Abstract In this paper we discuss recent significant developments in the field of venom research, specifically the emergence of top-down proteomic applications that allow achieving compositional resolution at the level of the protein species present in the venom, and the absolute quantification of the venom proteins (the term “protein species” is used here to refer to all the different molecular forms in which a protein can be found. Please consult the special issue of Jornal of Proteomics “Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts” published in 2016, vol. 134, pages 1-202). Challenges remain to be solved in order to achieve a compact and automated platform with which to routinely carry out comprehensive quantitative analysis of all toxins present in a venom. This short essay reflects the authors’ view of the immediate future in this direction for the proteomic analysis of venoms, particularly of snakes.

Abstract This work offers a general overview on the evolving strategies for the proteomic analysis of snake venoms, and discusses how these may be combined through diverse experimental approaches with the goal of achieving a more comprehensive knowledge on the compositional, toxic, and immunological characteristics of venoms. Some recent developments in this field are summarized, highlighting how strategies have evolved from the mere cataloguing of venom components (proteomics/venomics), to a broader exploration of their immunological (antivenomics) and functional (toxicovenomics) characteristics. Altogether, the combination of these complementary strategies is helping to build a wider, more integrative view of the life-threatening protein cocktails produced by venomous snakes, responsible for thousands of deaths every year.

Abstract Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient’s lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.

A doença de Castleman (DC) é uma desordem linfoproliferativa policlonal, também conhecida como hiperplasia nodular gigante ou hiperplasia angiofolicular linfoide. Esta é uma doença rara que está frequentemente associada ao vírus da imunodeficiência humana (HIV) e ao herpes vírus 8 (HHV-8). Os achados histopatológicos encontrados na DC sugerem uma intensa resposta aos estímulos antigênicos observada em várias doenças associadas com ativação imune, como a artrite reumatoide. Um fator importante implicado na patogênese da DC é a produção autônoma da interleucina-6 (IL-6). Nessa doença, as manifestações clínicas estão relacionadas aos níveis de IL-6, e a remoção cirúrgica dos linfonodos acometidos ou a utilização de anticorpos anti-IL-6 fazem regredir os sintomas. Descrevemos um caso da DC multicêntrica em uma mulher jovem, não associada à infecção pelo vírus HHV-8 ou à imunossupressão. Uma breve revisão da literatura se segue à descrição do caso clínico.

Castleman's disease (CD) is a polyclonal lymphoproliferative disorder also known as giant nodular hyperplasia or angiofollicular lymph node hyperplasia. It is a rare disease often associated to human immunodeficiency virus (HIV) and human herpes virus 8 (HHV-8). Histopathological findings in Castleman's disease suggest an exaggerated response to antigenic stimuli seen in other diseases associated with immune activation, such as rheumatoid arthritis. An important aspect of its pathogenesis is the autonomous production of interleukin-6 (IL-6). In this disease, the clinical manifestations are associated to IL-6 serum levels, and surgical removal of the compromised lymph nodes or use of anti-IL-6 antibodies can slow down the symptoms. We describe a multicentric Castleman's disease in a young woman not associated to HHV-8 virus infection or immunosuppression. A short review of the literature follows the description of this clinical case.

El presente trabajo ofrece una breve visión de cómo se ha enfrentado el problema del ofidismo en Costa Rica, y en especial, de cómo la investigación científica y tecnológica ha contribuido a tal fin. Se resumen los orígenes de la lucha antiofídica en el país y de la creación del Instituto Clodomiro Picado en la Universidad de Costa Rica, en 1970. Se describen las primeras etapas de su labor, y la evolución de distintas líneas de investigación sobre serpientes, venenos y antivenenos, que por espacio de cuatro décadas han contribuido a que en el país y en la región centroamericana, existan recursos terapéuticos adecuados y suficientes para enfrentar el ofidismo.

The present work offers a brief overview on how the problem of ophidism has been confronted in Costa Rica, and especially, on how scientific and technological research has contributed to this goal. The origins of the antiophidic struggle in the country and the creation of the Instituto Clodomiro Picado, at the University of Costa Rica in 1970, are briefly summarized. The first stages of its work are described, as well as the evolution of different research areas on snakes, venoms and antivenoms; which have contributed during four decades to the existence of adequate and sufficient therapeutic resources to tackle ophidism in Costa Rica and in the Central American region.

