RESUMEN
"Black widow" spiders belong to the genus Latrodectus and are one of the few spiders in the world whose bite can cause severe envenomation in humans and domestic animals. In Argentina, these spiders are distributed throughout the country and are responsible for the highest number of bites by spiders of toxicological sanitary interest. Here, we studied the toxicity and some biochemical and immunochemical characteristics of eighteen venom samples from Latrodectus spiders from eight different provinces of Argentina, and the neutralization of some of these samples by two therapeutic antivenoms used in the country for the treatment of envenomation and by a anti-Latrodectus antivenom prepared against the venom of Latrodectus mactans from Mexico. We observed important toxicity in all the samples studied and a variation in the toxicity of samples, even in those from the same region and province and even in the same Latrodectus species from the same region. The therapeutic antivenoms efficiently neutralized all the venoms studied.
Asunto(s)
Antivenenos/uso terapéutico , Venenos de Araña/toxicidad , Animales , Argentina , Araña Viuda Negra , Femenino , Geografía , Ratones , Venenos de Araña/antagonistas & inhibidoresRESUMEN
Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Proteínas de Artrópodos/antagonistas & inhibidores , Araña Reclusa Parda/enzimología , Diseño de Fármacos , Fosfolipasa D/antagonistas & inhibidores , Venenos de Araña/antagonistas & inhibidores , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Bencimidazoles/farmacología , Araña Reclusa Parda/genética , Araña Reclusa Parda/patogenicidad , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hemólisis/efectos de los fármacos , Humanos , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Necrosis , Fosfolipasa D/química , Fosfolipasa D/genética , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Piperidinas/farmacología , Conejos , Proteínas Recombinantes/genética , Piel/efectos de los fármacos , Piel/patología , Picaduras de Arañas/tratamiento farmacológico , Picaduras de Arañas/enzimología , Venenos de Araña/química , Venenos de Araña/genética , Suramina/farmacologíaRESUMEN
An important step in the development of therapeutic antivenoms is the pre-clinical testing using in vivo methods to assess their neutralizing potency. For spider antivenoms (Loxosceles species), horse serum potency against the necrotizing activities of Loxosceles intermedia crude venom is currently tested in rabbits. These procedures are time consuming and involve a large number of animals. The aim of this study was to develop an in vitro method to assess the neutralizing potency of anti-Loxosceles sera. We first demonstrated that it was not possible to establish a correlation between the ELISA antibody reactivity of horse anti-Loxosceles serum and their neutralizing potency. We then showed that the antivenoms recognized several peptide epitopes from different regions of SMase-D proteins, which are toxic antigens from Loxosceles venoms. The recognition of some peptides was observed only when high neutralizing potency sera was used. Based on these results, three peptides (peptide 1, DNRRPIWNLAHMVNA and peptide 3, DFSGPYLPSLPTLDA corresponding to residues 2-16 and 164-178, respectively, of SMase-1 protein from Loxosceles laeta, and peptide 2, EFVNLGANSIETDVS corresponding to residues 22-36 of A1H - LoxGa protein from Loxosceles gaucho and LiD1 protein from L. intermedia) were selected. The peptides were synthesized, coupled to bovine serum albumin (BSA), and used as antigens in indirect ELISA to test their reactivity with horse anti-Loxosceles serum of varying neutralizing potencies. We found certain assay conditions that discriminated between the high and low neutralizing potency sera. This study introduced an in vitro and peptide-based neutralization assay for anti-Loxosceles antivenoms.
Asunto(s)
Antivenenos/biosíntesis , Antivenenos/farmacología , Diseño de Fármacos , Pruebas de Neutralización/métodos , Venenos de Araña/antagonistas & inhibidores , Arañas/química , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Biología Computacional , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Epítopos/metabolismo , Caballos/sangre , Sueros Inmunes/metabolismo , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Albúmina Sérica Bovina , Arañas/enzimologíaRESUMEN
Loxosceles spiders are found globally, especially in South and North America. In Brazil, approximately 10,000 cases of Loxosceles spp. spider bites are reported annually. Herein we analyzed 81 patients diagnosed as either cutaneous or cutaneous-hemolytic loxoscelism, in a geographical area where most accidents are caused by Loxosceles gaucho, and we report their clinical and laboratory data obtained during week 1 and 2 after the bite. Massive hemolysis was noticed in only 2 cases, but high serum bilirubin and LDH levels, suggestive of hemolysis, were noticed in 25 cases on admission. Anemia was not frequent (14.7%), and reticulocytosis was particularly noticed during week 2 (in 56% of patients). High D-dimer levels were suggestive of endothelial cell activation and intravascular thrombin generation, but thrombocytopenia was noticed in only 17.6% of patients in week 1. Acute kidney injury (AKI) only occurred in patients with massive hemolysis. The definitive diagnosis of overt disseminated intravascular coagulation (DIC) could not be established on admission. Fever was associated with the presence of hemolysis (p = 0.03). Altogether, these findings provide evidence that mild hemolysis is frequent in loxoscelism and suggest that AKI is uncommon, exclusively occurring in patients with massive hemolysis.
