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Resumen Introducción: dentro de los insectos del orden lepidóptero, la familia Saturniidae tiene más de 2.400 especies, allí se encuentran Hylesia y Lonomia. Objetivo: presentar dos casos de pacientes con accidente lonómico, el manejo y desenlace para reconocer la importancia de estos eventos en las regiones silvestres de la Orinoquia colombiana. Casos clínicos: paciente de 8 años proveniente de área rural quien refería contacto en región palmar de mano derecha con gusanos quién 48 horas después de dicho contacto presentó equimosis en extremidades, flictena sangrante en talón derecho, cefalea, escalofríos y artralgias, además se evidenciaron tiempos de coagulación prolongados. Paciente de 13 años de características similares a las del caso previamente descrito sin presentar manifestaciones clínicas, pero que presentaba tiempos de coagulación prolongados. Se consideró que cursaba con accidente lonómico por lo que se aplicaron 5 ampollas de suero antilonómico polivalente a cada una sin registrar reacciones adversas. En ambos casos cursaron con evolución clínica adecuada con disminución a rangos de seguridad de tiempos de coagulación. Conclusiones: el veneno lonómico actúa en la cascada de coagulación produciendo manifestaciones hemorrágicas de gravedad variable. El suero antilonómico es el único tratamiento eficaz, a pesar de estar disponible desde hace más de 20 años en Brasil hay un 5% de progresión a síndromes hemorrágicos severos y un 1.5 a 2% de mortalidad. A pesar de tener gran relevancia clínica en las Américas existe subregistro, es importante conocer sus manifestaciones y el manejo para así poder evitar complicaciones mortales. Abstract Background: among the insects of the order Lepidoptera, the family Saturniidae has more than 2,400 species, there are Hylesia and Lonomia. Objective: to present two cases of patients with lonomic accident, the management and outcome to recognize the importance of these events in the wild regions of the Colombian orinoquia. Clinical case: an 8-year-old patient from a rural area who refers to contact in the palmar region of the right hand with worms for more than 48 hours, presents ecchymosis in the extremities, bleeding flictena in the right heel, headache, chills and arthralgias. Clotting times are performed which are prolonged. A 13-year-old patient who also referred contact without presenting clinical manifestations but presenting prolonged clotting times. They are considered to occur due to a lonomic accident, so 5 ampoules of polyvalent antilonomic serum are administered to each one without registering adverse reactions. They have an adequate clinical evolution with a decrease in the safe ranges of clotting times. Conclusions: the lonomic venom acts in the coagulation cascade producing hemorrhagic manifestations of variable severity. Antilonomic serum is the only effective treatment, despite being available for more than 20 years in Brazil, there is a 5% progression to severe hemorrhagic syndromes and 1.5 to 2% mortality. Despite being of great clinical relevance in the Americas, there is an underreporting, it is important to know its manifestations and management in order to avoid fatal complications.

ilustraciones, fotografías acolor El veneno de las serpientes de coral, del género Micrurus, es conocido por generar un efecto neurotóxico. Este efecto es similar entre las diferentes especies de este género, sin embargo, aún no se conoce completamente la eficacia de la neutralización cruzada de los antivenenos. En el presente estudio evaluamos los efectos de los venenos de tres serpientes de coral colombianas, de las especies M. lemniscatus, M, medemi y M, sangilensis. Determinando la capacidad de inhibir la neurotransmisión, en una preparación de nervio frénico y diafragma de ratas Wistar. Se evaluaron los efectos del veneno a dosis de 1, 10 y 50 µg/ml y se evaluó la capacidad neutralizante del antiveneno anticoral polivalente (AAP) del Instituto Nacional de Salud (INS), frente a la dosis de 10 µg/ml de cada especie. Los 3 venenos generaron bloqueos neuromusculares dependientes de las dosis en comparación con los controles. Siendo el veneno de M. lemniscatus el que tuvo una actividad neurotóxica más rápida, seguido de M. sangilensis y finalmente de M. medemi. Así mismo los 3 venenos fueron neutralizados con éxito por el AAP del INS. Siendo el veneno de M. medemi el que más porcentaje de neutralización tuvo, seguido del veneno de M, sangilensis y finalmente del veneno de M, lemniscatus. Demostrando así que el AAP del INS tiene una capacidad neutralizante y polivalente. A pesar de ser fabricado con venenos de serpientes de coral distintas a las involucradas en este estudio. (Texto tomado de la fuente) Coral snake’s venoms of the genus Micrurus are characterized by peripheral paralysis neurotoxicity. A similar neurotoxic effect is induced by the venom of most members of this genus, yet the efficaciousness of cross species venom/anti-venom treatment has not been thoroughly investigated. In this study we evaluated the effects of the venom of three Colombian coral snakes, species M. lemniscatus, M. medemi, and M. sangilensis, and its ability to inhibit neurotransmission in the phrenic nerve and diaphragm of Wistar rats. Venom effects were evaluated in doses of 1, 10, and 50 µg / ml. Further, the neutralizing capacity of the polyvalent anticoral antivenom (PAA) of the National Institute of Health (NIH) was evaluated against a venom dose of 10 µg / ml of each species. All 3 venoms generated a significant, dose-dependent, neuromuscular block compared to controls with M. lemniscatus exhibiting the fastest neurotoxic response followed by M. sangilensis, and M. medemi. Finally, the NIH PAA was able to completely neutralize M. medemi and partially neutralize both M. sangilensis and M. lemniscatus. Thus, we have demonstrated that the INS AAP has neutralizing and polyvalent capacity despite being manufactured with venoms from coral snakes not involved in this study. El Departamento Administrativo de Ciencia, Tecnología e Innovación Minciencias, es la entidad encargada de promover las políticas públicas para fomentar la ciencia, la tecnología y la innovación en Colombia por medio del Proyecto 2104777-58348, Contrato 686 de 2018 COLCIENCIAS-INS: “Caracterización bioquímica y biológica del veneno de las corales colombianas Micrurus medemi, Micrurus sangilensis y Micrurus lenmiscatus y su neutralización con el antiveneno anticoral polivalente producido por el INS” que permitió el desarrollo y culminación de este proyecto de tesis. Instituto Nacional de Salud, permitió el uso de instalaciones, reactivos, animales de laboratorio y uso de salas del bioterio de barrera ABSL2 para el entrenamiento y desarrollo de pruebas preliminares para el desarrollo y culminación de este proyecto de tesis. La Universidad Nacional de Colombia, permitió el uso de instalaciones, reactivos, animales de laboratorio y uso de salas del bioterio para el entrenamiento y desarrollo de pruebas preliminares para el desarrollo y culminación de este proyecto de tesis. Maestría Magister en Ciencias – Farmacología

