Avanços sobre vacinas e anti-soros contra veneno de serpentes e estudo de epítopos para a família de toxinas fosfolipase A2

Animal venoms are produced by specialized glands into their predators and injected in the prey, releasing secretions rich in toxins that are harmful to health. Those venomous animals trigger countless accidents around the world. These include snakebite accidents, which have been included by the World Health Organisation (WHO) in the list of neglected tropical diseases. This public health problem is caused by snakes whose toxins include proteins such as phospholipases A2 (PLA2), enzymes that are widely present in venom and trigger various physiological effects. The treatment recommended by the Ministry of Health is the application of antiophidic serum, which is manufacture based on hyperimmunization carried out on horses capable of neutralizing the action of the venom. However, this procedure has some disadvantages that end up hampering its benefits and effectiveness for victims. Thus, new methods of producing antivenom have been developed with the aim of creating alternative therapies that can improve and minimize problems. Consequently, in silico studies of immunobiologicals are becoming an option to facilitate the development of new antivenoms. This project therefore proposes the use of computational tools to predict epitopes in snake venoms, with a focus on the PLA2 family. Extracting PLA2 protein sequences from databases such as UniProt and the Immune Epitope Database in order to make predictions and classify epitope candidates. As a result, we obtained 17 candidate positions based on in silico analyses and experimental evidence from the literature. It is expect that these approaches will support studies in this area, as they will become an useful tool for developing the next generation of antivenoms.

As peçonhas animais são produzidas por glândulas especializadas em seus predadores, as quais são injetadas nas presas, liberando secreções ricas em toxinas prejudiciais à saúde. Estes animais peçonhentos desencadeiam inúmeros acidentes no mundo todo. Dentre eles, estão os acidentes ofídicos que passaram a ser incluídos pela Organização Mundial da Saúde (OMS) na lista das doenças tropicais negligenciadas. Esse problema de saúde pública é ocasionado pelas serpentes que apresentam na composição de suas toxinas proteínas como as fosfolipases A2 (PLA2), uma das enzimas mais presentes em peçonhas, as quais desencadeiam diversos efeitos fisiológicos. O tratamento preconizado pelo Ministério da Saúde é a aplicação de soro antiofídico, o qual é fabricado a partir da hiperimunização realizada em cavalos capazes de neutralizar a ação do veneno. Entretanto, esse procedimento possui algumas desvantagens que podem diminuir seus benefícios e eficácia às vítimas. Dessa forma, novas elaborações na produção de antiveneno vêm sendo desenvolvidas com intuito de criar terapias alternativas que gerem melhorias e minimizem os problemas. Consequentemente, estudos in silico de imunobiológicos se tornam opções para facilitar o desenvolvimento de novos antivenenos. Em virtude disto, neste projeto propõe o uso de ferramentas computacionais para predição de epítopos em venenos de serpentes com foco na família PLA2. Extraindo sequências proteicas de PLA2 de bancos de dados como UniProt e Immune Epitope Database, a fim de realizar as predições e classificar os candidatos a epítopos. Como resultado obtivemos 17 posições candidatas com base nas análises in sílico e evidências experimentais da literatura. Espera-se que essa abordagem possa auxiliar estudos nessa área, uma vez que se torna uma ferramenta útil para o desenvolvimento da próxima geração de antivenenos.

Anti-Metalloproteases: production and characterization of polyclonal IgG anti-F2 fraction antibodies purified from the venom of the snake bitis arietans

Bitis arietans is a medically important snake found in Sub-Saharan Africa. The envenomation is characterized by local and systemic effects, and the lack of antivenoms aggravates the treatment. This study aimed to identify venom toxins and develop antitoxins. The F2 fraction obtained from Bitis arietans venom (BaV) demonstrated the presence of several proteins in its composition, including metalloproteases. Titration assays carried out together with the immunization of mice demonstrated the development of anti-F2 fraction antibodies by the animals. The determination of the affinity of antibodies against different Bitis venoms was evaluated, revealing that only BaV had peptides recognized by anti-F2 fraction antibodies. In vivo analyses demonstrated the hemorrhagic capacity of the venom and the effectiveness of the antibodies in inhibiting up to 80% of the hemorrhage and 0% of the lethality caused by BaV. Together, the data indicate: (1) the prevalence of proteins that influence hemostasis and envenomation; (2) the effectiveness of antibodies in inhibiting specific activities of BaV; and (3) isolation and characterization of toxins can become crucial steps in the development of new alternative treatments. Thus, the results obtained help in understanding the envenoming mechanism and may be useful for the study of new complementary therapies.

