Extraction and quantification of antivenomous antibodies in chicken eggs against scorpion venom / Extração e quantificação de anticorpos entivenenos em ovos de galinha contra veneno de escorpião

Avian-derived IgY is thought to be the best therapy for scorpion bites concerning low-level side effects. The present study analyzed a hypothesis about the neutralization of scorpion venom Androcotonus australis through antibodies produced in the egg yolks of chickens. The venom used for inoculation was obtained from Androctonus australis (yellow fat-tailed scorpion) from southern Punjab, Pakistan. The lethal dose of LD50 against scorpion venom was calculated in chickens and mice. Safe doses were given to egg-laying chickens to produce IgY antibodies. The antivenom IgY antibodies were extracted from the egg yolks of immunized chicken using the polyethylene glycol (PEG) method. Moreover, IgY was confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Ouchterlony double immunodiffusion assay test. The antibody titers were evaluated by the enzyme-linked immunosorbent assay (ELISA). The neutralisation capacity of extracted anti-scorpion antibodies was tested on mice. The calculated LD50 of scorpion venom for chicken and mice was 4 mg/kg and 2.5 mg/kg, respectively. SDS-PAGE and Ouchterlony double immunodiffusion confirmed the presence of IgY against scorpion venom. The maximum titer value of specific IgY produced against scorpion venom was 3.5 ug/ml. A concentration of 220 ul/LD50 was effective to neutralize 1 mg of scorpion venom. It is suggested that IgY obtained from egg yolks is safe against targeted venom and can be used as an effective alternative to equine IgG antibodies against scorpion envenoming.

Acredita-se que a IgY derivada de aves seja a melhor terapia para picadas de escorpião em relação aos efeitos colaterais. O presente estudo teve como objetivo analisar uma hipótese sobre a neutralização do veneno do escorpião Androcotonus australis através de anticorpos produzidos na gema de ovos de galinhas. O veneno usado para inoculação foi obtido de Androctonus australis (escorpião amarelo de cauda gorda) do sul de Punjab, Paquistão. A dose letal de LD50 contra veneno de escorpião foi calculada em galinhas e camundongos. Doses seguras foram dadas a galinhas poedeiras para produzir anticorpos IgY. Os anticorpos antiveneno IgY foram extraídos das gemas de ovos de galinhas imunizadas pelo método do polietilenoglicol (PEG). Além disso, a IgY foi confirmada por eletroforese em gel de poliacrilamida e dodecil sulfato de sódio (SDS-PAGE) e pelo teste de imunodifusão dupla de Ouchterlony. Os títulos de anticorpos foram avaliados pelo ensaio imunoenzimático (ELISA). A capacidade de neutralização dos anticorpos anti-escorpião extraídos foi testada em camundongos. A LD50 calculada do veneno de escorpião para galinhas e camundongos foi de 4 mg/kg e 2,5 mg/kg, respectivamente. SDS-PAGE e imunodifusão dupla Ouchterlony confirmaram a presença de IgY contra veneno de escorpião. O valor máximo do título de IgY específico produzido contra veneno de escorpião foi de 3,5 ug/ml. Uma concentração de 220 ul/LD50 foi considerada eficaz para neutralizar 1 mg de veneno de escorpião. Sugere-se que a IgY obtida da gema do ovo seja segura contra o veneno direcionado e possa ser usada como uma alternativa eficaz aos anticorpos IgG equinos contra o envenenamento por escorpiões.

Picadura de escorpión en Bolivia: una revisión crítica de la literatura

Las picaduras son frecuentes y se consideran un verdadero problema de salud pública. Objetivo: describir las especies de escorpiones, propiedades del veneno, fisiopatología, manifestaciones clínicas, diagnóstico y su manejo en la unidad de cuidados intensivos. Metodología: se realizó una búsqueda de la literatura en las bases de datos SciELO, LILACS, Scopus, PubMed-MedLine, Google Académico, así como en los servicios ClinicalKeys, se extrajo la información relevante, se seleccionaron aquellos estudios de tipo serie de casos, artículos originales o revisiones narrativas, de alcance y sistemáticas. Desarrollo: en Bolivia, fue descrita la especie Tityus (Tityus) sorataensis. El 66 al 90 por ciento de las picaduras tienen signos y síntomas limitados a dolor local, parestesias y cambios en la piel. Los efectos sistémicos se hacen evidentes a los 30 minutos y por lo general, dentro de las cuatro horas posteriores a la picadura. El tratamiento puede ser empírico, aplicar medidas generales, manejo del dolor y si está presente algunas complicaciones como edema pulmonar, choque cardiogénico es necesario su manejo en una sala de cuidados intensivos. Conclusiones: la intoxicación por picadura de escorpión, es rara, es un reto terapéutico, parece ser recomendable la administración del antídoto (antiveneno) junto al tratamiento de sostén. un mejor conocimiento de los escorpiones, puede alentar el interés en realizar nuevas investigaciones.

Bites are frequent and are considered a real public health problem. Objective: describe the species of scorpions, properties of the venom, pathophysiology, clinical manifestations, diagnosis and its management in the intensive care unit. Methodology: a literature search was carried out in the SciELO, LILACS, Scopus, PubMed-MedLine, Google Scholar databases, as well as in the ClinicalKeys services, in the period February-April 2023. Relevant information was extracted, They selected case series studies, original articles or narrative, scoping and systematic reviews. Development: in Bolivia, the species Tityus (Tityus) sorataensis was described. 66 to 90 percent of bites have signs and symptoms limited to local pain, paresthesias, and skin changes. Systemic effects become apparent within 30 minutes and generally within four hours of being bitten. The treatment can be empirical, apply general measures, pain management and if some complications are present such as pulmonary edema, cardiogenic shock, it is necessary to manage it in an intensive care room. Conclusions: Scorpion sting poisoning is rare, it is a therapeutic challenge, it seems to be advisable to administer the antidote (antivenoin) together with supportive treatment. a better understanding of scorpions may encourage interest in further research.

Métodos alternativos para determinação da potência de veneno e antiveneno botrópico: aplicações e perspectivas para o controle da qualidade / Alternative methods for potency evaluation for Bothrops venom and antivenom: applications and perspectives for quality control

Introdução: Acidentes com animais peçonhentos são classificados como doenças tropicais negligenciadas e são atualmente a mais frequente causa de intoxicação em humanos no Brasil. O único tratamento disponível é a rápida administração de antivenenos específicos e de qualidade garantida. Para assegurar a eficácia e a segurança desses produtos, são realizados ensaios de determinação da potência in vivo para veneno e antiveneno, desde as etapas de produção até sua liberação final. Apesar dos diversos estudos sobre métodos   alternativos ao ensaio murino, nenhum método foi efetivamente validado. Objetivo: Compilar os métodos alternativos desenvolvidos para os antivenenos botrópicos, avaliando sua disponibilidade, perspectivas e aplicações em laboratórios de produção e controle da qualidade. Método: Foi realizada uma busca nas bases PubMed, BVS e Scopus entre novembro de 2021 e junho de 2022. Foram identificados 89 trabalhos, dos quais 31 foram selecionados de acordo com os critérios de elegibilidade. Resultados: Nos métodos alternativos identificados, observamos a preferência de 42,80% dos estudos por metodologias que utilizem linhagens celulares como método alternativo aos ensaios murinos, sendo que a maioria destes trabalhos 58,30% optou pela linhagem celular Vero. Conclusões: Pela diversidade das toxinas encontradas em cada gênero de serpentes, entende-se que é de extrema importância que o ensaio de potência dos antivenenos tenha como base a avaliação e a quantificação precisa da inibição da atividade biológica dos venenos. Ensaios de citotoxicidade são amplamente utilizados e têm acumulado evidências de sua adequação como importante ferramenta alternativa ao ensaio murino para o controle da qualidade de veneno e antiveneno antibotrópico.