OBJECTIVE: To analyze mortality due to Hymenoptera stings in Costa Rica during 1985-2006. METHODS: Records of deaths due to Hymenoptera stings in 1985-2006 were retrieved from Instituto Nacional de Estadística y Censos (National Statistics and Census Institute). Mortality rates were calculated on the basis of national population reports, as of 1 July of each year. Information for each case included age, gender, and the province in which the death occurred. In addition, reports of Hymenoptera sting accidents received by the Centro Nacional de Intoxicaciones (National Poison Center, CNI) in 1995-2006 were obtained to assess exposure to these insects. RESULTS: Over the 22-year period analyzed, 52 fatalities due to Hymenoptera stings were recorded. Annual mortality rates varied from 0-1.73 per 1 million inhabitants, with a mean of 0.74 (95% confidence interval: 0.46-0.93). The majority of deaths occurred in males (88.5%), representing a male to female ratio of 7.7:1. A predominance of fatalities was observed in the elderly (50 years of age and older), as well as in children less than 10 years of age. The province with the highest mortality rate was Guanacaste. The CNI documented 1 591 reports of Hymenoptera stings (mostly by bees) in 1995-2006, resulting in an annual average of 133 cases, with only a slight predominance of males over females (1.4:1). CONCLUSIONS: Stings by Hymenoptera, mostly by bees, constitute a frequent occurrence in Costa Rica that can be life-threatening in a small proportion of cases, most often in males and the elderly. The annual number of fatalities fluctuated from 0-6, averaging 2.4 deaths per year. Awareness should be raised not only among the general population, but also among health care personnel that should consider this risk in the clinical management of patients stung by Hymenoptera.

OBJETIVO: Analizar la mortalidad por picaduras de himenópteros en Costa Rica en el período de 1985 a 2006. MÉTODOS: Se obtuvieron los registros de picaduras de himenópteros entre 1985 y 2006 del Instituto Nacional de Estadísticas y Censos. Las tasas de mortalidad se calcularon a partir de los datos de la población nacional para el 1 de julio de cada año. La información de cada caso abarcaba la edad, el sexo y la provincia en la que ocurrió la muerte. Además, para evaluar la exposición a estos insectos se contó con las notificaciones de accidentes por picaduras de himenópteros del Centro Nacional de Intoxicaciones (CNI) de 1995 a 2006. RESULTADOS: En el período de 22 años analizado se registraron 52 muertes por picaduras de himenópteros. La tasa anual de mortalidad varió entre 0 y 1,73 por millón de habitantes, con una media de 0,74 (intervalo de confianza de 95%: 0,46 a 0,93). La mayoría de las muertes correspondieron a hombres (88,5%), con una razón hombre:mujer de 7,7:1. Predominaron los casos letales en adultos mayores (50 años o más) y niños menores de 10 años. La provincia con la mayor mortalidad fue Guanacaste. El CNI documentó 1 591 casos de picaduras de himenópteros (principalmente abejas) entre 1995 y 2006, para un promedio anual de 133 casos, con un ligero predominio de hombres sobre mujeres (1,4:1). CONCLUSIONES: Las picaduras de himenópteros, principalmente de abejas, son sucesos frecuentes en Costa Rica que pueden amenazar la vida en una reducida proporción de casos, principalmente hombres y adultos mayores. El número anual de muertes varió entre 0 y 6, con un promedio de 2,4 muertes anuales. Se debe fomentar la conciencia no solo en la población general sino también en el personal especializado de salud, quienes deben tomar en cuenta este riesgo al atender a los pacientes con picaduras de himenópteros.