Asunto(s)
Hidrolasas Diéster Fosfóricas/toxicidad , Enfermedades de la Piel/diagnóstico , Picaduras de Arañas/diagnóstico , Venenos de Araña/toxicidad , Arañas , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/etiología , Adolescente , Adulto , Anciano , Anemia/inducido químicamente , Anemia/etiología , Animales , Antivenenos/uso terapéutico , Bilirrubina/sangre , Brasil , Niño , Preescolar , Coagulación Intravascular Diseminada/inducido químicamente , Coagulación Intravascular Diseminada/etiología , Femenino , Hemólisis/efectos de los fármacos , Humanos , L-Lactato Deshidrogenasa/sangre , Masculino , Persona de Mediana Edad , Reticulocitosis/efectos de los fármacos , Piel/efectos de los fármacos , Piel/patología , Enfermedades de la Piel/etiología , Enfermedades de la Piel/terapia , Picaduras de Arañas/complicaciones , Picaduras de Arañas/terapia , Venenos de Araña/antagonistas & inhibidores , Adulto JovenRESUMEN
The venom of Loxosceles intermedia (Li) spiders is responsible for cutaneous lesions and other clinical manifestations. We previously reported that the monoclonal antibody LimAb7 can neutralize the dermonecrotic activity of crude Li venom. In this study, we observed that this antibody recognizes several proteins from the venom dermonecrotic fraction (DNF), including LiD1. Identifying the epitope of such a neutralizing antibody could help designing immunogens for producing therapeutic sera or vaccination approaches. To this aim, two sets of 25- and 15-mer overlapping peptides that cover the complete amino acid sequence of LiD1 were synthesized using the SPOT technique. None of them was recognized by LimAb7, suggesting that the epitope is discontinuous. Then, the screening of four peptide phage-display libraries yielded four possible epitope mimics that, however, did not show any obvious similarity with the LiD1 sequence. These mimotopes, together with a 3D model of LiD1, were used to predict with the MIMOP bioinformatic tool the putative epitope region (residues C197, Y224, W225, T226, D228, K229, R230, T232 and Y248 of LiD1) recognized by LimAb7. This analysis and the results of alanine-scanning experiments highlighted a few residues (such as W225 and D228) that are found in the active site of different SMases D and that may be important for LiD1 enzymatic activity. Finally, the only mimotope NCNKNDHLFACW that interacts with LimAb7 by SPOT and its analog NSNKNDHLFASW were used as immunogens in rabbits. The resulting antibodies could neutralize some of the biological effects induced by crude Li venom, demonstrating a mimotope-induced protection against L. intermedia venom.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Antitoxinas/sangre , Arácnidos , Epítopos/inmunología , Venenos de Araña/antagonistas & inhibidores , Vacunas de Subunidad/inmunología , Animales , Mapeo Epitopo , Femenino , Biblioteca de Péptidos , Perciformes , Conejos , Venenos de Araña/toxicidadRESUMEN
Envenomation by spiders of the genus Phoneutria ("banana spider") may be lethal, especially in children. The only available specific treatment is the use of antivenom, which is produced by only one laboratory in the world. In this study we report the development of an equine F (ab')2 experimental antivenom raised against the venom of Phoneutria nigriventer. The antivenom neutralized the venom of spiders from different regions of Argentina (Misiones and Jujuy), the development of envenomation symptoms in experimental animals was totally inhibited. These results show that local production of this type of antivenom is possible. Independence of production is important since international acquisition is always conditioned by the availability of stock surplus from the sole producer.