Introduction: Accidents with venomous animals are classified as neglected tropical diseases and are currently the most frequent cause of intoxication in humans in Brazil. The only available treatment is the rapid administration of specific, quality-assured antivenoms. To ensure the efficacy and safety of these products, in vivo potency determination tests for venom and antivenom are performed during the production stages, until final release. Despite several studies on alternative methods to the murine assay, no method has been effectively validated. Objective: To compile alternative methods developed for Bothrops antivenoms, assessing the availability of the methods and the prospects and applications in Bothrops venom and antivenom production and quality control laboratories. Method: A search was conducted in PubMed, BVS, and Scopus databases between November 2021 and June 2022. 89 articles were identified, of which 31 were selected according to the eligibility criteria. Results: We observed in the alternative methods identified a preference of 42.80% of the studies for methodologies that use cell lines as an alternative method to the murine assays, and most of these works (58.30%) opted for a VERO cell line. Conclusions: Due to the diversity of toxins found in each genus of snakes, it is understood that the potency assay for antivenoms should be based on the evaluation and precise quantification of the inhibition of biological activity of venoms. Cytotoxicity assays are widely used and have been accumulating evidence of their suitability as an important alternative tool to the murine assay for quality control for Bothrops venom and antivenom.

Oxidative stress and changes in hematological and biochemical biomarkers in Crotalus durissus terrificus (South American rattlesnake) venom-induced acute kidney injury in rats / Estrés oxidativo y alteraciones en biomarcadores hematológicos y bioquímicos en lesión renal aguda inducida por el veneno de Crotalus durissus terrificus (cascabel de America del Sur) en ratones

Abstract Acute kidney injury (AKI) is the major cause of mortality following bites by the South American rattlesnake Crotalus durissus terrificus. We investigated the early onset of Crotalus durissus terrificus venom-induced AKI in rats within 2 h of venom injection and its attenuation by antivenom. Several biomarkers were used to monitor AKI in the absence or presence of antivenom. Male Wistar rats were divided into five groups (n=5 each): G1, rats injected with saline (control); G2, rats injected with venom (6 mg kg-1, intraperitoneally) and euthanized after 2 h to evaluate AKI; G3 and G4, rats injected with 0.9% sterile saline or antivenom 2 h after venom, respectively, and monitored until death or up to 24 h post-venom, and G5, rats injected with antivenom alone and monitored for 24 h. Blood, urine and renal tissue samples were collected immediately after death to assess oxidative stress, hematological and biochemical alterations, and renal histological damage. Venom caused AKI within 2 h (G2) that persisted for up to 8.2 ± 1.6 h (G3), as confirmed by increases in blood urea, creatinine, and renal proteinuria; these increases were attenuated by antivenom. There were no changes in blood protein concentrations in G2 and G3, whereas there were increases in blood reduced glutathione, glutathione peroxidase, and plasma TBARS (but not in catalase) that were attenuated to varying extents by antivenom. There were no marked changes in platelets or leukocytes, but an increase in erythrocytes after 8.2 h with venom alone was attenuated by antivenom. Renal glomerular and tubular damage was greatest after 2 h post-venom groups alone was attenuated by antivenom. Renal glomerular and tubular damage was greatest after 2 h post-venom and declined thereafter. Venom caused early-onset AKI, with variable effects on lipid peroxidation and oxidative stress. Antivenom attenuated the AKI, as shown by the decrease in blood urea and the normalization of proteinuria, without protecting against lipid peroxidation.

Resumen La injuria o lesión renal aguda (LRA) es la mayor causa de mortalidad debido a las mordeduras por cascabeles Crotalus durissus terrificus. Se estudió la instalación precoz de LRA, en ratas, inducida por el veneno de Crotalus durissus terrificus después de 2 h de su inoculación y la atenuación por el antiveneno. Se utilizaron diversos biomarcadores para monitorear LRA en ausencia o presencia del antiveneno. Ratas Wistar machos fueron divididos en 5 grupos (n=5 por grupo): G1, ratas inoculadas con solución salina (control); G2, ratas inoculadas con veneno (6 mg kg-1 dosis, vía intraperitoneal), y sacrificadas después de 2 h para evaluar LRA; G3 y G4, ratas inoculadas con 0.9% de solución salina esterilizada o antiveneno luego de 2 h después de inoculado el veneno, respectivamente, y monitoreadas hasta su muerte o hasta 24 h después de inoculado el veneno; y G5, ratas inoculadas con antiveneno solo y monitoreadas durante 24 h. Las muestras de sangre, orina, y tejido renal fueron colectadas inmediatamente después de la muerte de los animales para evaluar estrés oxidativo, alteraciones hematológicas y bioquímicas, y daño histológico renal. El veneno causó LRA dentro de las 2 h (G2) persistiendo durante más de 8,2 ± 1,6 h (G3), estando esto confirmado por el incremento de urea sanguínea, creatinina, y proteinuria renal; estos aumentos disminuyeron con la aplicación del antiveneno. No se observaron alteraciones en las concentraciones de proteínas sanguíneas en G2 y G3, mientras que se encontraron incrementos en glutatión reducido sanguíneo, glutatión peroxidasa y TBARS plasmática (pero no en catalasa), que disminuyeron con la aplicación del antiveneno aunque en diferente grado. No ocurrieron alteraciones marcadas de plaquetas o leucocitos, mientras que el aumento de glóbulos rojos observado luego de 8,2 h de la inoculación con veneno, disminuyó con el antiveneno. El daño renal glomerular y tubular fue más importante luego de 2 h de la inoculación con veneno y posteriormente disminuyó. El veneno causó LRA precoz a las 2 h, con efectos variables sobre la peroxidación lipídica y el estrés oxidativo. El antiveneno redujo el daño renal, conforme lo demostrado por la disminución en la urea sanguínea y por la normalización de la proteinuria, aunque no se observó protección contra la peroxidación lipídica.

A new approach of immunotherapy against Crotalus snakes envenoming: ostrich (Struthio camelus) egg yolk antibodies (IgY-technology) / Un nuevo enfoque de inmunoterapia contra el envenenamiento de serpientes Crotalus: anticuerpos de yema de huevo de avestruz (Struthio camelus) (tecnología IgY)

Abstract Crotalid envenomation is a neglected collective health problem involving many countries in America, which need secure and inexpensive snake anti-venom treatments. Here, high antibody titers (IgY) were raised in the Ostrich (Struthio camelus) egg yolk by immunizing with the venom of Venezuelan venomous Crotalus snakes. Ostriches were immunized with a pool of venoms from common rattlesnake (Crotalus durissus cumanensis), Uracoan rattlesnake (Crotalus vegrandis), Guayana rattlesnake (Crotalus durissus ruruima) and black rattlesnake (Crotalus pifanorum). The anti-snake venom antibodies were prepared from egg yolk by the water dilution method, enriched by the addition of caprylic acid (CA) and precipitation with ammonium sulfate at 30% (W/V). The purity and molecular mass of the final product was satisfactory, yielding a single ∼ 175 kDa band in SDS-PAGE gels ran under non-reducing conditions. In the immunoblot analysis, specific binding of the antivenom was observed with most venom proteins. The LD50 was 16.5 g/mouse (825 μg/kg body weight). High titers of IgY against Crot/pool venom were shown by ELISA. The median effective dose (ED50) was 19.66 mg/2LD50. IgY antibodies neutralized efficiently the Crot/pool venom lethality. As far as we know, this is the first anti-snake venom produced in ostriches, which could make this technology an affordable alternative for low-income countries, since it is likely to produce manteniabout 2-4 g of IgY per ostrich egg. Hence, almost 400 g of IgY can be purified from only one ostrich during a year. In addition, there are enormous differences in the cost of investment in the maintenance of horses, from the points of view of infrastructure, feeding and veterinary care, in which the cost can reach USD 100 per animal per day, compared to a maintenance cost of USD 146 per month per producing bird. These results are encouraging and could easily be extrapolated to the manufacturing of other antivenoms and antitoxins as well, as they could be applied to the manufacturing of potential diagnostic tools.