Animais peçonhentos e sua trajetória secular para as ações de saúde: do histórico à aplicação / Venomous animals and their secular trajectory for health actions: from history to application / Los animales venenosos y su trayectoria secular para las acciones de salud: de la historia a la aplicación

O livro Os animais peçonhentos na saúde pública expõe um vasto histórico de acidentes envolvendo problemas de saúde provocados por animais peçonhentos. Os autores resgatam trabalhos e vivências, descritos por Louis Pasteur e Vital Brazil, fundamentais para o desenvolvimento de processos da ciência translacional como compreendida hoje, no século XXI. A obra é marcada pela defesa da necessidade de políticas públicas coerentes que sejam capazes de promover ações de capacitação, prevenção, diagnóstico, tratamento e reabilitação dos pacientes.

The book Os animais peçonhentos na saúde pública presents a vast history of accidents involving health problems caused by venomous animals. The authors rescue works and experiences, described by Louis Pasteur and Vital Brazil, fundamental for the development of translational science processes as understood today, in the 21st century. The work is marked by the defense of the need for coherent public policies capable of promoting training, prevention, diagnosis, treatment and rehabilitation of patients.

El libro Os animais peçonhentos na saúde pública expone una vasta historia de accidentes que involucran problemas de salud causados por animals venenosos. Los autores rescatan obras y experiencias, descritas por Louis Pasteur y Vital Brazil, fundamentales para el desarrollo de los procesos de ciencia translacional tal como se entienden hoy, en el siglo XXI. El libro está marcado por la defensa de la necesidad de políticas públicas coherentes capaces de promover la formación, la prevención, el diagnóstico, el tratamiento y la rehabilitación de los pacientes.

Instituto Butantan and Instituto Clodomiro Picado: a long-standing partnership in science, technology, and public health

Instituto Butantan (São Paulo, Brazil) and Instituto Clodomiro Picado (San José, Costa Rica) are public institutions devoted to scientific and technological research, production of antivenoms and other immunobiologicals, and a variety of public health interventions aimed at confronting the problem of snakebite envenoming in their countries and elsewhere. In the context of the 120th anniversary of Instituto Butantan, this work describes the historical developments in the relationship between these institutions, which has evolved into a solid cooperation platform in science, technology, and public health. The relationship between Instituto Butantan and Costa Rica started early in the 20th century, with the provision of Brazilian antivenoms to Costa Rica through the coordination of Instituto Butantan and the health system of Costa Rica, with the leadership of Clodomiro Picado Twight. After the decade of 1980, a prolific collaborative network has been established between Instituto Butantan and Instituto Clodomiro Picado (founded in 1970) in the areas of scientific and technological research in pharmacology, biochemistry, experimental pathology, immunology, and public health, as well as in antivenom development, production, preclinical evaluation, and quality control. In addition, both institutions have played a key role in the integration of regional efforts in Latin America to create a network of public institutions devoted to antivenom production and quality control, in close coordination with the Pan American Health Organization (PAHO). This long-standing partnership is an example of a highly productive south-south cooperation under a frame of solidarity and public well-being.

Landscape of research, production, and regulation in venoms and antivenoms: a bibliometric analysis / Panorama de la investigación, la producción y la regulación de venenos y antivenenos: un análisis bibliométrico / Panorama de pesquisa, produção e regulação de venenos e antivenenos: análise bibliométrica

ABSTRACT Objectives. To assess the productivity and visibility in research, clinical studies, treatment, use and production of antivenoms against poisonous snakes, scorpions and spiders. Methods. Bibliometric analysis of research and other activities. Articles on venoms and antivenoms published between 2000 and 2020 were retrieved from the Scopus database. The records were analyzed by bibliometric indicators including number of documents per year, journals, authors, and citation frequency. VOSviewer® v.1.6.13 was used to construct bibliometric networks for country co-authorships and co-occurrence of terms. Results. Australia, Brazil, Costa Rica and India were among the six top countries with most documents and were selected for more detailed analysis. Costa Rica was the country with the largest percentage of its publications dedicated to antivenom production and venomics. Only a few papers dealt with the issues of quality, safety, and efficacy of antivenoms or the role of the national regulatory authorities. The use of VOSviewer® allowed visualization through joint publications of networking between countries. Visualization by co-occurrence of terms showed differences in the research carried out. Conclusions. Working in a collaborative and coordinated manner these four countries could have a major impact on envenoming globally. Attention should be given not only to antivenom production but also to strengthening regulatory oversight of antivenom products.