Se evaluaron los efectos tóxicos de los venenos de cinco serpientes costarricenses en cuanto a su capacidad tripanocida contra dos cepas de Trypanosoma cruzi y sus efectos en cuanto a los mecanismos de muerte celular. Los venenos de Bothrops asper, Bothriechis schlegelii, Crotalus durissus durissus, Atropoides nummifer y A. picadoi, mostraron actividad tripanocida contra las formas de epimastigoto, amastigoto y tripomastigoto. Los venenos de B. asper y de A. nummifer presentaron la más alta citotoxicidad para las células Vero. Los de B. asper y B. schlegelii presentaron la más alta actividad en los epimastigotos de la cepa CL, mientras que los venenos de B. asper y el de A. nummifer fueron más eficientes contra los epimastigotos de la cepa Jennifer. El veneno de B. schlegelii produce un efecto proliferativo en las células Vero; mientras que el de C. d. durissus produce el mismo efecto en los epimastigotos de la cepa CL, ambos a la concentración de 2,5 μg/mL. Los valores de CI50 mostraron que se requieren menores cantidades contra los amastigotos en relación con los epimastigotos. Los venenos de B. asper y B. schlegelii presentan la más alta actividad contra los amastigotos de ambas cepas. Con los tripomastigotos sanguíneos de la cepa GA, los cinco venenos ocasionaron una disminución de la motilidad en los diferentes tiempos de exposición, pero el veneno de A. nummifer, en las concentraciones más bajas, mostró una actividad más marcada en comparación con los otros veneno. En cuanto a los efectos de los venenos, mediados por los grados de apoptosis, necrosis o proliferación celular, se observó que estos fenómenos se presentan y tienen relación con el tipo de veneno, su concentración y el tiempo de exposición.

The trypanocide effect of venoms from five Costa Rican species of snakes was evaluated against two strains of Trypanosoma cruzi and their cellular toxic effects were likewise observed. The venoms of Bothrops asper, Bothriechis schlegelii, Crotalus durissus durissus, Atropoide nummifer and A. picadoi showed evident trypanocide action against epimastigotes, amastigotes and trypomastigotes. The venoms of B. asper and B. schlegelii were shown to be the most active against the epimastigotes of the CL strain, whereas those of B. asper and A. nummifer were more effective against the epimastigotes of the Jennifer strain. The venoms of B. schlegelii and C.d. durissus, at the lowest concentrations of 2.5 μg/mL, were able to trigger a proliferative effect on Vero cells and epimastigotes of the CL strain, respectively. The IC50 values showed that lower amounts of venoms are necessary in order to inhibit amastigotes as compared to epimastigotes. The venoms of B. asper and B. schlegelii exhibited the highest activity against amastigotes of both T. cruzi strains. All venoms were able to arrest motility of blood trypomastigotes of the GA strain at different times and the most active in this case was A. nummifer venom. The toxic effects of the venoms measured by the degree of apoptosis, necrosis and cell proliferation that they produced showed that all these events occur and are related to the type of venom, its concentration and exposure time.

El veneno de la serpiente Bothrops asper induce graves alteraciones de la coagulación en los humanos accidentalmente envenenados. Sin embargo, se han realizado pocos estudios para identificar los componentes del veneno que interactúan con el sistema hemostático. En el presente trabajo, fraccionamos el veneno de B. asper para investigar la posible presencia de inhibidores de la agregación plaquetaria. Empleando una combinación de técnicas cromatográficas (filtración en gel, intercambio aniónico y cromatografía líquida de alto desempeño en fase reversa), aislamos una proteína acídica de cadena simple, con una masa molecular de ~8 kDa, estimada mediante electroforesis en gel de poliacrilamida con SDS. Su secuencia de aminoácidos N-terminal muestra una alta similitud con la de disintegrinas aisladas de diferentes venenos de serpiente, las cuales se unen a integrinas celulares como el receptor de fibrinógeno GPIIb/IIIa de las plaquetas. La proteína purificada ejerce una potente acción inhibitoria sobre la agregación in vitro de plaquetas humanas estimuladas con ADP, con una IC 50 estimada en 50 nM. Esta actividad biológica, sumada a las características bioquímicas observadas, demuestran que la proteína aislada del veneno de B. asper es una disintegrina, a la cual denominamos "both-rasperina". Esta es la primera disintegrina aislada de una especie de vipéridos de Centroamérica.