Resumen El envenenamiento por crotálidos es un problema de salud colectiva desatendido, que involucra a muchos países del continente americano, los cuales necesitan tratamientos seguros y económicos. En este trabajo, se obtuvieron títulos altos de anticuerpos (IgY) producidos en yema de huevo de avestruz (Struthio camelus) mediante la inmunización con el veneno de serpientes venezolanas del genero Crotalus. Se inmunizaron avestruces con una colección de veneno de serpientes de cascabel común (Crotalus durissus cumanensis), cascabel de Uracoa (Crotalus vegrandis), cascabel de Guayana (Crotalus durissus ruruima) y cascabel negra (Crotalus pifanorum). Los anticuerpos anti-veneno de serpiente se prepararon a partir de yema de huevo por el método de dilución en agua, enriquecidos mediante la adición de ácido caprílico (CA), seguido de una precipitación con sulfato de amonio al 30% (P/V). La pureza y masa molecular de los anticuerpos (IgY) se definieron mediante ensayos de SDS-PAGE nativos y las masas moleculares se establecieron electroforéticamente, obteniéndose una única banda de IgY de ∼ 175 kDa. El análisis de inmunotransferencia mostró la unión específica del antiveneno con la mayoría de las proteínas del veneno. La DL50 fue de 16,5 μg/ratón (825 μg / kg de peso corporal); Se mostraron títulos altos de IgY contra el veneno de Crot / pool mediante ELISA. La dosis mediana efectiva (DE50) fue de 19,66 mg/2 LD50. Los anticuerpos IgY neutralizaron eficazmente la letalidad del veneno de Crot / pool. Hasta donde sabemos, se trata del primer antídoto de serpiente producido en avestruces, lo que podría abaratar la producción de este tratamiento en países del tercer mundo. Ya que es probable que se obtengan alrededor de 2-4 g de IgY por huevo de avestruz. Por lo tanto, se podrían purificar casi 400 g de IgY de un solo avestruz durante un año. Asimismo, debido a las enormes diferencias en el costo de inversión en el mantenimiento de los caballos desde el punto de vista de infraestructura, alimentación y atención veterinaria, en los que el costo puede llegar a los 100 USD por día, frente a los 146 USD por mes de mantenimiento de la producción de aves. Estos resultados abren un campo terapéutico, para la fabricación de otros antivenenos contra un amplio espectro de toxinas y también como probables herramientas de diagnóstico.

Novas perspectivas na pesquisa e desenvolvimento de antivenenos

Snakebite envenoming has a significant impact on public health due to its severity and frequency. It is estimated that worldwide, 5.4 million snake bites occur annually and, as a consequence, thousands of deaths, amputations and other permanent disabilities. Based on the various difficulties and limitations in the production and use of antivenom, new research, methods of obtaining, developing and improving these biopharmaceuticals are proposed. Among them are the use of human antibodies, antibody fractions, aptamers, small inhibitory molecules and the use of “omics” technologies for cross-reactivity research. The present work aims to address the main impacts of snakebite in the world, the problems related to the current treatment and which guidelines can be adopted with innovative strategies in order to minimize this global public health problem. The study was carried out through a narrative review, with a qualitative approach and exploratory depth, on the theme of the main perspectives for the future in the fight against snakebite envenoming. It was possible to observe that for the generation of these new biopharmaceuticals, actions such as investments, whether governmental or from the private sector, and a better knowledge of the snakes/venoms of each region are necessary.

O envenenamento por serpentes impacta de maneira expressiva na saúde pública devido sua gravidade e frequência. Estima-se que no mundo, 5,4 milhões de picadas de serpentes ocorram anualmente e como consequência, milhares de mortes, amputações e outras incapacidades permanentes. Baseado nas diversas dificuldades e limitações na produção e utilização do antiveneno, novas pesquisas, métodos de obtenção, desenvolvimento e melhorias desses biofármacos são propostas. Entre elas estão a utilização de anticorpos humanos, frações de anticorpos, aptâmeros, pequenas moléculas inibidoras e a utilização de tecnologias “ômicas” para pesquisa de reatividade cruzada. O presente trabalho tem como objetivo abordar os principais impactos do ofidismo no mundo, os problemas relacionados ao tratamento atual e quais diretrizes podem ser adotadas com estratégias inovadoras, a fim de minimizar esse problema mundial de saúde pública. O estudo foi realizado através de uma revisão narrativa, de abordagem qualitativa e profundidade exploratória, sobre a temática das principais perspectivas para o futuro no combate ao ofidismo. Foi possível observar que para a geração desses novos biofármacos, ações como investimentos, sejam governamentais ou do setor privado e o melhor conhecimento das serpentes/venenos de cada região são necessárias.

Toxicological, enzymatic, and immunochemical characterization of Bothrops asper (Serpentes: Viperidae) reference venom from Panama / Caracterización toxicológica, enzimática e inmunoquímica del veneno de referencia de Bothrops asper (Serpentes: Viperidae) de Panamá

Introduction: It is estimated that 2 000 snakebites occur in Panama every year, 70 % of which are inflicted by Bothrops asper. Objective: To determine the biochemical and toxicologic effects and to assess the immunochemical characteristics of a reference pool of B. asper venom representative of Panama. Methods: The reference venom was prepared as a homogeneous mixture of the venoms obtained from 78 adult snakes collected in four geographic areas of Panama. Enzymatic and toxicological activities were assessed. The electrophoretic pattern was studied by SDS-PAGE. Immunoreactivity of various antivenoms was analyzed by Western blot. Results: B. asper reference venom has lethal, hemorrhagic, myotoxic, edema-forming, coagulant, defibrinating, proteinase and phospholipase A2 activities. SDS-PAGE showed the presence of protein bands with molecular weights ranging from 8 to 70 kDa, with the presence of predominant bands at ≈ 15 kDa and ≈ 30 to 66 kDa, which likely correspond to phospholipases A2 and metalloproteinases, respectively. Immunoblotting showed a high degree of recognition by various antivenoms, especially by antivenoms from Colombia and Costa Rica. Conclusions: Following recommendations by the World Health Organization, this reference venom of B. asper of Panama will become a useful tool for the preclinical evaluation of antivenoms distributed in this country.