ilustraciones, graficas Las lectinas son glicoproteínas de origen no inmune, que reconocen carbohidratos con diferentes afinidades, por lo tanto, tienen un amplio espectro de estudio y aplicación, en diferentes campos como el médico, por ejemplo, en terapias antirretrovirales y antitumorales. Paralelamente, son de gran importancia en el estudio de formación de complejos lectina–carbohidrato, así como las interacciones proteína carbohidrato, que conducen a diferentes respuestas biológicas. Se encuentran en todos los organismos desde virus hasta humanos, aunque han sido muy bien estudiadas en animales y plantas superiores. Su alta distribución en los diferentes reinos muestra su importancia en los procesos celulares, que se dan por interacciones célula–célula, hospedero–patógeno, y planta–simbionte, entre otras. En el caso de los artrópodos, las funciones fisiológicas de las lectinas aún no están establecidas, sin embargo, hay evidencia de su importancia en la respuesta inmune, metamorfosis, diferenciación, muda, entre otras. Con respecto al estudio de las lectinas en venenos, es muy poco lo que se menciona en las revisiones y en términos generales es muy escaso. Los estudios se han limitado en su mayoría a las lectinas del veneno de serpientes (85.9%) y en menor proporción peces (5,6%), arañas y escorpiones (2,8%). Puesto que, a partir de la glándula de escorpión, se ha registrado solamente una secuencia hipotética para la lectina de Tityus obscurus (TyobL), obtenida mediante estudios de transcriptómica. Además, la única lectina estudiada a partir del veneno es la de Buthus occitanus, especie del sur de Vietnam, que se caracteriza por reconocer residuos de Fuc>> D-Glc > L-Rham= D-xyl, y solo se conoce su análisis de aminoácidos y peso molecular de subunidades. Por consiguiente y teniendo en cuenta que no existen estudios enfocados en lectinas presentes en veneno de escorpión, y que además no se conoce la secuencia ni la estructura terciaria, con el desarrollo de este trabajo se detectaron nuevas lectinas (TymaLs), a partir del veneno de Tityus macrochirus, una especie endémica del departamento de Cundinamarca, las cuales se caracterizaron por ser glicoproteínas de alto peso molecular (>100 kDa), reconocer residuos de lactosa y/o azúcares acetilados, y estar compuestos por monómeros de pesos moleculares ~ 15 kDa. Para complementar el conocimiento acerca de estas lectinas, se llevaron a cabo estudios de predicción estructural con la secuencia (nt) propuesta para la lectina de T.obscurus (TyobL), los resultados obtenidos por SWISS MODEL muestran que es una lectina tipo taquilectina (TL), similar a la isolectina 5a de Tachypleus tridentatus purificada de la hemolinfa del cangrejo (Texto tomado de la fuente). Lectins are proteins of no immune origin belonging to a diverse animal or plant origin group and are characterized by irreversibly binding a given monosaccharide or oligosaccharide. These properties have made them essential molecules in studying carbohydrate structure and function. Studying glycans’ function and how they can regulate biological processes is one of the most rapidly growing fields in biochemistry and molecular biology; this ranges from coagulation to viral and bacterial infection processes acting on different types of cells. Lectins have been clues in the knowledge in this area which will lead to developing new alternatives in treating diseases. Advances in proteomics have also generated growing interest in understanding how glycans participate in the multiple interactions at the cellular level where glycoproteins play a particular role. The most well-known interactions leading to biological responses are protein-protein or protein-carbohydrate ones, while eventual carbohydrate-carbohydrate interactions have not been considered relevant to date; experimental evidence has been presented which has implicated them in biological processes including cellular traffic, host-pathogen interactions, embryogenesis, spermatogenesis, fertilization, nervous system development, and angiogenesis. Many studies related to Arthropods´ lectins are carried out although physiological functions have not been established yet, there is evidence about the role immune system, metamorphosis, and differentiation, among others. However, regarding venom lectins, scarce information is found in the bibliography. Mainly studies have been mostly snakes (85.9%) and, to a lesser extent, fish (5.6%), spiders, and scorpions 82.8%). Only one hypothetical sequence has been registered for Tityus obscurus lectin (TyobL) obtained in transcriptomics studies. Additionally, only one lectin from Buthus occitanus venom was isolated and characterized for recognizing Fuc>> D-Glc > L-Rham= D-xyl residues, aminoacidic and carbohydrate analysis and molecular weight were studied too. Thus, limited structural information is available about its primary and tertiary structure. Likewise, their sequence in nucleotides or amino acid is generally unknown preventing their production in recombinant form. Considering that no studies are focusing on the detection, isolation, and biochemical characterization of lectins from scorpion venom, in this work, an endemic specie from the department of Cundinamarca, Tityus macrochirus (TymaLs) were studied to detect and isolate new lectins (TymacLs), which had specificity towards lactose residues (β-D-Gal (1-4)-β-D-GlcNAc–O-R) and acetylated carbohydrates. These lectins are glycoproteins with molecular weight higher (>100 kDa), and 15 kDa for monomers forms. To deepen the knowledge of these lectins, we chose the sequence from T. obscurus, for carrying out structural prediction studies. These results obtained by SWISS-MODEL showed that it is a tachylectin-like lectin (TL), from Tachypleus tridentatus crab hemolymph. Maestría Magíster en Ciencias - Bioquímica Estudios de Lectinas en venenos animales