The venom of Bothrops asper induces severe coagulation disturbances in accidentally envenomed humans. However, only few studies have been conducted to identify components that interact with the hemostatic system in this venom. In the present work, we fractionated B. asper venom in order to investigate the possible presence of inhibitors of platelet aggregation. Using a combination of gel filtration, anion-exchange chromatography, and reverse-phase high performance liquid chromatography, we isolated an acidic protein which shows a single chain composition, with a molecular mass of ~8 kDa, estimated by SDS-polyacrylamide gel electrophoresis. Its N-terminal sequence has high similarity to disintegrins isolated from different snake venoms, which are known to bind to cellular integrins such as the GPIIb/IIIa fibrinogen receptor on platelets. The purified protein exerted potent aggregation inhibitory activity on ADP-stimulated human platelets in vitro, with an estimated IC50 of 50 nM. This biological activity, together with the biochemical characteristics observed, demonstrate that the protein isolated from B. asper venom is a disintegrin, hereby named "bothrasperin". This is the first disintegrin isolated from Central American viperid snake species.

El presente estudio analiza las publicaciones realizadas por autores de instituciones de Costa Rica e incluídas en el Science Citation lndex (SCI), durante el trienio 1999-2001. Se encontró 722 referencias, distribuidas en 328 revistas, de las cuales el 90.7% correspondían a artículos originales de investigación. La productividad total de publicaciones en el SCI durante 1999-2001 continúa con la tendencia ligeramente creciente observada para 1980-1998, tanto en cifras absolutas, como en su ajuste a la población. La contribución de la Revista de Biología Tropical a estas cifras correspondió al 10.0%, 8.9% y 19.1%, respectivamente, en los años 1999, 2000 y 2001. La distribución temática de los artículos no mostró cambios marcados en relación con el patrón descrito para 1980-1998, con un aporte mayoritario de las ciencias biomédicas (33.3%) y biológicas (27.5%), seguido por las ciencias agronómicas (15.5%), química (13.6%), física (5.0%), geología (3.6%) matemáticas (1.5%). El análisis de las citaciones de publicaciones individuales (hasta el 15 de julio del 2002), mostró que solo el 45.2% de los artículos habían recibido el menos una citación. Se recopilaron los diez artículos más citados de este grupo de referencias, las cuales variaron entre 26 y 114 citas. El promedio de citaciones por artículo en el período estudiado fue de 2.60 y el número promedio de autores por artículo fue de 2.92. Al igual que durante 1980-1998, la Universidad de Costa Rica se mantuvo como la institución de mayor aporte de publicaciones en el SCI durante 1999-2001, con un promedio del 50.0% de la producción del país. El porcentaje de publicaciones realizadas sin coautoría de instituciones foráneas mostró una nivelación de la tendencia decreciente descrita para 1980-1998, estabilizándose durante el trienio 1999-2001 alrededor de un 25-30%.

This study analyzed the publications of authors from Costa Rican institutions that were included in the Science Citation Index (SCI) during 1999-2001. Out of the 722 references detected, distributed in 328 journals, 90.7% corresponded to original research articles. The total productivity figures showed a moderately increasing trend, both in absolute and population-adjusted terms, in concordance with previous data recorded for the period 1980-1998. The contribution of Revista de Biología Tropical to these figures corresponded to 10.0%, 8.9%, and 19.1%, for the years 1999, 2000, and 2001, respectively. The subject distribution of articles followed a similar pattern to that describes for 1980-1998, with a predominante of biomedical (33.3%) and biological (27.5%) sciences, followed by agronomical (15.5%) sciences, chemistry (13.6%), physics (5.0%), geological sciences (3.6%), and mathematics (1.5%). Citation analyses for individual publications (up to July 15, 2002) revealed that only 45.2% of the articles had been cited at least once. The ten most cited references were analyzed, varying from 26 to 114 citations. The average citation per article was 2.60, and the average number of authors per article was 2.92. In agreement with data from 1980-1998, the University of Costa Rica appeared as the institution with highest productivity of SCI publications during 1999-2001, with a contribution of 50.0%. The percentage of publications performed without the participation of foreign co-authors showed a change in its decreasing trend of 1980-1998, stabilizing near the range of 25-30% during the period 1999-2001.