Introducción: Se estima que 2 000 mordeduras de serpiente ocurren en Panamá cada año, el 70 % de las cuales son infligidas por Bothrops asper. Objetivo: Determinar los efectos bioquímicos y toxicológicos y evaluar las características inmunoquímicas del veneno de referencia de B. asper representativo de Panamá. Métodos: El veneno de referencia se preparó como una mezcla homogénea de los venenos obtenidos de 78 serpientes adultas recolectadas en cuatro áreas geográficas de Panamá. Se evaluaron las actividades enzimáticas y toxicológicas. El patrón electroforético se estudió mediante SDS-PAGE. La inmunoreactividad de varios antivenenos se analizó mediante transferencia de Western. Resultados: El veneno de referencia de B. asper tiene actividades letales, hemorrágicas, miotóxicas, formadoras de edema, coagulantes, desfibrinante, proteolítica y de fosfolipasa A2. El análisis de SDS-PAGE mostró la presencia de bandas de proteínas con pesos moleculares que varían de 8 a 70 kDa, con la presencia de bandas predominantes a ≈ 15 kDa y ≈ 30 a 66 kDa, que probablemente corresponden a fosfolipasas A2 y metaloproteinasas, respectivamente. La inmunotransferencia mostró un alto grado de reconocimiento por varios antivenenos, especialmente por antivenenos de Colombia y de Costa Rica. Conclusiones: Siguiendo las recomendaciones de la Organización Mundial de la Salud, este veneno de referencia de B. asper de Panamá se convertirá en una herramienta útil para la evaluación preclínica de antivenenos distribuidos en este país.

Análisis de casos de araneísmo a nivel global en el período 2000-2020 / Analysis of cases of araneism at a global level in the period of 2000-2020

RESUMEN INTRODUCCIÓN Aunque son temidas, sólo algunas arañas han sido asociadas con una clínica severa en humanos. Este estudio se propuso revisar los informes de casos y series de casos sobre mordeduras de arañas para informar el estado actual del problema. MÉTODOS Se realizó una revisión sistemática de la literatura en varias bases de datos, sin umbral de fecha para la búsqueda. Se limitó la estrategia de búsqueda a los artículos publicados en portugués, francés, inglés y español. Los estudios elegibles fueron informes de casos y series de casos que reportaron desenlaces en humanos causados por mordeduras de arañas. Se extrajo información a nivel de paciente y a nivel de estudio. RESULTADOS La búsqueda arrojó 10 683 estudios. Se incluyeron 248 artículos, que reportaban 351 pacientes; de ellos, un 54% eran hombres. Los síntomas más frecuentes fueron locales. Se documentó la muerte de 17 pacientes (4,85%). Las arañas de los géneros Loxosceles y Latrodectus causaron la mayoría de los accidentes. Al comparar el uso o no de antiveneno en los géneros Loxosceles o Latrodectus, no hubo diferencias entre la longitud de estancia hospitalaria. DISCUSIÓN La mordedura de diferentes especies de arañas de todo el mundo puede causar graves consecuencias para la salud humana, especialmente las de los géneros Loxosceles y Latrodectus. Aunque la mortalidad no es elevada, estos casos deben ser rápidamente diagnosticados y tratados.

ABSTRACT INTRODUCTION Although spiders are feared, only a few of them have been associated with severe outcomes in humans. This study aimed at reviewing case reports and case series on spider bites to inform the current state of the problem. METHODS A systematic literature review was conducted in several databases, without search date limit. The search strategy was limited to articles published in Portuguese, French, English and Spanish. Eligible studies were case reports and case series that reported outcomes in humans caused by spider bites. Patient-level and study-level information was extracted. RESULTS The literature search yielded 10 683 studies. A total of 248 articles were included, reporting 351 patients; 54% of them were male. The most frequently reported signs and symptoms were local. The death of 17 patients was documented (4.85%). Spiders from the genera Loxosceles and Latrodectus caused most of the accidents. No difference in hospital stay length in Latrodectus or Loxosceles envenomation with or without antivenom was found. DISCUSSION The bite of different species of spiders around the world can cause serious consequences to human health, especially spiders from the genera Loxosceles and Latrodectus. Even as mortality is not high, spider bites must be quickly diagnosed and treated properly

Mordedura de Phalotris lemniscatus (Duméril, Bibron & Duméril, 1854) (Squamata, Dipsadidae) en Uruguay / Phalotris lemniscatus (Duméril, Bibron & Duméril, 1854) (Squamata, Dipsadidae) bites in Uruguay

Phalotris lemniscatus es la única especie representante del género Phalotris en Uruguay. Esta especie tiene una amplia distribución que incluye Uruguay, Rio Grande do Sul en Brasil y el norte de Argentina que se extiende a las áreas fronterizas con Bolivia y Paraguay. Aunque este ofidio no es agresivo, se registraron dos accidentes en Uruguay en los que se observó acción local y sistémica del veneno. Los mismos ocurrieron en las manos después de manipulación excesiva y prolongada de los ejemplares. Localmente presentaron edema leve, pero en términos sistémicos el veneno de Phalotris causó alteraciones en la coagulación. Los pacientes se recuperaron totalmente después de 3 días. Serán necesarios más estudios para establecer una terapia adecuada para los envenenamientos graves provocados por esta especie.

Phalotris lemniscatus is the only species representative of Phalotris genus in Uruguay. This species has a wide distribution that includes Uruguay, Rio Grande do Sul in Brazil and northern Argentina extending to the areas bordering Bolivia and Paraguay. Although this snake is not aggressive, there were two snakebite accidents in Uruguay. They occurred on the hands after excessive and prolonged handling of the specimens. Locally they showed mild edema, but in systemically Phalotris venom caused alterations in blood coagulation. The patients recovered completely after 3 days. More studies will be required to establish an adequate therapy for Phalotris severe envenomations.

Alteraciones hemostáticas provocadas por el veneno de la culebra mapanare (Porthidium lansbergii hutmanni) en pacientes venezolanos / Haemostatic alterations caused by the venom of Lansberg's hognosed pitviper (Porthidium lansbergii hutmanni) in Venezuelan patients

Objective: to describe the haemostatic characteristics of the venom as well as the potency appraisal of the polyvalent antiophidic serum against haemotoxicity from Porthidium lansbergii hutmani experimental envenomation. Methods: Evaluation was performed of the venom's lethality, haemorrhagic activity, effects on coagulation and platelet aggregation, proteolytic activity, and neutralization by the commercial antivenom available in the country. Results: Several components with haemostatic activities were found in Porthidium l. hutmanni venom when a study of fibrinogenolytic, haemorrhagic and proteolytic activities was conducted of a pool of P.l.h venom. Porthidium l. hutmanni venom lacked the coagulant and defibrinating activities that are characteristic of bothropic venoms. Porthidium l. hutmanni venom showed very high haemorrhagic and anticoagulant activities. These findings could be related to the presence of multiple metalloproteases, which was evidenced in this study, and also the possible presence of phospholipases or other anticoagulant activity proteins that were not defined here. They inhibited platelet aggregation, suggesting that the venom had some proteins with marked effects on haemostasis. The commercial antivenom proved to be of little effectiveness in neutralizing the crude venom haemorrhagic activity. Conclusions: These toxins cause many physiopathological alterations in bitten patients, creating a clinical picture characterized by oedema, local and systemic haemorrhages, and even necrosis, comparable to that seen in bothropic envenomation. Porthidium l. hutmanni venom has no in vitro procoagulant activity, typical of bothropic venoms, suggesting there are variances in its protein conformation. Porthidium l. hutmanni venom is used for horse immunization. However, in order to preserve the patient's life, it is necessary to improve the immunization process to produce antivenom containing high avidity and specificity antibodies against the major toxins present in this venom. Porthidium l. hutmanni venom has demonstrated being a venom with high lethal, haemorrhagic, proteolytic and procoagulant activities, whose description will have enormous utility among clinicians who deal with these accidents in its geographical distribution areas(AU)