RESUMEN Objetivos. Evaluar la productividad y la visibilidad en la investigación, los estudios clínicos, el tratamiento, el uso y la producción de antivenenos contra las picaduras de serpientes, arañas y escorpiones venenosos. Métodos. Análisis bibliométrico de la investigación y de las otras actividades. Se tomaron los artículos sobre venenos y antivenenos publicados entre el 2000 y el 2020 en la base de datos de Scopus. Estos documentos se analizaron mediante indicadores bibliométricos como el número de documentos por año, revistas, autores o frecuencia en las citas. Se utilizó VOSviewer® v.1.6.13 para crear una red bibliométrica para coautorías de países y coapariciones de términos. Resultados. Australia, Brasil, Costa Rica e India estaban entre los seis primeros países con más documentos y se seleccionaron para un análisis más detallado. Costa Rica fue el país con el mayor porcentaje de sus publicaciones dedicadas a la producción de antivenenos y la venómica. Solo unos pocos artículos trataban los temas de la calidad, la seguridad y la eficacia de los antivenenos, o la función de las autoridades regulatorias nacionales. Gracias a VOSviewer® pudimos visualizar las publicaciones conjuntas de las colaboraciones entre países. La visualización por la coaparición de términos arrojó diferencias en la investigación realizada. Conclusiones. Si estos cuatro países trabajasen de forma colaborativa y coordinada, podrían tener una repercusión mayor en los envenenamientos por picaduras en el mundo. El foco no debe ponerse solo en la producción de antivenenos, sino también en fortalecer la supervisión regulatoria de estos productos.

RESUMO Objetivos. Avaliar a produtividade e visibilidade em pesquisa, estudos clínicos, tratamento, uso e produção de antivenenos contra peçonhas de serpentes, escorpiões e aranhas. Métodos. Análise bibliométrica de pesquisas e outras atividades. Artigos sobre venenos e antivenenos publicados entre 2000 e 2020 foram obtidos da base de dados Scopus. O conteúdo foi analisado segundo indicadores bibliométricos, como número de artigos por ano, periódicos, autores e frequência de citação. Utilizou-se o software VOSviewer® v.1.6.13 para construir redes bibliométricas de coautoria de países e co-ocorrência de termos. Resultados. Austrália, Brasil, Costa Rica e Índia figuraram entre os seis principais países com o maior número de artigos e, assim, foram selecionados para uma análise mais aprofundada. A Costa Rica teve a maior porcentagem de publicações dedicadas à produção de antivenenos e pesquisa em venômica. Apenas um pequeno número de artigos tratou de questões relacionadas à qualidade, segurança e eficácia dos antivenenos ou ao papel das autoridades nacionais reguladoras. O software VOSviewer® permitiu visualizar, através das publicações conjuntas, as redes formadas entre diferentes países. A visualização por co-ocorrência de termos revelou diferenças nas pesquisas realizadas. Conclusões. Trabalhando de forma colaborativa e coordenada, esses quatro países tiveram uma influência importante em nível mundial no campo de acidentes por animais peçonhentos. Deve-se atentar não apenas à produção de antivenenos, mas também ao fortalecimento da fiscalização regulatória destes.

Envenenamiento ofídico en Costa Rica: logros y tareas pendientes / Snakebite envenoming in Costa Rica: achievements and pending tasks

Resumen Objetivo: Efectuar un análisis de los logros en Costa Rica en cuanto al problema de los envenenamientos por mordeduras de serpientes, y señalar tareas pendientes para reducir aún más el impacto de esta patología. Metodología: Se efectuó una revisión de bibliografía relacionada con el estudio del envenenamiento ofídico en Costa Rica y con los avances efectuados en el tema, en el país. Paralelamente, se identificaron aspectos que requieren atención en el manejo de esta enfermedad tropical desatendida. Conclusiones: Desde las primeras décadas del siglo XX, se han realizado avances significativos en la comprensión y manejo del problema de los envenenamientos por mordedura de serpiente en Costa Rica. Se ha trabajado desde una visión integral que incluye: investigación científico-tecnológica, producción y distribución de antivenenos, esfuerzos en prevención, capacitación de las personas profesionales de la salud en el diagnóstico y tratamiento, y docencia de grado y posgrado en el tema. El país ha asumido un papel de liderazgo a nivel internacional, tanto en el plano académico como en la provisión de antivenenos a muchos países. No obstante, aún quedan aspectos del problema que requieren nuevos esfuerzos en nuestro medio, sobre todo en lo referente a la atención de las consecuencias biomédicas, psicológicas, sociales y económicas que sufren las personas afectadas.

