A novel activity on thymocytes cells exerted by the rattlesnake (Crotalus durissus cumanensis) venom. / Nueva actividad de los timocitos estimulada por el veneno de la serpiente de cascabel (Crotalus durissus cumanensis)

INTRODUCTION: The thymus is active mainly during the neonatal and pre-adolescent periods. OBJECTIVE: To test naïve thymocytes proliferation and monocytes stimulation. MATERIALS AND METHODS: We collected fresh thymus tissue from neonate mice after surgery. Suspension cells were coated onto Ficoll-Hypaque support. The obtained cells (thymocytes) were cultured measuring the proliferation of naïve T cells stimulated by Crotalus durissus cumanensis (Cdc) venom at sub-lethal doses (20 ng). Then, we supplemented the wells with AlamarBlue™ and incubated them for 5 h to test their proliferation. Mononuclear cells from mice peripheral blood were collected and layered onto the support of the Ficoll-Hypaque solution. We added the thymocytes actively dividing (25 x 105 cells) from cultures stimulated with Cdc venom at 20 ng/well to cultured monocytes freshly obtained from the Ficoll-Hypaque separation. Both cell populations were incubated for 36 h until monocytes matured to macrophages. RESULTS: The naïve thymocytes rapidly proliferated after stimulation with the Cdc venom (NTCdc) and these successively induced the maturation and function of monocytes progenitor cells to mature macrophages, which ingested Chinese ink. CONCLUSIONS: The naïve thymocytes proliferated by stimulation with the Cdc venom and subsequently the NT/Cdc induced the rapid maturation and function of monocytes progenitor cells becoming mature macrophages with their phenotypic characteristics.

Introducción. El timo es activo principalmente durante los períodos neonatal y preadolescente. Objetivo. Probar la proliferación de los timocitos tempranos y la estimulación de monocitos que producen. Materiales y métodos. Se recogió tejido de timo fresco después de la cirugía de ratones recién nacidos. La suspensión de células se colocó sobre un soporte de Ficoll-Hypaque. Las células obtenidas (timocitos) se cultivaron y se midió la proliferación de células T vírgenes estimuladas por el veneno de Crotalus durissus cumanensis (Cdc) en dosis subletales (20 ng). A continuación, se agregó AlamarBlue™ a los pocillos y se incubaron durante 5 horas para evaluar la proliferación. Se recogieron células mononucleares de sangre periférica de ratones y se colocaron sobre un soporte de solución de Ficoll-Hypaque. Los timocitos que se dividieron activamente (25 x 105 células) a partir de los cultivos estimulados con veneno de Cdc (20 ng/pocillo) y se agregaron a los cultivos de monocitos recién obtenidos de la separación en la solución de Ficoll-Hypaque. Ambas poblaciones celulares se incubaron durante 36 horas hasta que los monocitos maduraron a macrófagos. Resultados. Los timocitos tempranos experimentaron una rápida proliferación estimulada por el veneno de Cdc (NTCdc) y, posteriormente, indujeron la maduración y la función de las células progenitoras de monocitos, los cuales maduraron a macrófagos, que se tiñeron con tinta china. Conclusiones. Los timocitos tempranos proliferaron con la estimulación del veneno de Cdc y, posteriormente, el NT/Cdc indujo la maduración rápida y la función de las células progenitoras de monocitos, transformándose en macrófagos con sus características fenotípicas.

A novel activity on thymocytes cells exerted by the rattlesnake (Crotalus durissus cumanensis) venom / Nueva actividad de los timocitos estimulada por el veneno de la serpiente de cascabel (Crotalus durissus cumanensis)