Objetivo: Describir las características hemostáticas del veneno y evaluar la potencia del suero polivalente antiofídico contra la hemotoxicidad provocada por el envenenamiento experimental por Porthidium lansbergii hutmanni. Métodos: Se realizó una evaluación de la letalidad, actividad hemorrágica, efectos en la coagulación y agregación plaquetaria, actividad proteolítica y neutralización por el antiveneno disponible comercialmente en el país. Resultados: Se encontraron varios componentes con actividad hemostática en el veneno de Porthidium l. hutmanni al realizarse un estudio de la actividad fibrinogenolítica, hemorrágica y proteolítica de una muestra de veneno de Porthidium l. hutmanni. El veneno de Porthidium l. hutmanni no mostró la actividad coagulante o defibrinante característica de los venenos botrópicos. El veneno de Porthidium l. hutmanni mostró una elevada actividad hemorrágica y anticoagulante. Estos resultados podrían estar relacionados con la presencia de múltiples metaloproteasas, la que quedó demostrado en el estudio, y también a la posible presencia de fosfolipasas u otras proteínas de actividad anticoagulante que no se definen en el mismo. La inhibición de la agregación plaquetaria sugiere que el veneno contiene algunas proteínas con un marcado efecto sobre la hemostasis. El antiveneno comercial mostró poca efectividad en la neutralización de la actividad hemorrágica del veneno crudo. Conclusiones: Estas toxinas provocan muchas alteraciones fisiopatológicas en las víctimas de mordeduras, creando un cuadro clínico caracterizado por edema, hemorragias locales y sistémicas e incluso necrosis comparable con la que ocurre en el envenenamiento botrópico. El veneno de Porthidium l. hutmanni no tiene la actividad procoagulante in vitro típica de los venenos botrópicos, lo que apunta a variaciones en su conformación proteica. El veneno de Porthidium l. hutmanni de utiliza en la inmunización de los caballos. Sin embargo, para preservar la vida del paciente, es necesario mejorar el proceso de inmunización con vistas a producir un antiveneno que contenga anticuerpos de elevada avidez y especificidad contra las principales toxinas presentes en el veneno. El veneno de Porthidium l. hutmanni ha mostrado ser un veneno de elevada actividad letal, hemorrágica, proteolítica y procoagulante, cuya descripción tendrá una enorme utilidad para los médicos que atienden esos accidentes en sus áreas de distribución geográfica(AU)

Creolin® administered by different pathways in rats experimentally poisoned with Bothrops jararaca venom / Creolina® administrada por diferentes vias em ratos envenenados experimentalmente com veneno de Bothrops jararaca

This study aimed to evaluate the effects of Creolin® when administered by different pathways in rats experimentally poisoned with Bothrops jararaca venom. In female Wistar rats, the Bothropic venom was inoculated intramuscularly, and then the rats were either treated with Creolin® (administered orally, topically, or intramuscularly), or with amixture of venom + Creolin® intramuscularly. Animals that received Creolin®, apart from the venom, by oral, topical, or intramuscular routes developed local symptoms and showed laboratory findings similar to those animals that received only the venom. Conversely, animals inoculated with the venom incubated with Creolin® showed no signs of local venom toxicity (necrosis or hemorrhage) and displayed hematological parameters within the normal range for the species. These results suggest that Creolin® exhibited an antiophidian effect only when it is mixed with the venom and administered intramuscularly.(AU)

Esse estudo objetivou avaliar os efeitos da Creolina® quando administrada por diferentes vias de acesso em ratos experimentalmente envenenados pela peçonha de Bothrops jararaca. Em ratas Wistar fêmeas foi inoculada a peçonha botrópica por via intramuscular, e em seguida as ratas foram tratadas com Creolina® (administrada oralmente, topicamente e intramuscularmente) ou a mistura de veneno + Creolina®. Os animais que receberam a Creolina®, além do veneno, por via oral, tópica e muscular desenvolveram a sintomatologia local e achados laboratoriais semelhantes ao grupo que recebeu apenas o veneno. De forma controversa, os animais inoculados com o veneno misturado a Creolina® não apresentaram sinais característicos da ação local do veneno (necrose, hemorragia) e apresentaram parâmetros hematológicos dentro da normalidade para espécie. Esses resultados sugerem que a Creolina® apresentou efeito antiofídico apenas quando misturada ao veneno e administrada intramuscularmente.(AU)

Creolin® administered by different pathways in rats experimentally poisoned with Bothrops jararaca venom / Creolina ® administrada por diferentes vias em ratos envenenados experimentalmente com veneno de Bothrops jararaca

ABSTRACT: This study aimed to evaluate the effects of Creolin® when administered by different pathways in rats experimentally poisoned with Bothrops jararaca venom. In female Wistar rats, the Bothropic venom was inoculated intramuscularly, and then the rats were either treated with Creolin® (administered orally, topically, or intramuscularly), or with amixture of venom + Creolin® intramuscularly. Animals that received Creolin®, apart from the venom, by oral, topical, or intramuscular routes developed local symptoms and showed laboratory findings similar to those animals that received only the venom. Conversely, animals inoculated with the venom incubated with Creolin® showed no signs of local venom toxicity (necrosis or hemorrhage) and displayed hematological parameters within the normal range for the species. These results suggest that Creolin® exhibited an antiophidian effect only when it is mixed with the venom and administered intramuscularly.

RESUMO: Esse estudo objetivou avaliar os efeitos da Creolina® quando administrada por diferentes vias de acesso em ratos experimentalmente envenenados pela peçonha de Bothrops jararaca. Em ratas Wistar fêmeas foi inoculada a peçonha botrópica por via intramuscular, e em seguida as ratas foram tratadas com Creolina® (administrada oralmente, topicamente e intramuscularmente) ou a mistura de veneno + Creolina®. Os animais que receberam a Creolina®, além do veneno, por via oral, tópica e muscular desenvolveram a sintomatologia local e achados laboratoriais semelhantes ao grupo que recebeu apenas o veneno. De forma controversa, os animais inoculados com o veneno misturado a Creolina® não apresentaram sinais característicos da ação local do veneno (necrose, hemorragia) e apresentaram parâmetros hematológicos dentro da normalidade para espécie. Esses resultados sugerem que a Creolina® apresentou efeito antiofídico apenas quando misturada ao veneno e administrada intramuscularmente.

Accidente ofídico en pacientes del Hospital Básico de Jipijapa (Manabí-Ecuador) / Snake accident in patients of the Basic Hospital of Jipijapa (Manabí-Ecuador)

Introducción: el accidente ofídico es el cuadro clínico producido por la mordedura de una serpiente venenosa. En el Ecuador se describen 41 especies de serpientes venenosas. Objetivo: caracterizar clínica y epidemiológicamente el accidente ofídico en pacientes del Hospital Básico de Jipijapa (Manabí-Ecuador). Método: se realizó un estudio observacional retrospectivo durante enero 2008 - abril 2012, a partir de las historias clínicas de los pacientes hospitalizados con diagnóstico de accidente ofídico o mordedura de serpiente en el Servicio de Medicina Interna del Hospital Básico de Jipijapa (Manabí-Ecuador). Resultados: del total de 78 pacientes, 61 (78,2%) recibieron suero antiofídico (SAO) y 17 pacientes (21,8%) no recibieron. No se encontró relación entre los días de hospitalización y el hecho de haber o no recibido SAO (p= 0,8), ni tampoco entre la prueba de coagulación positiva o negativa con el tiempo de hospitalización (p= 0,7). Se encontró una correlación baja entre la variable prueba de coagulación y el grado de mordedura, siendo estadísticamente significativo (p= 0,04). Se observó incremento de mordeduras en ciertos meses, en relación con los hábitos migratorios y reproductivos de las serpientes. Conclusiones: existió dificultad en la aplicación de protocolos de atención para accidente ofídico y si bien esto no repercutió en la morbilidad y mortalidad, puede afectar en la optimización de los recursos disponibles.