Asunto(s)
Antivenenos/biosíntesis , Venenos de Araña/antagonistas & inhibidores , AnimalesRESUMEN
Antibodies raised against recombinant Loxosceles intermedia dermonecrotic protein isoform 1 (rLiD1) display neutralizing capacity for the L. intermedia whole venom. We previously found that an immunodominant continuous B-cell epitope, recognized by these antibodies corresponds to a region of the protein known to be involved in the active site. In this study, we extend previous work by preparing a 27-residue synthetic replica of this epitope ((25)NLGANSIETDVSFDDNANPEYTYHGIP(51)) and using it as an immunogen in mice and rabbits. The immunization process induced antibodies that protected mice from a lethal dose of L. intermedia crude venom and rabbits against the dermonecrotic effects of rLiD1. An Ala scan of the epitope indicated that 4 residues, E44, Y45, T46 and Y47, are essential (over 70% decrease in binding upon replacement with alanine) for antibody recognition. The possible mechanisms of neutralization are discussed in light of these findings.
Asunto(s)
Antígenos/química , Antígenos/inmunología , Antivenenos/farmacología , Hemorragia/inducido químicamente , Fragmentos de Péptidos/inmunología , Venenos de Araña/inmunología , Venenos de Araña/toxicidad , Secuencia de Aminoácidos , Animales , Antivenenos/biosíntesis , Edema/inducido químicamente , Edema/patología , Ensayo de Inmunoadsorción Enzimática , Epítopos , Femenino , Hemorragia/sangre , Esquemas de Inmunización , Dosificación Letal Mediana , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Necrosis/inducido químicamente , Necrosis/patología , Pruebas de Neutralización , Conejos , Proteínas Recombinantes , Enfermedades de la Piel/inducido químicamente , Enfermedades de la Piel/patología , Venenos de Araña/antagonistas & inhibidoresRESUMEN
Loxoscelism is a necrotic-hemolytic syndrome caused by bites of brown spiders belonging to the genus Loxosceles. Many approaches for the treatment of Loxosceles poisoning have already been proposed, among which administration of specific antivenom is thought to be the more specific. We have evaluated the use of peptides as immunogen to raise in rabbits an antibody response that could protect animals from a challenge by the Loxtox isoform LiD1, one of the main toxic component of Loxosceles intermedia venom. Six antigenic regions of LiD1 were mapped by using the SPOT method. The corresponding peptides were further chemically synthesized, mixed, and used as immunogens in rabbits. Control animal received recombinant LiD1 alone or together with peptides. We found that the rabbit antibody response to peptides was cross-reactive with LiD1, although only one peptide from the mix of six was immunogenic. The dermonecrotic, hemorrhagic and oedema forming activities induced by LiD1 in naïve rabbits were inhibited by 82%, 35% and 35% respectively, by preincubation of LiD1 with anti-peptide antibodies prepared from immunized rabbits. Animals that were immunized with peptides or LiD1r, were found to be protected from the dermonecrotic, hemorrhagic and oedema forming activities induced by a challenge with LiD1. The protection conferred by peptides was, however, lower than that provided by the peptide protein combination or by the full-length protein. These results encourage us in the utilization of synthetic peptides for therapeutic serum development or vaccination approaches.
Asunto(s)
Epítopos/inmunología , Mordeduras y Picaduras de Insectos/inmunología , Hidrolasas Diéster Fosfóricas/inmunología , Venenos de Araña/antagonistas & inhibidores , Venenos de Araña/inmunología , Arañas , Animales , Edema/prevención & control , Mapeo Epitopo , Femenino , Hemorragia/prevención & control , Necrosis/prevención & control , Conejos , Vacunas Sintéticas/inmunologíaRESUMEN
A 17-year-old male was envenomated on the right forearm by a black widow spider that had presumably traveled in a packaged dishwasher and been shipped from Mexico to Michigan. The patient experienced vomiting and severe pain in his abdomen and chest approximately 30 min after being bitten. He received 6000 units (1 vial) of Latrodectus antivenin intravenously about 7 h after he was envenomated. He did not experience significant improvement in his symptoms after the administration of antivenin and additional antivenin was not given. The patient was hospitalized for 7 days and still was complaining of intermittent episodes of pain in his chest and lower back 3 weeks after envenomation. To avoid prolonged symptomatology and hospitalization, additional Latrodectus antivenin should be given promptly to those individuals whose symptoms are not ameliorated after 1 vial.