Snake venoms comprise a highly complex mixture of proteins, and there is also a high interspecific and intraspecific variability in their composition, even in the same region. Our aim was to compare the composition of the venoms of Bothrocophias myersi, Crotalus durissus, and Bothrops asper, snakes from the Colombian Andean region by Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). The venoms were given to the research group under an agreement with Fundación Zoológica de Cali. The venoms pool was obtained by manual extraction, lyophilized and frozen. The venom protein was quantified by direct measurement with Nanodrop® 280 nm. The protein composition was established by RP-HPLC, using a Lichosper 100 RP, C18 column (250X4 mm) with a pore size of 5|m, as well as by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The highest quantity of protein was found in the venom of B. myersi (108.6 mg/ mL) followed by C. durissus (78.1 mg/mL) and B. asper (74.1 mg/mL). All venoms showed bands of 15 and 50 KDa by using SDS-PAGE. B. myersi venom chromatogram exhibited 16 peaks by RP-HPLC. We conclude that the composition of the three venoms is quite similar, being phospholipase A2 the common protein therein, and together with metalloproteinases they were the most abundant protein families in the venom of B. myersi. SDS-PAGE and RP-HPLC techniques allow a first approach to the profile of the venoms, which in turn could clarify the clinical syndrome produced. RESUMEN Los venenos de las serpientes comprenden una mezcla compleja de proteínas, y existe una alta variabilidad interespecífica e intra-específica en su composición, incluso en la misma región. Nuestro objetivo fue comparar la composición de los venenos de Bothrocophias myersi, Crotalus durissus y Bothrops asper de la región andina de Colombia, mediante cromatografía líquida de alta eficiencia en fase reversa (RP-HPLC). Los venenos fueron entregados al grupo de investigación mediante un convenio con la Fundación Zoológica de Cali. El pool de venenos fue obtenido por extracción manual, liofilizado y congelado. La proteína de los venenos fue cuantificada por Absorbancia 280nm por medición directa con Nanodrop®. La composición proteica se estableció por RP-HPLC, utilizando una columna Lichosper 100 RP, C18 (250X4 mm) con un tamaño de poro de 5|jm, así como por electroforesis en gel dodecil sulfato de sodio-poliacrilamida (SDS-PAGE). La mayor cantidad de proteínas se encontró en el veneno de B. myersi (108.6 mg/mL), seguido de C. durissus (78.1 mg/mL) y B. asper (74.1 mg/mL). Todos los venenos mostraron bandas de 15 y 50 KDa por SDS-PAGE. El cromatograma de B. myersi exhibió 16 picos por RP-HPLC. Concluimos que la composición de los tres venenos es bastante similar, siendo la fosfolipasa A2 la proteína común en estos y junto con las metaloproteinasas fueron las familias de proteínas más abundantes en el veneno de B. myersi. Las técnicas de SDS-PAGE y el RP-HPLC permiten un primer acercamiento al perfil de los venenos, lo que a su vez podría contribuir a esclarecer el síndrome clínico producido.

Every year, in Brazil, about one hundred thousand accidents with venomous animals occur, resulting in approximately two hundred and twenty deaths. The main animals involved are scorpions, spiders, bees, wasps and snakes. Frequency is determined by different factors such as temperature, humidity, rainfall, ecological imbalance, expansion of urban and agricultural areas. Children are more vulnerable due to their inherent curiosity, added to the lack of risk and the condition of the immune system, which is still in formation, which enhances gravity. This study analyzed the epidemiological profile of pediatric accidents involving venomous animals in the municipality of Chapecó, SC, from 2014 to 2018. Method: Retrospective study of accidents involving venomous animals registered in the Epidemiological Surveillance Notification Disease Information System. Of the 489 notifications recorded, the spiders caused 24.34%. In 2014, 177 cases were reported, approximately one case every two days, the year with the highest incidence. The months of January and February were more significant by the total number of notifications. Limbs extremities were the hardest hit. The male gender was the most affected (55%). The highest frequency occurred in urban areas (79.8%). There were no deaths. The municipality of Chapecó reveals a high incidence of accidents with venomous animals, showing the need to establish services and campaigns about the risk, especially in the months with higher temperatures. With an emphasis on local reality, it is important that the population be alerted and precautions taken, especially with children. Cada año, en Brasil, hay alrededor de cien mil accidentes por animales venenosos, resultando en aproximadamente doscientas veinte muertes. Los principales animales involucrados son escorpiones, arañas, abejas, avispas y serpientes. La frecuencia está determinada por diferentes factores como temperatura, humedad, precipitaciones, desequilibrio ecológico, expansión de áreas urbanas y agrícolas. Los niños son más vulnerables debido a su curiosidad inherente, sumada al desconocimiento sobre el riesgo y la condición del sistema inmunológico, que aún está en formación, lo que potencia la gravedad. Este estudio tuvo como objetivo analizar el perfil epidemiológico de los accidentes pediátricos por animales venenosos en la ciudad de Chapecó, SC, entre 2014 y 2018. Se trata de un análisis retrospectivo de los accidentes por animales venenosos registrados en el Sistema de Informação de Agravos de Notificação na Vigilância Epidemiológica. De las 489 notificaciones registradas, las arañas causaron un 24,34%. En 2014 se registraron 177 casos, aproximadamente un caso cada dos días, el año de mayor incidencia. Los meses de enero y febrero fueron más significativos en cuanto al número total de notificaciones. Las extremidades de las fueron los lugares más afectados. Los hombres fueron los más afectados (55%). La mayor frecuencia ocurrió en áreas urbanas (79,8%). No hubo muertos. El municipio de Chapecó revela una alta incidencia de accidentes con animales venenosos, mostrando la necesidad de establecer servicios y campañas, especialmente en los meses con temperaturas más altas. Con énfasis en la realidad local, es importante que la población esté alerta y que se tomen precauciones, especialmente con los niños. Todos os anos, no Brasil, ocorrem cerca de cem mil acidentes por animais peçonhentos, resultando em aproximadamente de duzentos e vinte óbitos. Os principais animais envolvidos são escorpiões, aranhas, abelhas, vespas e serpentes. A frequência é determinada por diferentes fatores como temperatura, umidade, pluviosidade, desequilíbrio ecológico, expansão das áreas urbanas e agrícolas. As crianças são mais vulneráveis devido à inerente curiosidade, somado ao desconhecimento do risco e a condição do sistema imunológico, ainda em formação, o que potencializa a gravidade. Este estudo objetivou analisar o perfil epidemiológico dos acidentes pediátricos por animais peçonhentos, no município de Chapecó, SC, entre 2014 a 2018. Trata-se de um estudo retrospectivo de análise dos acidentes por animais peçonhentos registrados no Sistema de Informação de Agravos de Notificação na Vigilância Epidemiológica. Das 489 notificações registradas, as aranhas causaram 24,34%. Em 2014 contabilizou-se 177 casos, aproximadamente um caso a cada dois dias, o ano de maior incidência. Os meses de Janeiro e Fevereiro tiveram maior significância por número total de notificações. As extremidades dos membros foram os locais mais atingidos. O sexo masculino foi o mais acometido (55%). A maior frequência se sucedeu nas zonas urbanas (79,8%). Não houve óbitos. O município de Chapecó revela elevada incidência de acidentes por animais peçonhentos, mostrando a necessidade de se estabelecer serviços e campanhas sobre o risco, em especial nos meses com temperaturas mais elevadas. Com ênfase na realidade local, é importante que a população seja alertada e que sejam adotadas precauções, especialmente com crianças.