Introduction: ophidian accident is the clinical picture caused by the bite of a venomous snake. Forty one species of poisonous snakes are described in Ecuador Objective: to describe clinically and epidemiologically the ophidian accident in patients of the Basic Hospital of Jipijapa (Manabí-Ecuador). Methods: a retrospective observational, descriptive study, based on medical records of hospitalized patients with a diagnosis of snake bite in Internal Medicine Basic Hospital, Jipijapa (Manabí-Ecuador) during January 2008 - April 2012 was done. Results: of the total 78 patients, 61 (78.2%) received antivenin and 17 patients (21.8%) did not receive. There was no relationship between days of hospitalization and the fact of having or not received antivenin, p=Ns (0.8) nor between test positive or negative coagulation time found hospitalization p=Ns (0.7). A low correlation was found between the coagulation test variable and the degree of bite, being statistically significant (p = 0.04). An increase of bites in certain months, in relation to the migratory and reproductive habits of the snakes was observed. Conclusions: there was difficulty in applying protocols of care for ophidian accidents and although this did not affect morbidity and mortality, it can affect the optimization of available resources.

Preclinical efficacy against toxic activities of medically relevant Bothrops sp. (Serpentes: Viperidae) snake venoms by a polyspecific antivenom produced in Mexico / Eficacia de un antiveneno poliespecífico producido en México para neutralizar, a nivel preclínico, las actividades tóxicas de venenos de Bothrops sp (Serpentes: Viperidae) de importancia médica

Abstract:The assessment of the preclinical neutralizing ability of antivenoms in Latin America is necessary to determine their scope of efficacy. This study was aimed at analyzing the neutralizing efficacy of a polyspecific bothropic-crotalic antivenom manufactured by BIRMEX in Mexico against lethal, hemorrhagic, defibrinogenating and in vitro coagulant activities of the venoms of Bothrops jararaca (Brazil), B. atrox (Perú and Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia), and B. asper (Costa Rica). Standard laboratory tests to determine these activities were used. In agreement with previous studies with bothropic antivenoms in Latin America, a pattern of cross-neutralization of heterologous venoms was observed. However, the antivenom had low neutralizing potency against defibrinogenating effect of the venoms of B. atrox (Colombia) and B. asper (Costa Rica), and failed to neutralize the in vitro coagulant activity of the venom of B. asper (Costa Rica) at the highest antivenom/venom ratio tested. It is concluded that, with the exception of coagulant and defibrinogenating activities of B. asper (Costa Rica) venom, this antivenom neutralizes toxic effects of various Bothrops sp venoms. Future studies are necessary to assess the efficacy of this antivenom against other viperid venoms. Rev. Biol. Trop. 65 (1): 345-350. Epub 2017 March 01.

ResumenEs necesario estudiar a nivel preclínico la capacidad neutralizante de los antivenenos producidos en América Latina, para conocer su espectro de cobertura. En este estudio se analizó la eficacia preclínica de un antiveneno poliespecífico botrópico-crotálico producido por BIRMEX, en México, para neutralizar los efectos letal, hemorrágico, desfibrinogenante y coagulante in vitro de los venenos de Bothrops jararaca (Brasil), B. atrox (Perú y Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia) y B. asper (Costa Rica). Se emplearon metodologías de laboratorio estándar en los análisis. En consonancia con estudios anteriores con diversos antivenenos botrópicos en América Latina, se observó un amplio patrón de neutralización de estos venenos heterólogos en la mayoría de los efectos estudiados. Sin embargo, el antiveneno mostró una baja capacidad neutralizante contra el efecto desfibrinogenante de los venenos de B. atrox (Colombia) y B. asper (Costa Rica) y no neutralizó la actividad coagulante in vitro del veneno de B. asper (Costa Rica) a la máxima razón antiveneno/ veneno empleada.

MAINTENANCE OF RED-TAIL CORAL SNAKE (Micrurus mipartitus / Mantenimiento en cautiverio de la coral rabo de ají (Micrurus mipartitus) y evaluación en la variabilidad individual de su veneno

Red-tail coral snake (Micrurus mipartitus) is a long and thin bicolor coral snake widely distributed in Colombia and is the coral that causes the majority of accidents in the Andean region, so it is important to keep this species in captivity for anti-venom production and research. However, maintaining this species in captivity is very difficult because it refuses to feed, in addition to the high mortality rate due to maladaptation syndrome. In this study a force feeding diet, diverse substrates for maintenance and a milking technique were evaluated. Additionally, individual variability of the venom was determined by High Performance Liquid Chromatography (HPLC), Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Coagulant, Anticoagulant and Hemolytic activities. The results of this study demonstrate that it was possible to increase the survival rate of this species in captivity and to determine some of the important factors in the maintenance. As to the individual variability of the venom, we found differences in number and intensity of peaks recovered by chromatography and also displayed variations in some of its biological activities.

La coral "rabo de ají" es una coral bicolor larga y delgada. Esta especie está ampliamente distribuida en Colombia y es la coral que causa el mayor número de accidentes en la región Andina, por esto es importante mantener esta especie en cautiverio con fines de producción de antivenenos e investigación. No obstante, el mantenimiento de esta especie en cautiverio es difícil, debido a que se rehúsan a alimentarse voluntariamente y a que presentan alta mortalidad por el denominado síndrome de mal adaptación. En este estudio se evaluaron varios sustratos para el mantenimiento, además de una dieta forzada y una técnica de ordeño. Adicionalmente, se evaluó la variabilidad individual del veneno a través de cromatografía liquida de alta eficiencia (HPLC), electroforesis (SDS-PAGE) y las actividades coagulante, anticoagulante y hemolítica indirecta. Los resultados de este estudio demostraron que fue posible incrementar la sobrevivencia de esta especie en cautiverio, así como determinar algunos factores importantes en su mantenimiento. A partir de la evaluación del veneno se encontraron diferencias en el número y en la intensidad de picos en la cromatografía, así como en algunas de sus actividades biológicas.

Accidentes causados por la oruga Lonomia obliqua (Walker, 1855): Un problema emergente / Accidents with caterpillar Lonomia obliqua (Walker, 1855). An emerging problem

Lonomia obliqua (Walker, 1855) es una mariposa nocturna de la familia Saturniidae, ampliamente distribuida en selvas tropicales de Sudamérica. Su larva (oruga) se caracteriza por poseer espículas ramificadas puntiagudas a lo largo de su cuerpo, que contienen una mezcla compleja de moléculas tóxicas en su interior. Cuando las espículas contactan con la piel de las personas, las toxinas ingresan pasivamente a través de la lesión, generando un envenenamiento caracterizado por manifestaciones no solo locales sino también sistémicas (fundamentalmente manifestaciones hemorrágicas). Debido al elevado número de casos que se produjeron en Brasil en las últimas décadas, el Instituto Butantan ha producido un antiveneno capaz de neutralizar los efectos deletéreos de los accidentes por contacto con L. obliqua. En Argentina, los accidentes por Lonomia son poco frecuentes y se limitan a la provincia de Misiones. Teniendo en cuenta que a la fecha no hay en la literatura descripciones de casos clínicos ocurridos en el país con tratamiento específico (antiveneno), el propósito del presente trabajo es comunicar seis casos de accidentes por contacto con orugas Lonomia que fueron atendidos en el Hospital SAMIC de Puerto Iguazú (Misiones, Argentina) durante el año 2014, y que fueron tratados con el suero antilonómico producido en Brasil. Se destaca la evolución rápida y favorable de todos los pacientes, por lo que se recomienda el uso de este antiveneno para tratar los casos de erucismo por Lonomia en la Argentina.