Asunto(s)
Antivenenos/uso terapéutico , Araña Viuda Negra , Picaduras de Arañas/fisiopatología , Adolescente , Animales , Estudios de Seguimiento , Humanos , Masculino , México , Michigan , Dolor/etiología , Manejo del Dolor , Picaduras de Arañas/terapia , Venenos de Araña/antagonistas & inhibidores , Factores de Tiempo , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Antivenenos/farmacología , Hidrolasas Diéster Fosfóricas/inmunología , Picaduras de Arañas/terapia , Venenos de Araña/antagonistas & inhibidores , Animales , Células Cultivadas , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Eritrocitos/efectos de los fármacos , Caballos , Humanos , Inmunoquímica , Pruebas de Neutralización , Hidrolasas Diéster Fosfóricas/metabolismo , Conejos , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Venenos de Araña/enzimología , Venenos de Araña/inmunología , Arañas/enzimología , Arañas/metabolismoRESUMEN
The ability of IgG(T) and IgGa subclasses--isolated by liquid chromatography from equine arachnidic antivenom (AAV)-to neutralize toxic activities of Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus venoms as well as to remove venom toxins from circulation was investigated. These subclasses showed similar antibody titers against L. gaucho, P. nigriventer and T. serrulatus venoms, and by immunoblotting few differences were observed in the recognition pattern of venom antigens. IgG(T) and IgGa neutralized 100% lethality induced by L. gaucho and 50% of P. nigriventer venom, but IgGa failed to neutralize T. serrulatus venom, in contrast to IgG(T). Both subclasses neutralized local reactions and dermonecrosis induced by L. gaucho venom in rabbits. In mice, IgG(T) and IgGa partially neutralized the edematogenic activity induced by P. nigriventer and T. serrulatus venoms, but only IgG(T) neutralized (ca. 81%) the nociceptive activity induced by T. serrulatus venom. Both subclasses failed to neutralize nociceptive activity induced by P. nigriventer venom. IgG(T) reduced the serum venom levels of animals injected with L. gaucho, P. nigriventer or T. serrulatus venoms, while IgGa solely reduced L. gaucho and P. nigriventer venoms levels. Our results demostrate that IgG(T) and IgGa subclasses neutralize toxic activities induced by P. nigriventer, T. serrulatus and L. gaucho venoms with different efficacies, as well as depurate these venoms from circulation.
Asunto(s)
Antivenenos/farmacología , Inmunoglobulina G/farmacología , Venenos de Escorpión/antagonistas & inhibidores , Venenos de Araña/antagonistas & inhibidores , Arañas/química , Animales , Antivenenos/química , Cromatografía Liquida , Caballos/inmunología , Inmunoglobulina G/aislamiento & purificación , Ratones , Hidrolasas Diéster Fosfóricas/inmunología , Hidrolasas Diéster Fosfóricas/toxicidad , Venenos de Escorpión/inmunología , Venenos de Escorpión/toxicidad , Venenos de Araña/inmunología , Venenos de Araña/toxicidad , Arañas/metabolismoRESUMEN
Loxoscelism or brown spider envenomation is the most important form of araneism in some countries and constitutes the third cause of accidents by venomous animals in Brazil. The treatment of Loxosceles bites is still controversial, with a variety of interventions proposed and tried, such as antivenom. The majority of clinical studies demonstrate a significant delay between a spider's bite and presentation for treatment, and this delay is thought to lead to an ineffective administration of a specific antivenom. Even in Brazil, where the antivenom therapy has been indicated more frequently than in other countries, there are still doubts about its real capacity to neutralize local and systemic effects of the envenomation and the ideal period for its administration. Thus, various studies in animal models have tried to correlate the time of envenomation with the application of the antivenom and the permanence of the venom in circulation or in dermonecrotic lesions. The purpose of this study was to evaluate the use of antivenom in loxoscelism treatment and to systematize the results of studies in animals and humans available in the last 30 years, making possible a more critical analysis of the efficacy of the antivenom or its therapeutic value in bites by spiders of the genus Loxosceles.