RESUMEN El veneno de los centípedos se produce en glándulas exocrinas especializadas y constituyen una mezcla compleja mayoritariamente de péptidos y proteínas, y de algunas moléculas de diferente naturaleza química, como serotonina, acetilcolina, lípidos y carbohidratos. Los constituyentes proteicos son considerados fisiopatológicamente más importantes que los no proteicos. Éstos son responsables de las actividades enzimáticas y neurotóxicas que le permite a los centípedos matar o inmovilizar a la presa, asistir en la digestión de la misma, y utilizarlo en su autodefensa. Dentro del orden Scolopendromorpha, Scolopendra gigantea se considera hasta ahora la de mayor tamaño reportada. Esta especie habita en las zonas selváticas y semidesérticas de Venezuela y es considerada como la causante de la mayoría de los accidentes graves por escolopendrismo pudiendo en algunos casos ocasionar la muerte del afectado. La composición proteica de estos venenos es muy variada, así como sus actividades biológicas. Además, es probable que contengan componentes que pudieran tener un significativo interés farmacéutico. Conocer la composición y mecanismos de acción de los componentes del veneno permitiría mejorar el diagnóstico y tratamiento médico de los casos de escolopendrismo, con la intención de disminuir la tasa de morbilidad y de discapacidad ocasionada por los centípedos en Venezuela. Pero la realidad es que es muy poco lo que se conoce con relación al veneno de S. gigantea. A pesar de representar un creciente problema de Salud Colectiva, se ha convertido en un problema olvidado. ABSTRACT Centipede venom is produced in specialized exocrine glands and constitutes a complex mixture, mostly of peptides and proteins, and other molecules of different chemical nature, such as serotonin, acetylcholine, lipids and carbohydrates. Protein constituents are considered pathophysiologically more important than non-protein ones. These are responsible for the enzymatic and neurotoxic activities that allow centipedes to kill or immobilize prey, assist in its digestion, and use them in self-defense. Within the order Scolopendromorpha, Scolopendra gigantea is the largest species so far reported. This species lives in the jungle and semi-desert areas of Venezuela and is considered to be the cause of most serious accidents due to scolopendrism, and in some cases it can cause the death of the affected person. The protein composition of these venoms is varied, as well as their biological activities. Furthermore, they are likely to contain components that could be of significant pharmaceutical interest. Knowing the composition and action mechanisms of the components of the venom would allow to improve the diagnosis and medical treatment of cases of scolopendrism, to reduce the morbidity and disability caused by centipedes in Venezuela. However, little is known regarding the venom of S. gigantea. Despite representing a growing collective health problem, it has become a neglected problem.

Hymenoptera venom allergy is associated with significant morbidity and deterioration in health-related quality of life, and risk of fatal systemic reactions. Although venom immunotherapy is safe and the only effective treatment in allergic individuals, some patients prefer not to pursue this treatment. Since 2011, when the 50% reimbursement was stopped, patients must fully support the cost of immunotherapy. This study aimed to ascertain the reasons why patients decline immunotherapy.A medical records review of all patients proposed to receive venom immunotherapy at an Allergy and Clinical Immunology Department in Porto, Portugal, between 2006 and 2015, followed by a phone interview to patients refusing treatment.A total of 83 subjects were enrolled, with a mean (± SD) age of 44.4 (14.7) years and 55 (66%) males; 27 refused venom immunotherapy between 2006 and 2015. Nineteen were interviewed and 14 of those stated price as the main reason for declining treatment. The only identified risk factor associated with immunotherapy refusal was being proposed after 2011 (OR: 3.29; 95% CI: 1.12 - 9.68; p = 0.03).The number of patients refusing venom immunotherapy doubled since reimbursement was withdrawn. Price was identified as the major obstacle to treatment completion. Immunotherapy proposal after reimbursement was stopped was associated with a 3-fold increase in the risk of refusing treatment.These findings show how economic decisions may have a detrimental effect on patient care, as immunotherapy refusal left them exposed to an avoidable life-threatening risk.Introdução: A alergia a veneno de himenópteros está associada a uma significativa morbilidade e diminuição da qualidade de vida, bem como a risco de reações alérgicas fatais. Apesar da imunoterapia com veneno de himenópteros ser um tratamento seguro e o único eficaz nesta patologia, alguns doentes decidem não o realizar. Desde 2011, quando a comparticipação de 50% terminou, o custo da imunoterapia é totalmente suportado pelos doentes. Este trabalho pretendeu identificar os motivos da recusa desta terapêutica. Material e Métodos: Revisão dos registos clínicos de todos os doentes propostos para imunoterapia com veneno de himenópteros num serviço de Imunoalergologia, no período 2006 - 2015, seguida de entrevista telefónica aos que a recusaram. Resultados: Foram incluídos 83 doentes, com uma idade média (± DP) de 44,4 (14,7) anos. Cinquenta e cinco (66%) eram homens; 27 recusaram imunoterapia entre 2006 e 2015. Dezanove foram entrevistados e 14 identificaram o preço como principal motivo de recusa. O único fator de risco identificado para a recusa de imunoterapia foi ser proposta depois de 2011 (OR: 3,29; 95% CI: 1,12 – 9,68; p = 0,03). Discussão: O número de doentes a recusar imunoterapia duplicou desde que a comparticipação foi retirada. O preço foi o principal obstáculo à realização do tratamento. Ser proposto após o término da comparticipação do tratamento aumentou em três vezes o risco de recusa. Conclusão: Estes achados revelam o impacto negativo de uma decisão económica na saúde e segurança destes doentes, já que a recusa da imunoterapia os manteve expostos a um risco de vida evitável.