Lonomia obliqua (Walker, 1855) is a moth from the family Saturniidae, widely distributed in tropical rainforests of South America. In its larval stage (caterpillar) it is characterized by bristles that cover the animal’s body. These structures are hard and branched spiny evaginations of the cuticle, underneath which a complex mixture of toxic molecules is stored. When spicules are brought into contact with the skin of people, toxins enter passively through the injury, causing not only local but also systemic poisoning (primarily hemorrhagic manifestations). When the whole animal is accidentally crushed, the insect’s chitinous bristles are broken and the venomous secretions penetrate the human skin, reaching the blood circulation. Due to the numerous registered cases of erucism in Southern Brazil, the Butantan Institute has produced an antivenom able to neutralize the deleterious effects produced by contact with L. obliqua caterpillar bristles. In Argentina, these kinds of accidents are rare and restricted to the province of Misiones. Taking into account that to date there is no report in this country about clinical cases submitted to a specific treatment (antivenom), our aim is to communicate here six cases of Lonomia caterpillar-induced bleeding syndrome that were treated in the Hospital SAMIC of Puerto Iguazú (Misiones, Argentina) during 2014 with the antilonomic serum produced in Brazil. It is worthy to note that all patients evolved favorably within the first few hours, and for this reason, the use of this antivenom is recommended to treat the cases of Lonomia erucism in Argentina.

Optimización en la recuperación de inmunoglobulinas, como molécula entera y fragmento F(ab´)2, en la producción de antivenenos / Optimization in the immunoglobulins recovery, as whole molecule and F(ab´)2 fragment, in the antivenim production

En la actualidad se utilizan, principalmente, dos métodos de purificación de anticuerpos a partir de plasmas equinos hiperinmunes para la producción de antivenenos a nivel industrial, obteniéndose preparaciones enriquecidas en moléculas de inmunoglobulinas G ó fragmentos F(ab´)2. Con ambos métodos, luego de la precipitación, se observa una importante pérdida de capacidad neutralizante en comparación con la capacidad neutralizante de los plasmas de partida. En este trabajo, se realizó el fraccionamiento de plasmas equinos hiperinmunes utilizando ácido caprílico con y sin digestión enzimática con pepsina. El objetivo del trabajo fue dar a conocer la proporción de recuperación de la capacidad neutralizante luego del fraccionamiento; resultando ésta menor cuando el plasma se trató enzimáticamente. Adicionalmente, se propuso establecer cuál sería la etapa responsable de la diferencia en la recuperación de anticuerpos entre una metodología y otra. Cuando se purificaron las inmunoglobulinas enteras, se recuperó aproximadamente un 53% de la capacidad neutralizante mientras que cuando la muestra se purificó luego de ser tratada enzimáticamente, se obtuvo alrededor del 30% de esa actividad. Una relación de similar magnitud se verifica en la recuperación de la masa de proteínas solubles luego de remover los contaminantes, entre una metodología y otra. La insolubilización del fragmento Fc generado durante la digestión sería el responsable de esa pérdida adicional de proteína y capacidad neutralizante.

Today two methods are mainly used for the purification of antibodies from hyperimmune equine plasma at industrial level obtaining enriched preparations of immunoglobulin G (IgG) molecules or F(ab´)2 fragments. In both methods, after the precipitation, an important loss in the neutralizing capability was observed compared to the one of the original plasma. In this work, we performed the fractionation of hyperimmune equine plasma using caprylic acid, with and without enzymatic digestion with pepsin. The aim was to explain the percentage of recovery of the neutralizing capability after the fractionation; which resulted minor when the plasma was enzymatically treated. Additionally, we intended to establish which stage, in the purification process, was the responsible for the difference in the antibody recovery between one methodology and the other. When entire immunoglobulins were purified, approximately 53% of the neutralizing capacity was recovered, but when the sample was purified after the enzymatic treatment, around the 30% of the activity was obtained. A ratio of similar magnitude is verified on the recovery of the soluble protein mass after the removal of contaminants, between the two methods. The insolubilization of the fragment Fc generated during digestion would be responsible for the additional loss of protein and neutralizing capacity.

Optimización en la recuperación de inmunoglobulinas, como molécula entera y fragmento F(ab´)2, en la producción de antivenenos / Optimization in the immunoglobulins recovery, as whole molecule and F(ab´)2 fragment, in the antivenim production

En la actualidad se utilizan, principalmente, dos métodos de purificación de anticuerpos a partir de plasmas equinos hiperinmunes para la producción de antivenenos a nivel industrial, obteniéndose preparaciones enriquecidas en moléculas de inmunoglobulinas G ó fragmentos F(ab´)2. Con ambos métodos, luego de la precipitación, se observa una importante pérdida de capacidad neutralizante en comparación con la capacidad neutralizante de los plasmas de partida. En este trabajo, se realizó el fraccionamiento de plasmas equinos hiperinmunes utilizando ácido caprílico con y sin digestión enzimática con pepsina. El objetivo del trabajo fue dar a conocer la proporción de recuperación de la capacidad neutralizante luego del fraccionamiento; resultando ésta menor cuando el plasma se trató enzimáticamente. Adicionalmente, se propuso establecer cuál sería la etapa responsable de la diferencia en la recuperación de anticuerpos entre una metodología y otra. Cuando se purificaron las inmunoglobulinas enteras, se recuperó aproximadamente un 53% de la capacidad neutralizante mientras que cuando la muestra se purificó luego de ser tratada enzimáticamente, se obtuvo alrededor del 30% de esa actividad. Una relación de similar magnitud se verifica en la recuperación de la masa de proteínas solubles luego de remover los contaminantes, entre una metodología y otra. La insolubilización del fragmento Fc generado durante la digestión sería el responsable de esa pérdida adicional de proteína y capacidad neutralizante.(AU)

Today two methods are mainly used for the purification of antibodies from hyperimmune equine plasma at industrial level obtaining enriched preparations of immunoglobulin G (IgG) molecules or F(ab´)2 fragments. In both methods, after the precipitation, an important loss in the neutralizing capability was observed compared to the one of the original plasma. In this work, we performed the fractionation of hyperimmune equine plasma using caprylic acid, with and without enzymatic digestion with pepsin. The aim was to explain the percentage of recovery of the neutralizing capability after the fractionation; which resulted minor when the plasma was enzymatically treated. Additionally, we intended to establish which stage, in the purification process, was the responsible for the difference in the antibody recovery between one methodology and the other. When entire immunoglobulins were purified, approximately 53% of the neutralizing capacity was recovered, but when the sample was purified after the enzymatic treatment, around the 30% of the activity was obtained. A ratio of similar magnitude is verified on the recovery of the soluble protein mass after the removal of contaminants, between the two methods. The insolubilization of the fragment Fc generated during digestion would be responsible for the additional loss of protein and neutralizing capacity.(AU)

Escorpionismo. Registro de consultas médicas en el Centro Nacional de Intoxicaciones: período 2000-2010 / Scorpionism. Registration of medical consultations at the National Poison Center: Period 2000-2010

El escorpionismo es un envenenamiento de etiología accidental producido por la inoculación del veneno de un alacrán o escorpión, que ocurre predominantemente en áreas urbanas, en el ámbito domiciliario o peridomiciliario. En nuestro país son tres las especies de escorpiones de interés médico-sanitario: Tityus trivittatus, T. confluens y T. bahiensis. El cuadro clínico se caracteriza por presentar dolor agudo con escaso compromiso cutáneo y manifestaciones sistémicas que pueden, ocasionalmente, causar la muerte, principalmente en niños. El objetivo de esta publicación es, dar a conocer el registro de consultas en el Centro Nacional de Intoxicaciones en el período comprendido entre Enero de 2000 a Diciembre de 2010.