Asunto(s)
Antivenenos/uso terapéutico , Inhibidores de Serina Proteinasa/uso terapéutico , Picaduras de Arañas/terapia , Venenos de Araña/antagonistas & inhibidores , Arañas , Animales , Humanos , Hidrolasas Diéster Fosfóricas/toxicidad , Serina Endopeptidasas , Venenos de Araña/toxicidad , Resultado del TratamientoRESUMEN
Accidents caused by brown spiders (Loxosceles genus) are frequent in Brazil and are associated with dermonecrotic lesions and, eventually, systemic reactions that may be lethal. The major species implicated with human envenoming have been: L. intermedia, L. gaucho and L. laeta. In this study we characterized the venom from Loxosceles similis, a species of spider normally found inside caves. L. similis venom was characterized by two-dimensional gel electrophoresis and enzymatic activity (dermonecrosis and haemolysis). The lethal dose to mice and the capacity of commercial anti-serum to neutralize this venom were also analysed. The cross-reactivity with anti-venoms against L. intermedia, L. laeta and L. gaucho were studied. Our results showed that this venom was able to induce severe dermonecrotic lesions and showed the presence of the bacteria Clostridium septicum in association with the fangs. In addition, we have cloned the DNA coding for a dermonecrotic protein (LsD1), using the genomic DNA of L. similis. The deduced amino acid sequence showed a toxin of approximately 31.2 kDa with an estimated pI of 7.37 and sequence similar to LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom, a synanthropic species of medical importance.
Asunto(s)
Hidrolasas Diéster Fosfóricas/aislamiento & purificación , Hidrolasas Diéster Fosfóricas/toxicidad , Piel/patología , Venenos de Araña/aislamiento & purificación , Venenos de Araña/toxicidad , Arañas/química , Animales , Secuencia de Bases , Western Blotting , Brasil , Clostridium/aislamiento & purificación , Reacciones Cruzadas/inmunología , Cartilla de ADN , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Hemólisis/efectos de los fármacos , Sueros Inmunes/farmacología , Dosificación Letal Mediana , Ratones , Datos de Secuencia Molecular , Necrosis , Hidrolasas Diéster Fosfóricas/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Venenos de Araña/antagonistas & inhibidores , Venenos de Araña/genética , Arañas/microbiologíaRESUMEN
Loxosceles spiders have a wide distribution in the temperate and tropical regions of the world. Loxoscelism is characterized by necrotic skin ulceration at the bite site and, less commonly, a systemic illness that may be fatal. The purpose of this study was to characterize and compare aspects of the major medically important Loxosceles spider venoms in a standardized manner, particularly considering their neutralization by two Brazilian antivenoms. By SDS-PAGE (12% acrylamide), Loxosceles deserta, Loxosceles gaucho, Loxosceles intermedia, Loxosceles laeta and Loxosceles reclusa venoms had similar electrophoretic profiles, with the major protein bands of 32-35 kDa. All venoms exhibited gelatinolytic, caseinolytic and fibrinogenolytic activities in vitro with a large array of proteases, mainly between 18.1 and 31.8 kDa. Most of these enzymes were metalloproteases as this activity was abolished by 1,10-phenanthroline. Hyaluronidase activity was detected in a protein band of approximately 44 kDa in all venoms. Sphingomyelinase activity was demonstrated in all five venoms. Antigenic cross-reactivity, by Western blotting, was also observed among all venoms studied using commercial equine antivenoms produced in Brazil (Institute Butantan and CPPI). These antivenoms recognized mainly components between 25 and 40 kDa in all venoms with several minor components of >89 kDa. Strong cross-reactivity was also seen among all venoms through the ELISA technique (titre range: 64,000-512,000). All venoms (5 microg doses) induced a similar local reaction when injected intradermally into the flank of rabbits, demonstrating dermonecrosis, hemorrhage, vasoconstriction, edema, and erythema. However, no reaction was observed when each venom was pre-incubated (1 h, 37 degrees C) with Brazilian commercial sera prior to injection. The antivenoms also abolished the sphingomyelinase activity in vitro, suggesting the venoms of the major medically important Loxosceles spider species have generally similar toxic and enzymatic characteristics. Thus, as Brazilian commercial antivenoms are able to neutralize the dermonecrosis induced by Loxosceles venoms of diverse geographical origin, clinical studies should be undertaken on the potential for a single global Loxosceles antivenom.