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. Resumen 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.

Cell-penetrating peptides (CPPs) comprise a class of short polypeptides that possess the ability to selectively interact with the cytoplasmic membrane of certain cell types, translocate across plasma membranes and accumulate in the cell cytoplasm, organelles (e.g., the nucleus and mitochondria) and other subcellular compartments. CPPs are either of natural origin or de novo designed and synthesized from segments and patches of larger proteins or designed by algorithms. With such intrinsic properties, along with membrane permeation, translocation and cellular uptake properties, CPPs can intracellularly convey diverse substances and nanomaterials, such as hydrophilic organic compounds and drugs, macromolecules (nucleic acids and proteins), nanoparticles (nanocrystals and polyplexes), metals and radionuclides, which can be covalently attached via CPP N- and C-terminals or through preparation of CPP complexes. A cumulative number of studies on animal toxins, primarily isolated from the venom of arthropods and snakes, have revealed the cell-penetrating activities of venom peptides and toxins, which can be harnessed for application in biomedicine and pharmaceutical biotechnology. In this review, I aimed to collate examples of peptides from animal venoms and toxic secretions that possess the ability to penetrate diverse types of cells. These venom CPPs have been chemically or structurally modified to enhance cell selectivity, bioavailability and a range of target applications. Herein, examples are listed and discussed, including cysteine-stabilized and linear, α-helical peptides, with cationic and amphipathic character, from the venom of insects (e.g., melittin, anoplin, mastoparans), arachnids (latarcin, lycosin, chlorotoxin, maurocalcine/imperatoxin homologs and wasabi receptor toxin), fish (pardaxins), amphibian (bombesin) and snakes (crotamine and cathelicidins).

The making of antibodies in animals can be demanding due to that several antigens, mostly of low molecular masses, provoke imperceptible immune response or are even totally nonimmunogenic. The transformation of non-immunogenic molecules into effective antigens represent an important immunological tasks. The crotamine from the rattlesnake Crotalus durissus cumanensis snake venom was purified by a Mono S HR 10/10 chromatography column and used to immunise C57/B mice, after to be polymerised with glutaraldehyde. The murine polyclonal antibodies directed against native crotamine-like (NCL) treated with glutaraldehyde and their product crotamine-like polymer (CLP) were generated by immunisation injecting CLP via lymph node cells. These antibodies were capable of detecting CLP in an enzyme-linked immunosorbent assay. The SDS-PAGE of NCL and CLP showed bands of molecular masses ~ 3 kDa and ~18 kDa, respectively. These results offer evidence that the polyclonal antibodies recognise specific putative original and post-polymerisation epitopes on the CLP molecule, which were maintained following the process of polymerisation. The results are discussed in relation to the preservation of a functional postpolymerisation epitopes on CLP. La producción de anticuerpos en animales puede ser una actividad ardua, debido a que muchos antígenos, principalmente los de baja masa molecular, provocan una respuesta inmune imperceptible o aún son totalmente no-inmunogénicos. La transformación de una molécula no inmunogénica, en un antígeno efectivo representa un importante reto inmunológico. La crotamina obtenida del veneno de la serpiente de cascabel (Crotalus durissus cumanensis) fue purificada a través de una columna de cromatografía Mono S HR 10/10 (Biorad, EUA) y usada para inmunizar ratones de la cepa C57/B, luego de ser polimerizada con glutaraldehido. Los anticuerpos policlonales dirigidos contra la crotamina nativa tratada con glutaraldehido, y su producto el polímero obtenido de la crotamina (CLP) se lograron mediante inmunización vía ganglios linfáticos con polímeros de CLP. Esos anticuerpos policlonales fueron capaces de detectar el CLP, en un ensayo de ELISA. Los perfiles de migración (SDS-PAGE) de la crotamina nativa y la CLP mostraron bandas de masa molecular ~ 3 kDa y ~18 kDa, respectivamente. Estos resultados ofrecen evidencia de que los anticuerpos policlonales reconocen epítopes específicos originales y posteriores a la polimerización en la molécula de CLP, que se mantuvieron luego del proceso de polimerización. Los efectos se discuten en relación con la preservación de epítopes funcionales post-polimerización en CLP. 173-179 rodriguezacosta1946@yahoo.es