Abstract Aim: To analyze the achievements made in Costa Rica in confronting the problem of snakebite envenomings, and to identify pending tasks to further reduce the impact of this pathology. Methods: A review of the literature on snakebite envenomings in Costa Rica was carried out, identifying the main achievements reached in the country. In parallel, issues that require renewed attention in the management of this neglected tropical disease were identified. Conclusions: Since the first decades of the 20th century, significant advances have been made in Costa Rica for understanding and confronting the problem of snakebite envenomings. An integrative perspective has been implemented, which includes scientific and technological research, production and distribution of antivenoms, prevention campaigns, training of health professionals in the diagnosis and treatment of envenomings, and teaching at graduate and undergraduate levels. Costa Rica has had a leading international role in this topic at the academic level, and by providing antivenoms to many countries. Nevertheless, there are issues that require further efforts, especially regarding the attention to the biomedical, psychological, social and economic consequences suffered by people affected by snakebites.

Toxinology in Colombia: contributions of ophidism/scorpionism program and other research groups / Toxinología en Colombia: contribuciones del programa de ofidismo/escorpionismo y de otros grupos de investigación

Background: Toxinology is a sub-field of toxicology dedicated to studying toxins produced by animals, plants and, microorganisms. In Colombia, during the last thirty years, this area has been mainly investigated by Ophidism/Scorpionism Program of Universidad de Antioquia. However, some other research groups have also contributed to our knowledge of venoms and toxins, as well as their related effects and treatments. Objective: to highlight the most significant findings in toxinology made by the Ophidism/Scorpionism Program and other research groups in Colombia. Methods: 119 papers dealing with the history of ophidiology and toxinology in Colombia were collected and analyzed. Results: some useful terms are described to understand toxinology and its scope. Also, a brief history of ophidiology is presented, spanning from the discovery of America until present-day findings. Finally, an overall description of several results related to toxin isolation, characterization, antivenoms, clinical trials, description of new species, proteomic and transcriptomic, among others. The nineteens were characterized by the study of snakebites, their clinic manifestations, and the use of antivenoms. In addition, the ethnopharmacological studies of medicinal plants used in snakebite treatments began to be explored. The 2000s included the newly ethnopharmacology, toxin isolation, clinical trials, inhibitor studies, scorpion venom characterization, and scorpion stings features. Finally, from 2010 until today, proteomic and transcriptomic gave the most important findings. Conclusions: Toxinology works in Colombia have contributed to our knowledge about endemic species, clinical manifestations of snakebite and scorpion stings, and the development of new therapeutic agents. However, we invite Colciencias and other funding agencies to assign more resources to support a higher number of researchers in this field, since snakebite is considered a neglected tropical disease by the World Health Organization, which needs more attention from governments and scholars. Finally, the venoms of some species and their possible mode of action are still unknown to us. Besides, given the complexity of venoms, we are not yet aware of the potential use of toxins in current biomedicine. Thus, studies in toxinology must continue.

Antecedentes: La Toxinología es el campo de la Toxicología que estudia las toxinas producidas por animales, plantas y microorganismos. En Colombia, durante los últimos treinta años, los estudios realizados en esta área han sido desarrollados principalmente por el Programa de Ofidismo/Escorpionismo de la Universidad de Antioquia. Sin embargo, otros grupos de investigación también han contribuido en el conocimiento de venenos, toxinas, efectos y tratamientos. Objetivo: Destacar los hallazgos más relevantes en toxinología realizados por el Programa de Ofidismo Escorpionismo y otros grupos de investigación en Colombia. Métodos: Se recopilaron 119 artículos referentes a la historia de la ofidiología y la toxinología en Colombia. Resultados: Se describieron algunos términos útiles para el entendimiento de la toxinología y sus alcances. Se construyó una breve historia de la ofidiología que inicia con el descubrimiento de América y finaliza con hallazgos recientes. Se realizó una amplia descripción de varios resultados relacionados con el aislamiento y caracterización de toxinas, antivenenos, ensayos clínicos, descripciones de nuevas especies, proteómica y transcriptómica, entre otras. Así, la década de los noventa se caracterizó por el estudio de las mordeduras de serpientes, sus manifestaciones clínicas, el uso de antivenenos y la exploración de la etnofarmacología asociada a las mordeduras de serpiente. La década del 2000 incluyó nuevamente etnofarmacología, el aislamiento de toxinas, ensayos clínicos, estudios sobre inhibidores de toxinas, caracterización de venenos y picaduras de escorpión. Finalmente, desde 2010 hasta hoy, la proteómica y transcriptómica aportaron los hallazgos más importantes. Conclusiones: Los estudios de Toxinología en Colombia han contribuido al conocimiento de especies endémicas, manifestaciones clínicas de mordeduras de serpientes y picaduras escorpiones, y el desarrollo de nuevos agentes terapéuticos. No obstante, se invita a Colciencias y a otras agencias de financiamiento a apoyar la investigación en este campo, ya que es considerada una enfermedad tropical desatendida por la Organización Mundial de la Salud y necesita mayor atención por parte del gobierno e instituciones académicas. Además, dada la complejidad de los venenos, se desconoce el uso potencial de las toxinas en la biomedicina actual. Así, se deben continuar realizando estudios en toxinología.