Asunto(s)
Antivenenos/metabolismo , Reacciones Cruzadas/efectos de los fármacos , Piel/patología , Venenos de Araña/toxicidad , Arañas/química , Animales , Western Blotting , Brasil , Caseínas/metabolismo , Reacciones Cruzadas/inmunología , Edema/inducido químicamente , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Eritema/inducido químicamente , Fibrinógeno/metabolismo , Gelatina/metabolismo , Ácido Hialurónico/metabolismo , Necrosis/inducido químicamente , Pruebas de Neutralización , Conejos , Especificidad de la Especie , Esfingomielina Fosfodiesterasa/metabolismo , Venenos de Araña/antagonistas & inhibidores , Venenos de Araña/enzimología , Venenos de Araña/inmunologíaRESUMEN
We have produced 13 mAbs for Loxosceles intermedia crude venom. Twelve were reactive against proteins of 32-35 kDa and one of these Li mAb(7) showed high neutralizing potency for the dermonecrotic activity of L. intermedia venom. This Li mAb(7) showed no cross-reactivity, with Loxosceles laeta (Brazil), L. laeta (Perú) and Loxosceles gaucho venoms. The mAbs were produced by immunization with the crude venom and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia whole venom or dermonecrotic fraction (DNF) as antigens coated onto microtitre plates. A sensitive two-site immunometric assay was designed and shown to be useful for identifying and quantifying DNF from L. intermedia in biological samples. The Li mAb(7) coated onto microtitre plates and hyperimmune horse anti-L. intermedia IgGs prepared by immunoaffinity chromatography and conjugated to horseradish peroxidase were used to set up a sandwich-type ELISA. Measurable absorbance signals were obtained with 0.2 ng of L. intermedia crude venom per assay.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antivenenos/farmacología , Venenos de Araña/antagonistas & inhibidores , Venenos de Araña/inmunología , Arañas , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Antivenenos/química , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Conejos , Picaduras de Arañas/sangre , Picaduras de Arañas/inmunología , Venenos de Araña/químicaRESUMEN
Neutralization of dermonecrotic and lethal activities and differences among the principal toxic proteins (32-35 kDa) of medically important Loxosceles spider venoms in Brazil (Loxosceles gaucho, Loxosceles laeta and Loxosceles intermedia) were studied using monoclonal antibodies (MAbs) produced against the dermonecrotic component (35 kDa) of L. gaucho venom. MAb titers were 512,000 to homologous venom, between 2000 and 64,000 for L. intermedia venom and between 1000 and 64,000 for L. laeta venom. By Western blotting, MAbs could recognize mainly the 35 kDa protein of L. gaucho venom and with less intensity the 35 kDa protein of L. intermedia venom. These MAbs also recognized weakly or did not recognize the 32 kDa component of L. laeta venom. Only MoALg1 showed high affinity for L. gaucho venom and neutralized in vivo 90-97% of the dermonecrotic activity, besides delaying the lethality induced by homologous venom. MoALg1 maintained its capacity to neutralize the dermonecrotic activity, even when administered (i.v.) 6h after envenoming (i.d.). All MAbs obtained failed to neutralize the toxic activities of the heterologous venoms.These results suggest that different epitopes are present in the protein responsible for the dermonecrotic activity of Loxosceles venoms, and confirm the participation of other venom components during the local reaction process. This study also confirms the importance of antibodies for neutralization of dermonecrotic activity, even when administered some hours after envenoming, and emphasizes the differences of composition and toxicity of medically important Loxosceles venoms. These findings must be considered in order to improve loxoscelism immunotherapy.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Piel/patología , Venenos de Araña/antagonistas & inhibidores , Venenos de Araña/toxicidad , Arañas/metabolismo , Animales , Anticuerpos Monoclonales/química , Western Blotting , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Inmunoquímica , Dosificación Letal Mediana , Ratones , Ratones Endogámicos BALB C , Necrosis , Especificidad de la Especie , Venenos de Araña/químicaRESUMEN
The haemodynamic alterations induced by the central and peripheral administration of the armed spider (Phoneutria nigriventer) venom (PNV) were investigated in anaesthetised rabbits. The intracerebroventricular injection of increasing doses of PNV (30 and 100 microg/kg) elicited a biphasic cardiovascular response characterised by a brief hypotension (1-3 min) followed by a marked and sustained (more than 30 min) increase in mean arterial pressure (61 +/- 5 and 61 +/- 10%, respectively) and in systemic vascular resistance (135 +/- 21 and 161 +/- 37%) accompanied by mild increases in cardiac contractility. Systemic alterations such as salivation and muscular fasciculation were also observed. At the opposite, the dose of 100 microg/kg of PNV injected intravenously produced only a hypotensive effect (29 +/- 4% decrease in mean arterial pressure) and a decrease in vascular resistance (38 +/- 5%). Nevertheless, a much higher dose of PNV (1 mg/kg) injected intravenously produced a hypertensive response analogous to the one observed upon central administration. The central hypertensive response induced by PNV was not affected by preteating the animals with selective antagonists of receptors of different neurotransmitters or endogenous mediators such as: acethylcoline muscarinic, bradykinin B2, angiotensin II AT1 receptors and also antagonists of the excitatory amino acid receptors of the central nervous system. Nevertheless, the intravenous pretreatment with the selective alpha1-adrenergic receptor antagonist prazosin significantly blunted the excitatory cardiovascular response evoked by the central injection of PNV. It is concluded that PNV can induce central as well as peripheral haemodynamic effects. The central component seems to be mediated by the activation of cardiovascular centres which in turn lead to an increase in the sympathetic outflow to the periphery, whereas the peripheral component can be accounted for either by direct activation of the vascular alpha1-adrenergic receptors or by catecholamine release from the sympathetic nerve endings.