The scorpionism is an accidental poisoning produced by the inoculation of the venom of a scorpion, which in Argentina occurs predominantly in urban areas, in the domestic environment or around the human habitat. In our country there are three species of scorpions of medical-health concern: Tityus trivittatus, T. confluens and T. bahiensis. The clinical picture is characterized by severe pain with limited cutaneous and systemic manifestations, can occasionally include death, especially in children. The aim of this publication is to present the consultation in the National Poison Center in the period from January 2000 to December 2010.

Atividades biológicas do veneno de serpentes Bothrops atrox capturadas na Floresta Nacional do Tapajós, Oeste do Pará / Biological activities of Bothrops atrox snake venom captured at Floresta Nacional do Tapajós, Western Pará

Bothrops atrox (Ba) snake is responsible for most of the snakebites in Brazilian Amazon region and its venom induces a variety of local and systemic effects. However, this venom is not included in the pool of immunization used to produce Bothrops antivenom. It is known that the composition of snake venoms and their biological activities can vary not only among species but also within the same species. This variation can be influenced by snake ontogenetic stage, sex or geographical origin. In this work we characterize the electrophoretic profile and coagulant, phospholipase, fibrino(geno)lytic, platelet aggregation, desfibrinogenanting, edematogenic and hemorrhagic activities of Ba venom. We also evaluated the cellular migration induced by this venom and verified the capability of Bothrops antivenom to neutralize the coagulant and desfibrinogenanting activities. Thus, we used the lyophilized crude venom from adult, males (3) and females (3) Ba snakes (n=6), captured at Floresta Nacional do Tapajós (FLONA), Santarém region (Belterra), Pará. Adult B. jararaca (Bj) venom from Instituto Butantan was used in order to compare the results obtained in vitro assays. The Ba and Bj venoms showed differences in the number and intensity of protein bands under reducing or nonreducing conditions. The values of minimum coagulant dose of Ba and Bj venoms, respectively, on bovine plasma (10.87 ± 0.42 and 35.89 ± 0.22 µg/mL) and fibrinogen (17.68 ± 1.67 and 68.25 ± 0.64 µg/mL) indicated that Ba venom was more coagulant. In relation to the activation of procoagulant factors by Ba venom, we found higher activity on factor II (636.71 ± 25.76 µmol p-nitroaniline/min/mg venom) in comparison to factor X (182.15 ± 4.87 µmol p-nitroaniline/min/mg venom). Ba and Bj venoms were able to hydrolyze phospholipids and directly degrade fibrin on dose-dependent, but with different intensities. Both venoms induced a quick degradation of human fibrinogen Aα chain, a slow hydrolysis of the Bβ chain and no degradation of the γ chain, but the Bj venom hydrolyzed the Bβ chain more slowly than Ba venom. Unlike the verified Bj venom, the Ba venom did not induce platelet aggregation. We also observed that Ba venom showed desfibrinogenanting, edematogenic and hemorrhagic activities and induced cellular migration. Interestingly, Bothrops antivenom neutralized the coagulant and desfibrinogenanting activities of Ba venom. Our data indicate variability between the biological activities of Ba venom from FLONA and Bj venom from Instituto Butantan, as well as those of Ba from other geographical origins. This variability may influence on the severity of clinical manifestations observed on victims of snake bites and the necessity or not of more effective antivenoms.

A serpente Bothrops atrox (Ba) é responsável pela maioria dos acidentes ofídicos na região amazônica brasileira e seu veneno induz uma variedade de efeitos locais e sistêmicos. No entanto, este veneno não está incluído no pool de imunização utilizado para a produção do antiveneno botrópico. Sabe-se também que a composição e as atividades biológicas dos venenos de uma mesma espécie de serpente podem apresentar variações ontogenética, sexual ou geográfica. Neste trabalho caracterizamos o perfil eletroforético e as atividades coagulante, fosfolipásica, fibrino(geno)lítica, agregante plaquetária, desfibrinogenante, edematogênica e hemorrágica do veneno de serpentes Ba. Avaliamos ainda a migração celular induzida por este veneno e verificamos a capacidade neutralizante do antiveneno botrópico sobre as atividades coagulante e desfibrinogenante. Para tanto, utilizamos o veneno bruto liofilizado de serpentes Ba (n=6), adultas, machos (3) e fêmeas (3) capturadas na Floresta Nacional do Tapajós (FLONA), região de Santarém (Belterra), Pará. O veneno de B. jararaca (Bj) adulta do Instituto Butantan foi utilizado para comparar os resultados obtidos nos ensaios in vitro. Os venenos de Ba e Bj apresentaram diferenças no número e intensidade das bandas proteicas em condições redutoras e não redutoras. Os valores da dose mínima coagulante dos venenos de Ba e Bj, respectivamente, sobre o plasma (10,87 ± 0,42 e 35,89 ± 0,22 µg/mL) e fibrinogênio bovino (17,68 ± 1,67 e 68,25 ± 0,64 µg/mL) indicam que o veneno de Ba é mais coagulante. Em relação à ativação dos fatores procoagulantes pelo veneno de Ba, verificamos atividade mais elevada sobre o fator II (636,71 ± 25,76 µmol p-nitroanilina/min/mg de veneno) em comparação à do fator X (182,15 ± 4,87 µmol p-nitroanilina/min/mg de veneno). Os venenos de Ba e Bj foram capazes de hidrolisar fosfolipídios e de degradar fibrina diretamente e de forma dosedependente, entretanto com intensidades diferentes. Ambos os venenos induziram uma rápida degradação da cadeia Aα do fibrinogênio humano e nenhuma degradação da cadeia γ; porém o veneno de Bj hidrolisou a cadeia Bβ mais lentamente que o de Ba. Ao contrário do que verificamos com o veneno de Bj, o veneno de Ba não induziu agregação plaquetária. Observamos ainda que o veneno de Ba apresentou atividades desfibrinogenante, edematogênica e hemorrágica, sendo capaz de induzir migração celular. Interessantemente, o antiveneno botrópico neutralizou as atividades coagulante e desfibrinogenante do veneno de Ba. Nossos dados indicam variabilidade entre as atividades biológicas dos venenos de Ba da FLONA e de Bj do Instituto Butantan, assim como com as de Ba de outras origens geográficas. Essa variabilidade pode repercutir na gravidade das manifestações clínicas observadas nas vítimas de acidentes ofídicos e na necessidade ou não de antivenenos mais efetivos.