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia As cobras assumem uma posição preponderante entre os animais venenosos, produzindo venenos que constituem complexas e potentes misturas, nos quais proteínas e enzimas se destacam pelas suas propriedades fármaco - toxicológicas. Os envenenamentos causados por estes são um inegável problema de saúde pública para o Homem, nomeadamente em países em desenvolvimento, levando a graves consequências que podem culminar em morte. A toxicidade resultante das variadas toxinas aí presentes pode afetar vários sistemas e órgãos do corpo humano, sendo os antivenenos a terapêutica de primeira linha para a maior parte das situações. Contudo, estas mesmas propriedades dos constituintes responsáveis pelos efeitos negativos mencionados, apresentam-se como uma potencial base para a conceção de agentes terapêuticos, aplicáveis em diversas áreas medicinais, o que se traduz em fármacos já aprovados e comercializados, resultado da mimetização das estruturas desses compostos, bem como na atual existência de outros fármacos e formulações em desenvolvimento. É neste paralelismo que se centra a presente revisão bibliográfica. Por outro lado, tal como previsto no plano de estudos do Mestrado Integrado em Ciências Farmacêuticas na Faculdade de Farmácia da Universidade de Coimbra, realizei os estágio curriculares, o culminar e complemento de quatros anos e meio de formação teórica que o antecedem, o espaço e oportunidade para aplicação e consolidação dos conhecimentos até aí adquiridos. Mais concretamente, em Farmácia Comunitária, enquanto área obrigatória, e em Indústria Farmacêutica, permitindo-me conhecer e experimentar mais do que um setor farmacêutico. Snakes take a predominant position among venomous animal species, producing venoms that are complex and powerful mixtures in which proteins and enzymes stand out for their pharmacological and toxicological properties. Poisonings caused by them are an undeniable public health problem for humans, especially in developing countries, leading to serious consequences that can culminate in death. The toxicity resulting from the various toxins present there can affect various systems and organs of the human body, as antivenom is the first-line therapy for most situations. However, these constituents properties, responsible for the negative effects mentioned, present themselves as a potential basis for the conception of therapeutic agents, applicable in several medicinal fields, which translates into already approved and commercialized drugs, as a result of the mimetization of the structures of these compounds, as well as in the current existence of other drugs and formulations in development. The present bibliographic review focuses this parallelism. On the other hand, as foreseen in the study programme of Integrated Master's Degree in Pharmaceutical Sciences, at Faculty of Pharmacy, University of Coimbra, I carried out the curricular internships, the culmination and complement of four and an half years of theoretical training that precede it, the space and opportunity for the application and consolidation of the knowledge acquired so far. More specifically, in Community Pharmacy, as a mandatory area, and in Pharmaceutical Industry, allowing me to know and experience more than a pharmaceutical sector.

A ação proteolítica das proteinases do veneno de serpentes têm sido determinada em um grande número de substratos sintéticos e naturais. Uma serino proteinase básica, ativa sobre a caseína e fibrinogênio foi purificada a partir do veneno de Bothrops moojeni usando um simples passo de cromatografia em coluna de CM Sepharose fast flow equilibrada com tampão Tris-HCl 0,05 M pH 7,0 em KC1 0,1 M e eluída com gradiente convexo de concentração ( 0,1 M - 0,45 M KCI) do mesmo tampão. A enzima , denominada M003, não é hemorrágica e apresenta somente traços de atividade coagulante. Substratos sintéticos ou cromogênicos (DL-BAPNA azocaseína e azoalbumina) não são hidrolisados pela M 003. Em gel de eletroforese em poliacrilamida pH 4,3, M 003 apresentou uma banda única Em eletroforese em gel de poliacrilamida com agentes desnaturantes, M 003 se comportou como uma proteína de cadeia simples com um peso molecular aparente de 26.600, na presença e ausência de p-mercaptoetanol. A enzima não contem carboidratos neutros, seu aminoácido N-terminal é a alanina e tem pi 7.8. A composição em aminoácidos mostrou 249 resíduos/mol, um alto conteúdo de aminoácidos hidrofílicos e 14 resíduos de meia-cistina, os quais possibilitam até 7 pontes de dissulfetos. A proteinase cliva a cadeia a mais rapidamente que a cadeia (3 do fibrinogênio bovino e não mostrou nenhum efeito sobre a cadeia 8. As atividades coagulante e proteolítica foram inibidas quando M 003 foi tratada com EDTA, P-mercaptoetanol e leupeptina. A atividade esterolítica de M 003 sobre -N-tosyl-Arginina metil éster (TAME) é de 29,66 pMol min'1 mg'1. A recuperação total em absorbância foi de aproximadamente 66 %, com M003 representando 1,42 % (p/p) do veneno bruto inicial. Proteolytic action of snake venom proteinases has been determined on a great number of natural and synthetic substrates. A basic serine proteinase active on casein and fibrinogen was purifíed from Bothrops moojeni venom using only one step by chromatography on CM Sepharose fast flow column previously equilibrated with 0.05 M, pH 7.0 Tris-HCl 0.1 M KC1 and eluted with a convex concentration gradient (0.1 M - 0.45 M KC1) of the same buffer. The enzyme, named M003, is not hemorrhagic and presents only traces of blood- clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by M 003. On polyacrylamide gel electrophoresis at pH 4.3, M 003 presented a single protein band. On sodium dodecyl sulfate-polyaciylamide electrophoresis M 003 behave as a single- chain protein with a mol. wts of 26,600 in the presence and absence of P- mercaptoethanol and pi 7.8. The enzyme does not contain neutra) carbohydrates and its N-terminal amino acid is alanine. The amino acid composition showed 249 residues/moles a high contents hydrophilic amino acids and 14 half-cys residues, which should account for 7 dissulfide bonds. The proteinase cleaves the A-a chain faster than the B-p of bovine fibrinogen and shows no effect on the 8-chain. Both coagulant and proteolytic activities where inhibited when the M003 was treated with EDTA, P-mercaptoethanol and leupeptin. Specific esterolytic activities of M 003 on alfa-N-tosyl-1- arginine methyl ester (TAME) are 29.64 pmol min-1 mg-1. Total absorbance recovery for column was around 66%. M 003 represented 1.42% (w/w) of the initial desiccated venom Dissertação (Mestrado)