Design and production of a recombinant hybrid toxin to raise protective antibodies against Loxosceles spider venom

Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen’s formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.

The history of antivenoms development: beyond Calmette and Vital Brazil

This review presents the main contributions to our knowledge regarding the development of antivenoms for therapeutic use in victims of venomous animal bites. We cover the progress of serum therapy since tetanus and diphtheria antitoxins in Germany and France until the current scenario of antivenom production worldwide. During these more than 120 years of antivenom development, many researchers contributed to establish what are nowadays the antivenoms used for therapeutic purpose. The history of antivenoms development is fascinating! This review aims to recognize all those who contributed to the establishment of new sera, new methodologies and saving lives: much more than Calmette and Vital Brazil.

Current challenges for confronting the public health problem of snakebite envenoming in Central America

Snakebite envenoming is a serious public health problem in Central America, where approximately 5,500 casesoccur every year. Panama has the highest incidence and El Salvador the lowest. The majority, and most severe,cases are inflicted by the pit viper Bothrops asper (family Viperidae), locally known as ‘terciopelo’, ‘barba amarilla’ or‘equis’. About 1% of the bites are caused by coral snakes of the genus Micrurus (family Elapidae). Despite significantand successful efforts in Central America regarding snakebite envenomings in the areas of research, antivenommanufacture and quality control, training of health professionals in the diagnosis and clinical management of bites,and prevention of snakebites, much remains to be done in order to further reduce the impact of this medicalcondition. This essay presents seven challenges for improving the confrontation of snakebite envenoming inCentral America. Overcoming these challenges demands a coordinated partnership of highly diverse stakeholdersthough inter-sectorial and inter-programmatic interventions.

Current challenges for confronting the public health problem of snakebite envenoming in Central America

Snakebite envenoming is a serious public health problem in Central America, where approximately 5,500 casesoccur every year. Panama has the highest incidence and El Salvador the lowest. The majority, and most severe,cases are inflicted by the pit viper Bothrops asper (family Viperidae), locally known as ‘terciopelo’, ‘barba amarilla’ or‘equis’. About 1% of the bites are caused by coral snakes of the genus Micrurus (family Elapidae). Despite significantand successful efforts in Central America regarding snakebite envenomings in the areas of research, antivenommanufacture and quality control, training of health professionals in the diagnosis and clinical management of bites,and prevention of snakebites, much remains to be done in order to further reduce the impact of this medicalcondition. This essay presents seven challenges for improving the confrontation of snakebite envenoming inCentral America. Overcoming these challenges demands a coordinated partnership of highly diverse stakeholdersthough inter-sectorial and inter-programmatic interventions.

Estudo de venenos de serpentes do complexo Bothrops, uma comparação da filogenia e da reatividade com antivenenos / Caracterization of the microvescles present in Crotalus durissus terrificus venom

