can also contribute to proteolytic tissue destruction.

Asunto(s)

Proteínas del Sistema Complemento/inmunología , Metaloproteinasa 2 de la Matriz/metabolismo , Enfermedades de la Piel/inducido químicamente , Esfingomielina Fosfodiesterasa/toxicidad , Picaduras de Arañas/inmunología , Picaduras de Arañas/patología , Venenos de Araña/toxicidad , Animales , Proteínas del Sistema Complemento/efectos de los fármacos , Eritrocitos/inmunología , Fibroblastos/enzimología , Fibroblastos/patología , Hemólisis , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Necrosis , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/patología , Conejos , Ovinos , Enfermedades de la Piel/inmunología , Enfermedades de la Piel/patología , Picaduras de Arañas/metabolismo , Arañas

RESUMEN

Envenomation by Loxosceles spiders causes two main clinical manifestations: cutaneous and systemic loxoscelism. The factors contributing to the severity of loxoscelism are not fully understood. We have analysed biochemical and toxicity variations in venom of L. laeta and L. intermedia, with the aim to find a correlation with the seriousness of loxoscelism. Differences in expression of proteins, glycoproteins and sphingomyelinase activity were observed between venom from male and female spiders and between venom from the two species. These differences were reflected in the toxicity of the venoms including the capacity to induce complement-dependent haemolysis, dermonecrosis and lethality. Comparative analysis of gender and species, showed that these biological activities were more prominent in venom from female spiders, especially from L. laeta. Antiserum raised against venom from females L. laeta spiders had the highest efficacy in neutralizing venoms of males and females of both species. These results indicate that the severity of loxoscelism depends, at least partially, on the species and sex of the spider and suggest that for accidents involving L. laeta an specific serum therapy is necessary. Furthermore, it emphasizes the efficacy of the antiserum produced against L. laeta female venom in neutralizing Loxosceles venoms from different species and gender.

Asunto(s)

Antivenenos/metabolismo , Piel/patología , Picaduras de Arañas/metabolismo , Venenos de Araña/química , Arañas/química , Análisis de Varianza , Animales , Antivenenos/uso terapéutico , Western Blotting , Electroforesis en Gel de Poliacrilamida , Eritrocitos/efectos de los fármacos , Femenino , Citometría de Flujo , Hemólisis/efectos de los fármacos , Humanos , Dosificación Letal Mediana , Masculino , Ratones , Ratones Endogámicos BALB C , Necrosis/inducido químicamente , Factores Sexuales , Especificidad de la Especie , Esfingomielina Fosfodiesterasa/metabolismo , Picaduras de Arañas/inducido químicamente , Picaduras de Arañas/tratamiento farmacológico , Venenos de Araña/toxicidad

RESUMEN

Loxosceles adelaida spiders (Araneae, Sicariidae) are found near and inside the caves in the Parque Estadual Turistico do Alto Ribeira (PETAR), Sao Paulo, Brazil, which are visited by thousands of tourists every year. Several Loxosceles species are a public health problem in many regions of the world, by causing severe dermonecrosis and/or complement dependent haemolysis upon envenomation. The aim of this study was to characterize the biochemical and biological properties of L. adelaida venom and evaluate the toxic potential of envenomation by this non-synanthropic Loxosceles species. The biological activities of the L. adelaida venom was compared to that of Loxosceles gaucho, a synanthropic species of medical importance in Brazil. L. adelaida venom showed a similar potential to induce haemolysis, dermonecrosis and lethality as L. gaucho venom. L. adelaida crude venom was purified, yielding a 31 kDa component endowed with haemolytic and dermonecrotic activities. In conclusion, we show here that the troglophile Loxosceles species, L. adelaida, commonly found in the complex of caves from PETAR, is potentially able to cause envenomation with the same gravity of those produced by synanthropic species.

Asunto(s)

Hemólisis/efectos de los fármacos , Piel/patología , Esfingomielina Fosfodiesterasa/toxicidad , Venenos de Araña/química , Arañas/química , Animales , Western Blotting , Brasil , Cromatografía Líquida de Alta Presión , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Eritrocitos/efectos de los fármacos , Citometría de Flujo , Dosificación Letal Mediana , Ratones , Necrosis/inducido químicamente , Conejos , Especificidad de la Especie , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Venenos de Araña/enzimología , Arañas/enzimología