RESUMEN Tradicionalmente las hojas de Oenothera rosea “chupasangre” son usadas para reducir los hematomas, por lo que, esta investigación estuvo dirigida a la evaluación de los componentes capaces de retardar la coagulación sanguínea. Del extracto etanólico de O. rosea se separó por cromatografía de capa fina en celulosa, usando etanol: agua (1:5), dos fracciones F-2 y F-5, que luego fueron preincubadas por 10 minutos con trombina y el veneno de la serpiente Lachesis muta, rico en Enzima Semejante a Trombina (EST) y luego se midió la actividad coagulante sobre plasma humano citratado y fibrinógeno bovino (Fb), así como el sustrato cromogénico BApNA. Los resultados mostraron que F-2 alargó el tiempo de coagulación sobre Fb en 58,58 % y el F-5 en 67,78 %, mientras que usando veneno los retardos fueron para F-2 10,67 % y F-5 36,27 %. Usando plasma, los valores fueron para F-2 34,14 % y F-5 70,59 %. Asimismo, empleando trombina la actividad amidolítica se redujo en F-2 48,48 % y F-5 67,32 %, mientras que con la EST de L. muta la inhibición de F-2 50,20 % y F-5 69,10%. Mediante estos ensayos in vitro se concluye que F-2 y F-5 podrían ser flavonoides anticoagulantes, con probable acción antitrombolítica. ABSTRACT Traditionally the leaves of Oenothera rosea "chupasangre" are used to reduce the bruising in humans. Therefore, this research was focused at evaluating of the fractions capable of retarding blood clotting. From the ethanolic extract of O. rosea separated by thin layer chromatography on cellulose using ethanol: water (1: 5), F-2 and F-5, wich were preincubated for 10 minutes with thrombin and the venom of the Lachesis muta snake rich in EST (Thrombin-like enzyme) and then coagulant activity was measured on citrated human plasma and bovine fibrinogen, as well as the BApNA chromogenic substrate. The results showed that F-2 lengthened the clotting time over Fb 58,58 % and F-5 67,78 %, while using poison the delays were for F-2 10,67 % and F-5 36,27 %. Using plasma, the values were for F-2 34,14 % and F-5 70,59 %. Likewise, by using thrombin the amidolytic activity was reduced by F-2 48,48 % and F-5 67,32 % while with the EST of L. muta the inhibition of F-2 50,20 % and F-5 69, 10%. These in vitro tests conclude that F-2 and F-5 will be anticoagulant flavonoids with probable antithrombolytic action.

This work aimed to understand better how the phylogenetic evolution of the genus Micrurus occurred within the american continent, answering questions such as when the genus originated, where it was the center of dispersion, and which path the species took until its current distribution. To do this, we first constructed a phylogeny based on the NADH sub-unit 4 gene, which revealed the group's emergence still during the Miocene, before what other related works say. The tricolor monads and triads color pattern species formed two monophyletic groups. By the divergence times presented by our phylogeny, we corroborate with the current hypothesis that the ancestral population of Micrurus entered South America through an arch of islands that connected North and South America during the Miocene, and that the genus had a South American origin. We also suggest that the creation of Central America with the closing of the Panama’s Isthmus; the uplift of the Andes Mountain Range; the closure of the Acre System forming the Amazon River; and the rise of the Brazilian Central Plateau were key factors for the evolution of Micrurus. Furthermore, our data indicate that the center of dispersion of the genus was in the north of South America, from where an ancestral population of the monad group migrated towards the south, colonizing the east coast of the continent and the Amazon Forest - after the closure of the System Acre - and a second population migrated towards Central and North America - after the closing of the Panama’s Isthmus. The triad group dispersed towards the south, colonizing from dry areas in the Caatinga and Brazilian Cerrado, to wet areas in the Amazon. The divergence of South American species was greatly influenced by the Andes uplift, which was probably responsible for vicariance events, separating species on the cis- and trans-Andes sides. In addition, we tested the hypothesis that the so-called aposematic coloration of the snakes of the genus Micrurus is co-evolving with the toxicity of their venom. And, this time, two phylogenies were built for each of the two main toxins from Micrurus' venom, PLA2 and 3FTx, to be compared with the NADH phylogeny that shows us how the group's evolutionary history occurred. Thus, when analyzing the groups formed in the NADH phylogeny we suggested that the color pattern of these snakes is a characteristic linked to the phylogenetic history of the group, and not so much to environmental factors. Likewise, the groups formed in the toxin trees made us suggest that the composition of the venom of these snakes also follows the evolutionary history of the group, rather than environmental factors. With these results, we suggest that the genes responsible for the expression of the coloration of coral snakes are closely linked to the genes encoding the toxins that make up their venom, and thus, these two characteristics may actually be in coevolution, and the coloration does, in fact, have an aposematic function in these snakes. Nenhuma Este trabalho teve como objetivo entender melhor como ocorreu a evolução filogenética do gênero Micrurus dentro do continente americano, respondendo às perguntas como quando o gênero se originou, onde foi o centro de dispersão, e qual caminho as espécies percorreram até sua atual distribuição. Para isso, primeiramente construímos uma filogenia baseada no gene NADH sub-unidade 4, que revelou o surgimento do grupo ainda no Mioceno, antes do que sugerem outros trabalhos relacionados. As espécies de padrão de coloração tricolor mônade e tríade formaram dois grupos monofiléticos. Pelos tempos de divergência apresentados pela nossa filogenia, nós corroboramos a atual hipótese de que a população ancestral de Micrurus entrou na América do Sul através de um arco de ilhas que conectou a América do Norte com a do Sul durante o Mioceno, e que o gênero teve uma origem sul-americana. Sugerimos também que a criação da América Central com o fechamento do Istmo do Panamá, o levantamento da Cadeia Montanhosa dos Andes, o fechamento do Sistema Acre formando o Rio Amazonas e o surgimento do Planalto Central Brasileiro foram fatores chave para a evolução de Micrurus. Ainda, nossos dados apontam que o centro de dispersão do gênero foi ao norte da América do Sul, de onde uma população ancestral do grupo mônade migrou em direção ao sul, colonizando a costa leste do continente e a Floresta Amazônica – após o fechamento do Sistema Acre – e uma segunda população migrou em direção à América Central e do Norte – após o fechamento do Istmo do Panamá. Já o grupo tríade se dispersou em direção ao sul, colonizando desde áreas secas na Caatinga e Cerrado brasileiro até áreas úmidas na Amazônia. A divergência das espécies sul-americanas foi bastante influenciada pelo levantamento dos Andes, que foi provavelmente responsável por eventos de vicariância, separando espécies nos lados cis- e trans-Andes. Além disso, foi testada a hipótese de se a coloração dita aposemática das cobras-corais do gênero Micrurus está em coevolução com a toxicidade de seu veneno. Para isso foram construídas duas filogenias para cada umas das duas principais toxinas do veneno de Micrurus, PLA2 e 3FTx, para serem comparadas com a filogenia do NADH que nos mostra como ocorreu a história evolutiva do grupo. Ao analisar os grupos formados na filogenia do NADH foi sugerido que o padrão de coloração dessas serpentes é uma característica ligada a história filogenética do grupo, e não tanto a fatores ambientais. Da mesma forma, os grupos formados nas árvores das toxinas nos fizeram sugerir que a composição do veneno dessas serpentes também segue a história evolutiva do grupo, em vez de fatores ambientais. Com esses resultados, nós sugerimos que os genes responsáveis pela expressão da coloração das cobras-corais estão intimamente ligados aos genes codificadores das toxinas que compõem seu veneno e, assim, essas duas características podem realmente estar em coevolução, e que a coloração tem, de fato, uma função aposemática nessas serpentes.