Frequently the venom composition has been considered as a reflex of snake phylogeny, with important implications for the production of antivenoms and treatment of envenomings. Snakes from Bothrops complex are responsible for most snake bites registered in Brazil, and their phylogenetic relationships are not yet completely clarified. Another important discussion is related to the efficacy of Bothrops Antivenom (SAB) in the treatment of envenoming by snakes whose venoms are not present in the immunization pool. Thus, the aim of this study was to analyze the composition of venoms from 6 species of snakes from Bothrops complex, and their correlation with snake phylogeny and reactivity with o SAB. In this way, venoms from Bothropoides jararaca, Bothropoides neuwiedi, Bothrops atrox, Bothrops jararacussu, Rhinocerophis cotiara and Rhinocerophis alternatus were analyzed by SDS-PAGE and reverse phase chromatography on HPLC C18 column. The reactivity of SAB with venoms and their fractions was performed by ELISA and western blot, followed by assessment of neutralization of lethality and hemorrhagic activity of the venoms of B. jararaca (present in immunization pool of SAB) and B. atrox (absent from the pool). The venoms showed distinct electrophoretic and chromatographic profiles, without apparent correlation with the snake phylogeny. Only venoms of the genus Rhinocerophis (R. alternatus and R. cotiara) showed relatively similar profiles. SAB recognized the venoms of different species with the same antibody titer of 640,000. Most of the fractions eluted from C18 column were recognized by SAB, especially those corresponding to snake venom metalloproteinases (SVMP) of class P-III. By SDS-PAGE, the venom of B. jararacussu presented the more distinct electrophoretic profile with predominance of phospholipase A2 (PLA2), while in the other venoms, the SVMPs were the predominant component. Two microlitters of antivenom were sufficient to protect 100 % of mice challenged with 3 LD50 of B. jararaca venom (105 µg), although for B. atrox venom (225 µg) the same effect was only obtained when doubling the antivenom amount. However, the SAB neutralized completely the hemorrhage induced by 10 µg of the both venoms, with the same volume of serum (4 µL). Thus, we conclude that SAB reacts similarly with 16 the same family of toxins from distinct venoms, regardless of the phylogeny of the snake or the presence of venom in the immunization pool employed in SAB production. This suggests that a Bothrops antivenom with greater efficiency and broad spectrum can be developed, if one considers the reactivity of antivenom to the different classes of toxins and not just the phylogeny of snakes.

Frequentemente a composição dos venenos tem sido considerada com um reflexo da filogenia das serpentes, com importantes implicações para a produção de antivenenos. As serpentes do complexo Bothrops são responsáveis pela grande maioria dos acidentes ofídicos registrados no Brasil e suas relações filogenéticas ainda não foram completamente esclarecidas. Outra discussão importante diz respeito à eficácia do Soro Antibotrópico (SAB) no tratamento dos envenenamentos ocasionados por serpentes cujos venenos não fazem parte do pool de imunização, utilizado em sua produção. Portanto, o objetivo deste trabalho foi analisar a composição dos venenos de 6 espécies de serpentes do complexo Bothrops e sua correlação com a filogenia da serpente e reatividade com o SAB. Para tanto, venenos de Bothropoides jararaca, Bothropoides neuwiedi, Bothrops jararacussu, Bothrops atrox, Rhinocerophis alternatus e Rhinocerophis cotiara foram analisados por SDS-PAGE e cromatografia de fase reversa em HPLC usando coluna C18. A reatividade do SAB com os venenos e suas frações foi analisada por ELISA e Western blotting, seguida por testes de neutralização da letalidade e da atividade hemorrágica induzida pelos venenos de B. jararaca (presente no pool de imunização do SAB) e B. atrox (ausente do pool). Os venenos mostraram distintos perfis eletroforéticos e cromatográficos, sem correlação aparente com a filogenia da serpente. Somente os venenos do gênero Rhinocerophis (R. alternatus e R. cotira) exibiram perfis relativamente semelhantes. O SAB reconheceu os venenos das seis espécies com um mesmo título de anticorpos (640.000). A maioria das frações eluídas da coluna C18 foram reconhecidas pelo SAB, principalmente as correspondentes à Metaloproteinases de Venenos de Serpentes (SVMPs) da classe P-III. Por SDS-PAGE, o veneno de B. jararacussu apresentou o perfil eletroforético mais distinto com predominância de PLA2, enquanto nos outros venenos as SVMPs foram os componentes predominantes. Dois microlitros de antiveneno foram suficientes para proteger 100 % dos camundongos desafiados com 3DL50 do veneno de B. jararaca (105 µg), embora para B. atrox (225 µg) o mesmo efeito tenha sido 14 obtido somente com o dobro do volume de antiveneno. Entretanto, o SAB neutralizou completamente a hemorragia induzida por 10 µg de ambos os venenos com um mesmo volume de soro (4 µL). Portanto, concluímos que o SAB reage similarmente com uma mesma família de toxinas de venenos distintos, independentemente da filogenia da serpente ou mesmo da presença do veneno no pool de imunização. Isso sugere que um antiveneno botrópico de maior espectro e eficiência pode ser desenvolvido, se for considerada a reatividade do antiveneno frente às diversas classes de toxinas e não apenas a filogenia da serpente.