Asunto(s)
Hemodinámica/efectos de los fármacos , Venenos de Araña/toxicidad , Antagonistas Adrenérgicos alfa/administración & dosificación , Antagonistas de Receptores de Angiotensina , Animales , Antihipertensivos/administración & dosificación , Antagonistas de los Receptores de Bradiquinina , Sistema Cardiovascular/efectos de los fármacos , Antagonistas Colinérgicos/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Inyecciones Intravenosas , Masculino , Prazosina/administración & dosificación , Conejos , Venenos de Araña/administración & dosificación , Venenos de Araña/antagonistas & inhibidoresRESUMEN
Spider bites due to Loxosceles intermedia are currently a major public health problem in South Brazil. About 3000 cases are reported annually. Specific treatment for spider bites is provided by the polyvalent anti-arachnidic antiserum produced by Butantan Institute, São Paulo, Brazil by immunizing horses with mixtures of venoms from Tityus serrulatus and T. bahiensis scorpions, as well as Phoneutria nigriventer and L. gaucho spiders. Due to the large amounts of the anti-arachnidic antivenom required and since L. intermedia venom has some biochemical and pharmacological variations, we have produced a specific anti-L. intermedia antivenom. This study shows that horses immunized with crude L. intermedia venom produced IgG antibodies that neutralized the dermonecrotic and lethal activities of the venom. The neutralizing potency of the anti-loxoscelic antivenom was compared with that of the anti-arachnidic antivenom. Our results indicate that both antivenoms were effective in terms of neutralization. However, the anti-loxoscelic antivenom was more efficient than the anti-arachnidic.
Asunto(s)
Anticuerpos/inmunología , Antivenenos/farmacología , Caballos/inmunología , Inmunoglobulina G/inmunología , Venenos de Araña/toxicidad , Animales , Brasil , Relación Dosis-Respuesta a Droga , Evaluación de Medicamentos/métodos , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Ratones , Pruebas de Neutralización , Venenos de Araña/antagonistas & inhibidores , Venenos de Araña/inmunologíaRESUMEN
The systemic symptoms, tissue lesions and release of cytokines were analysed in four isogenic mouse strains with distinct haplotypes injected with various doses of Loxosceles intermedia spider venom. The estimated LD50 were 24.5 microg for C57Bl/6, 17.6 microg for BALB/c, 6.3 microg for C3H/HeJ and 4.6 microg for A/Sn mice. Prostration, acute cachexia, hypothermia, neurological disorders and hemoglobinuria were the signals preceding death. Accumulation of eosinophilic material inside the proximal and distal renal tubules and acute tubular necrosis were the most common histopathological findings. Death was prevented by previous treatment of venom with specific antivenom serum. The protein F35 purified from the whole venom retained the ability to induce the symptoms of the whole venom. The cytokines tumor necrosis factor (TNF), interleukins IL-6 and IL-10 and the radical nitric oxide were detected in serum at different levels after venom injection. These findings indicate that the state of shock produced in mice by whole endotoxin-free L. intermedia venom or by its purified fraction, protein F35, mimics the endotoxemic shock, that susceptibility to the systemic effects of the venom varies among mice of different haplotypes and that the pattern of in vivo cytokine release resembles that of endotoxemic shock.