The venom characterization of Lansberg’s hognose viper (Porthidium lansbergii hutmanni) was described using a combination of SDS-PAGE are 1 and 2d electrophoresis, MALDI TOF/TOF and tandem mass spectrometry (LC-MS / MS), as well as some proteolytic activities. This study shown the existence of metallo and serine proteases, cysteine-rich proteins (CRISPs), and L-amino acid oxidases (LAAOs), which appeared in variable amounts. It was also described, for the first time in this venom, the presence of the glutaminil-cyclase (vQC) that had been only before observed in species of Crotalus atrox, C. godmani, C. d. terrificus and M. fulvius of the American continent, but not in Porthidium genus. The presence of toxins with a gelatinolytic and caseinolytic activity was also defined. The crude venoms are excellent sources of enzymatic and non-enzymatic actions, which should be purified and characterized with the aim of their possible use as therapeutic agents, in antitumor and haemostatic treatments. se realizó utilizando una combinación de electroforesis (SDSPAGE) de una y dos dimensiones, por MALDI TOF / TOF y espectrometría de masas en tándem (LC-MS/MS), así como algunas actividades proteolíticas usando caseína y gelatina. Este estudio reveló la existencia de metalo y serino proteasas, proteínas ricas en cisteína (CRISP) y L-aminoacido oxidasas (LAAOs), que aparecieron en cantidades variables. También se describió, por primera vez en este veneno, la presencia de la glutaminilciclasa (vQC) que solo se había observado anteriormente en especies de Crotalus atrox, C. godmani, C. durissus terrificus y M. fulvius del continente americano, pero no en el género Porthidium. También se definió la presencia de toxinas con actividades gelatinolítica y caseinolítica. Los venenos crudos son una excelente fuente de acciones enzimáticas y no enzimáticas, que deben ser purificadas y caracterizadas con el objetivo de su posible uso como agentes terapéuticos, en tratamientos antitumorales y hemostáticos. 207-220 mariupinex@gmail.com rodriguezacosta1946@yahoo.es

Se ha purificado una enzima coagulante del veneno de la serpiente Bothrops brazili denominada enzima similar a trombina (TLE) mediante tres pasos cromatográficos sucesivos sobre Sephadex G-75, DEAE Sephadex A-50 y Sephadex G-50, empleando buffer Tris-HCl 0,05 M pH 8,5. La enzima fue purificada 15,9 veces con un rendimiento del 28,6 % y por PAGE-SDS se obtuvo una sola banda proteica de 48 kDa, tanto en condiciones reductoras como no reductoras usando 2β-Mercaptoetanol. Se trata de una proteína con actividad coagulante, tanto sobre plasma humano citratado como sobre fibrinógeno bovino. La enzima mostró actividad amidolítica sobre el sustrato cromogénico Benzoil-Arginil-p-Nitroanilina (BApNA) y la potencia coagulante sobre el fibrinógeno bovino fue calculada en 121 unidades NIH de trombina/mg. La enzima fue inhibida por PMSF y por el inhibidor de tripsina de soya, por lo que se trata de una serinoproteasa; el pH óptimo para la actividad amidolítica fue de 8,5 y la proteína fue estable al tratamiento térmico solo hasta los 40 ºC. La dosis defibrinogenante mínima fue 8 μg/g de ratón y mediante pruebas de inmunodifusión doble se observó inmunorreactividad con respecto al suero antibotrópico polivalente del INS A coagulant enzyme from Bothrops brazili snake venom called thrombin-like enzyme was purified by three successive chromatographic steps on Sephadex G-75, DEAE Sephadex A-50 and Sephadex G-50 using 0.05M Tris-HCl buffer pH 8.5. The enzyme was purified 15.9 times with a yield of 28.6% and by PAGE-SDS a single protein band of 48 kDa was obtained both in reducing and non-reducing conditions using 2β-Mercaptoethano., It is a unicatenary protein with coagulant activity on both citrated human plasma and bovine fibrinogen. The enzyme showed amidolytic activity on the chromogenic substrate Benzoyl-Arginyl-p- Nitroaniline (BApNA) and the coagulant potency on bovine fibrinogen was calculated on 121 NIH units of thrombin / mg. The enzyme was inhibited by PMSF and the soybean trypsin inhibitor, therefore, it is a serine protease; the optimum pH for the amidolytic activity was 8.5 and the protein was stable to heat treatment only up to 40 °C. The minimal defibrinogenating dose was 8 μg / g of mouse and by double immunodiffusion tests immunoreactivity was observed with respect to INS polyvalent antibothropic serum