Cross-neutralization of the coagulant activity of Crotalus durissus terrificus venom from the northeast of Argentina by bivalent bothropic antivenom

Cross-neutralization of Crotalus durissus terrificus venom coagulant activity was tested using bivalent horse antivenom against Bothrops alternatus and Bothrops diporus venoms. Our in vitro and in vivo experiments showed that bothropic antivenom neutralizes the thrombin-like activity of crotalic snake venom and this cross-reaction was demonstrated by immunoassays either with whole venom or a purified thrombin-like enzyme. These results suggest common antigenic properties and, consequently, similar molecular structure among venom thrombin-like enzymes. Besides, they provide information that could be further used in the development of new antivenom formulations.

Ethnobotanic study of Randia aculeata (Rubiaceae) in Jamapa, Veracruz, Mexico, and its anti-snake venom effects on mouse tissue

In Mexico, medicinal plants are widely used. The use of Randia aculeata by healers against snakebites has never been scientifically tested in relation to possible effects on blood parameters and muscle tissue damage. Interviews were carried out in Jamapa, Veracuz, Mexico, with local residents to collect information about the traditional use of Randia aculeata. In this locality, seven pieces of fruit from the plant are mixed in a liter of alcohol, and then administered orally against snakebites. By using histological techniques and a murine model, we explored its cytoprotective properties against the effects of Crotalus simus and Bothrops asper venoms. Possible protections provided by the plant against tissue damage to skeletal and cardiac muscles and against the typical loss of red blood cells were analyzed. Randia aculeata caused an increase in microhematocrit and total hemoglobin, parameters that are often decremented in association with the loss of red blood cells, which is a characteristic effect of animal venom. Randia aculeata was also shown to protect against the lowering of platelet levels caused by Bothrops asper venom. Finally, Randia aculeata produced a partial inhibition of necrosis following administration of snake venom in skeletal and myocardial muscles. The present results provide solid evidence for the traditional use of Randia aculeata against snakebites, as demonstrated by protection against muscular tissue damage and the diminution of red blood cells.

Envenenamientos por mordeduras de serpientes en América Latina y el Caribe: Una visión integral de carácter regional / Snakebite poisoning in Latin America and the Caribbean: An integral view from a regional perspective

Se analiza la situación de los envenenamientos por mordeduras de serpiente en América Latina y el Caribe, los cuales representan un importante problema de Salud Pública en la región. Esta patología afecta, fundamentalmente, a la población rural de nuestros países, y tiene un alto impacto en sectores desatendidos por los programas de salud. Estos envenenamientos son causados, en su gran mayoría, por especies de la familia Viperidae, especialmente del género Bothrops. Existe un conglomerado de laboratorios públicos y privados productores de antivenenos en la región, aunque en algunos casos la producción no satisface las necesidades de algunos países, por lo que los antivenenos deben ser importados de países vecinos. Las investigaciones científicas y tecnológicas efectuadas en América Latina han generado un gran bagaje de conocimiento sobre las serpientes y sus venenos, así como sobre la clínica de los envenenamientos y el perfil de eficacia y seguridad de los antivenenos, a niveles preclínico y clínico. Pese a los indudables logros obtenidos en el manejo de esta enfermedad en la región, se debe redoblar esfuerzos para garantizar:

(a) un mejor conocimiento de los venenos y sus efectos; (b) una visión más realista de la incidencia de estos envenenamientos; (c) un mejoramiento cualitativo y cuantitativo en la producción de antivenenos; (d) un mejor control de calidad de los antivenenos que se importan en algunos países; (e) una más adecuada distribución de los antivenenos en la región, especialmente en zonas rurales de alta incidencia de envenenamientos; (f) una mayor capacitación del personal de salud en el tratamiento de estos envenenamientos, incluyendo el correcto uso de antivenenos; (g) un seguimiento y atención a las personas que han sufrido secuelas como producto de estos accidentes; y (h) programas comunitarios de prevención y atención de esta patología. Estas tareas deben ser enfrentadas con una filosofía de equidad, solidaridad y cooperación en la región, con la participación de múltiples protagonistas a muy diversos niveles.

The public health problem of envenomings induced by snakebites in Latin America and the Caribbean is analyzed in this work. This pathology affects predominantly the rural population and has a high impact on regions where the provision of health services is insufficient. The majority of envenomings are inflicted by species of the genera Bothrops and Crotalus, classified in the family Viperidae. There are several laboratories in the region which manufacture antivenoms for the treatment of these envenomings, although the volume of production in some cases does not fulfill the national demand and, consequently, antivenoms have to be imported. A significant body of knowledge has been gained in the taxonomy of the snakes and the biochemistry, toxicology and immunology of venoms, as well as in the preclinical and clinical performance of antivenoms. Despite significant advances in the control of this neglected tropical disease in Latin America, there are pending tasks in the region, such as:

(a) To improve our knowledge on snakes and their venoms; (b) to assess the actual incidence and mortality of snakebite envenomings; (c) to increase the volume of antivenom produced and, in some cases, to improve the quality of antivenoms; (d) to improve the national quality control laboratories; (e) to develop more effective strategies of distribution of antivenoms, especially to remote rural areas where snakebites are frequent; (f) to foster permanent education programs for the health staff in charge of the treatment of these envenomings; (g) to follow up and provide support to people that suffer physical or psychological sequelae as a consequence of these envenomings; and (h) to strengthen community programs aimed at improving the prevention and adequate management of snakebites. This conglomerate of tasks should be approached with a philosophy of solidarity, integration and cooperation in the region, with the involvement of multiple actors and institutions.

What is the optimum concentration of m-cresol in antivenoms?

Antivenoms against snake and scorpion envenomations are usually equine in nature and composed mostly of F(ab')2; additionally, phenol and m-cresol are mainly employed for their preservation. Although there is no study on this subject, m-cresol is utilized by most manufacturers in a concentration that ranges from 0.15 to 0.35 g percent. Decreasing the concentration of m-cresol to its minimal effective level may protect victims from its toxic effects and keep the antivenom stable during its shelf life without forming any aggregates. In the present work, different concentrations of m-cresol, ranging from 0.1 to 0.35 g percent, were used with some selected batches of snake and scorpion antivenoms. A low concentration of 0.15 g percent showed an acceptable preserving result that complies perfectly with antimicrobial specifications stated by the British Pharmacopoeia. Tested antivenoms (in 12 batches), when kept in a cold room for 39 months (more than their shelf life), retained their physical, chemical and microbiological activities according to the specifications of pharmacopeias. The present data demonstrated that reduction of m-cresol concentration to 0.15 g percent in case of equine F(ab')2 antivenoms will improve safety of such preparations and preserve their stability during their shelf life.(AU)

Polyvalent horse F(Ab`)2 snake antivenom: Development of process to produce polyvalent horse F(Ab`)2 antibodies anti-african snake venom

A method to obtain polyvalent anti-Bitis and polyvalent-anti-Naja antibodies was developed by immunizing horses with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambicacrude venoms. Antibody production was followed by the ELISA method during the immunization procedure. Once the desired anti-venom antibody titers were attained, horses were bled and theimmunoglobulins were separated from the sera by (NH4)2SO4 precipitation, cleaved with pepsin and filtered through a 30 kDa ultrafiltration membrane. F(ab´)2 fragments were further purified by Q-Fast Flow chromatography, concentrated by molecular ultrafiltration and sterilized by filtration through 0.22 m membranes. The resulting F(ab´)2 preparations were rich in intact L and in pieces of H IgG(T) chains, as demonstrated by electrophoresis and Western blot and exhibited high antibody titers, as assayed bythe ELISA method. In addition, the preparations possess a significant capacity to neutralize the lethalityof venoms, as estimated by ED50 determination in mouse assay and are free of toxic substances, pyrogen and bacterial or fungal contaminations.

Cross - reactivity of horse monovalent antivenoms to venoms of ten bothrops species

Horses were immunized with B. alternatus, B. atrox, B. cotiara, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B, moojeni, B. neuwiedi and B. pradoi venoms. Antibodies recognizing the venom antigenic components were either immunochemically detected by the precipattion methods or biologically by the assays measuring the venomns indirect hemolytic and lethal toxic activities . Specific and cross - reacting antivenoms.Modifications in the serum electrophoretic patterns characterized by a reduction of the albumin peak and by a correspondent increase of the y-globulins with a pattent or no modification of the α or β globulins were found in these sera.

Cavalos foram imunizados com veneno de B. alternatus, B. atrox, B. cotiara, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B, moojeni, B. neuwiedi and B. pradoi. Anticorpos específicos para componentes antigênicos dos venenos foram detectados pelos métodos imuno- enzimático, dupla-difusão e precipitação quantitativa enquanto que os anticorpos neutralizantes foram analisados pelos métodos da hemólise indireta em placas e pela neutralização de seus efeitos letais. Anticorpos, tanto específicos como dando reações cruzadas com venenos botrópicos foram encontrados em todos os dez soros monovalentes. Modificações nos padrões eletroforéticos, caracterizadas por uma redução no pico da albumina e por um correspondente aumento das γ. globulinas com modificações ora acentuadas ora pouco perceptíveis nas frações das α e β globolinas, foram detectadas todos esses soros.