RESUMEN

Loxoscelism is the clinical condition produced by the venom of spiders belonging to the genus Loxosceles, which can be observed as two well-defined clinical variants: cutaneous loxoscelism and systemic or viscerocutaneous loxoscelism. We have recently identified, purified and characterised the toxins (sphingomyelinases) from Loxosceles intermedia venom that are responsible for all the local (dermonecrosis) and systemic effects (complement dependent haemolysis) induced by whole venom. In the present study, we have cloned and expressed the two functional sphingomyelinases isoforms, P1 and P2, and shown that the recombinant proteins display all the functional characteristics of whole L. intermedia venom, e.g., dermonecrotic and complement-dependent hemolytic activities and ability of hydrolyzing sphingomyelin. We have also compared the cross-reactivities of antisera raised against the toxins from different Loxosceles species and show here that the cross-reactivity is high when toxins are from the same species (P1 and P2 from L. intermedia) but low when the toxins are from different species (L. intermedia versus L. laeta). These data suggest that in order to obtain a suitable comprehensive neutralizing antiserum using the recombinant toxin as an immunogen, a mixture of the recombinant toxins from the different species has to be used. The use of anti-recombinant toxin antisera may have clinical benefits to those individuals displaying acute loxoscelic lesions.

Asunto(s)

Hidrolasas Diéster Fosfóricas/genética , Esfingomielina Fosfodiesterasa/genética , Venenos de Araña/genética , Arañas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Reacciones Cruzadas , Datos de Secuencia Molecular , Familia de Multigenes , Hidrolasas Diéster Fosfóricas/inmunología , Hidrolasas Diéster Fosfóricas/metabolismo , Filogenia , Esfingomielina Fosfodiesterasa/inmunología , Esfingomielina Fosfodiesterasa/metabolismo , Venenos de Araña/inmunología , Venenos de Araña/metabolismo , Arañas/inmunología , Arañas/metabolismo

RESUMEN

The present paper deals with the phlebotomine species captured during the period from January 1998 to June 2000 in 12 caves located in the Serra da Bodoquena, situated in the south central region of Mato Grosso do Sul State, Brazil. Three of the caves are situated further north (in Bodoquena county), seven in the central area (Bonito county) and two in the south (Jardim county). These last two caves and three of those in Bonito are located at the west side of the ridge. Eighteen species of phlebotomines were captured within the caves: Brumptomyia avellari (Costa Lima, 1932), Brumptomyia brumpti (Larrousse, 1920), Brumptomyia cunhai (Mangabeira, 1942), Brumptomyia galindoi (Fairchild & Hertig, 1947), Evandromyia corumbaensis (Galati, Nunes, Oshiro & Rego, 1989), Lutzomyia almerioi Galati & Nunes, 1999, Lutzomyia longipalpis (Lutz & Neiva, 1912), Martinsmyia oliveirai (Martins, Falcão & Silva, 1970), Micropygomyia acanthopharynx (Martins, Falcão & Silva, 1962), Micropygomyia peresi (Mangabeira, 1942), Micropygomyia quinquefer (Dyar, 1929), Nyssomyia whitmani (Antunes & Coutinho, 1939), Psathyromyia campograndensis (Oliveira, Andrade-Filho, Falcão & Brazil, 2001), Psathyromyia punctigeniculata (Floch & Abonnenc, 1944), Psathyromyia shannoni (Dyar, 1929), Pintomyia kuscheli (Le Pont, Martinez, Torrez-Espejo & Dujardin, 1998), Sciopemyia sordellii (Shannon & Del Ponte, 1927) and Sciopemyia sp. A total of 29,599 phlebotomine sandflies was obtained. Lutzomyia almerioi was absolutely predominant (91.5 per cent) over the other species on both sides of the Bodoquena ridge, with the exception of the southern caves in which it was absent. It presents summer predominance, with nocturnal and diurnal activities. The species breeds in the caves and was captured during daytime both in the dark area and in the mouth of the caves. Martinsmyia oliveirai, the second most frequent sandfly, also presents a summer peak and only predominated over the other species in one cave, in which there were human residues.

Asunto(s)

Animales , Dípteros , Ecología , Psychodidae

RESUMEN

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.

Asunto(s)

Venenos de Araña/genética , Arañas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Activación de Complemento/efectos de los fármacos , ADN Complementario/genética , Eritrocitos/efectos de los fármacos , Expresión Génica , Glicoforinas/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Necrosis , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/toxicidad , Picaduras de Arañas/etiología , Venenos de Araña/enzimología , Venenos de Araña/toxicidad , Arañas/enzimología , Arañas/patogenicidad

RESUMEN

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.

Asunto(s)

Activación de Complemento/efectos de los fármacos , Hemólisis/efectos de los fármacos , Fosfolipasa D/farmacología , Esfingomielina Fosfodiesterasa/farmacología , Venenos de Araña/farmacología , Animales , Anexina A5/metabolismo , Sitios de Unión , Corynebacterium pseudotuberculosis , Relación Dosis-Respuesta Inmunológica , Membrana Eritrocítica/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/inmunología , Glicoforinas/metabolismo , Humanos , Arañas