Abstract. Introduction: The thymus is active mainly during the neonatal and pre-adolescent periods. Objective: To test naïve thymocytes proliferation and monocytes stimulation. Materials and methods: We collected fresh thymus tissue from neonate mice after surgery. Suspension cells were coated onto Ficoll-Hypaque support. The obtained cells (thymocytes) were cultured measuring the proliferation of naïve T cells stimulated by Crotalus durissus cumanensis (Cdc) venom at sub-lethal doses (20 ng). Then, we supplemented the wells with AlamarBlue™ and incubated them for 5 h to test their proliferation. Mononuclear cells from mice peripheral blood were collected and layered onto the support of the Ficoll-Hypaque solution. We added the thymocytes actively dividing (25 x 105 cells) from cultures stimulated with Cdc venom at 20 ng/well to cultured monocytes freshly obtained from the Ficoll-Hypaque separation. Both cell populations were incubated for 36 h until monocytes matured to macrophages. Results: The naïve thymocytes rapidly proliferated after stimulation with the Cdc venom (NTCdc) and these successively induced the maturation and function of monocytes progenitor cells to mature macrophages, which ingested Chinese ink. Conclusions: The naïve thymocytes proliferated by stimulation with the Cdc venom and subsequently the NT/Cdc induced the rapid maturation and function of monocytes progenitor cells becoming mature macrophages with their phenotypic characteristics.

Resumen. Introducción. El timo es activo principalmente durante los períodos neonatal y preadolescente. Objetivo. Probar la proliferación de los timocitos tempranos y la estimulación de monocitos que producen. Materiales y métodos. Se recogió tejido de timo fresco después de la cirugía de ratones recién nacidos. La suspensión de células se colocó sobre un soporte de Ficoll-Hypaque. Las células obtenidas (timocitos) se cultivaron y se midió la proliferación de células T vírgenes estimuladas por el veneno de Crotalus durissus cumanensis (Cdc) en dosis subletales (20 ng). A continuación, se agregó AlamarBlue™ a los pocillos y se incubaron durante 5 horas para evaluar la proliferación. Se recogieron células mononucleares de sangre periférica de ratones y se colocaron sobre un soporte de solución de Ficoll-Hypaque. Los timocitos que se dividieron activamente (25 x 105 células) a partir de los cultivos estimulados con veneno de Cdc (20 ng/pocillo) y se agregaron a los cultivos de monocitos recién obtenidos de la separación en la solución de Ficoll-Hypaque. Ambas poblaciones celulares se incubaron durante 36 horas hasta que los monocitos maduraron a macrófagos. Resultados Los timocitos tempranos experimentaron una rápida proliferación estimulada por el veneno de Cdc (NTCdc) y, posteriormente, indujeron la maduración y la función de las células progenitoras de monocitos, los cuales maduraron a macrófagos, que se tiñeron con tinta china. Conclusiones. Los timocitos tempranos proliferaron con la estimulación del veneno de Cdc y, posteriormente, el NT/Cdc indujo la maduración rápida y la función de las células progenitoras de monocitos, transformándose en macrófagos con sus características fenotípicas.

Immunotoxicological effects triggered by the rattlesnake Crotalus durissus cumanensis, mapanare (Bothrops colombiensis) venoms and its purified fractions on spleen and lymph nodes cells.

Purpose: The snakes in Venezuela vary in their different venom composition amid the species. In this sense, studies have been carried out elucidating mechanisms related to their immunostimulatory and/or immunosuppressive effects in vitro, measuring inhibition or stimulation on the mice spleen and lymph nodes lymphocytes under the rattlesnake (Crotalus durissus cumanensis) (Cdc) and mapanare (Bothrops colombiensis) crude venoms actions, and also its purified fraction crotoxin (CTX) (Cdc) and a semi-purified fraction (SPF) (Bc) activities. Material and methods: The stimulation of lymphocyte proliferation was carried out in the presence or absence of Concanavalin A (ConA) and lipopolysaccharides (LPS). Results: The lymphocyte response was measured by the Alamar Blue® (Resazurin) assay, observing that the Crotalus crude venom increased basal proliferation in the spleen and lymph nodes, being also increased with ConA and LPS. CTX slightly decreased the proliferative response in the presence of mitogens. Both Bc venom and its SPF fraction had no significant effect on basal proliferation in the spleen and lymph nodes, but a decrease in the response with ConA was observed. These results suggest that CTX has an inhibitory action on lymphocyte proliferation, while Cdc crude venom has a stimulatory action on T and B cell populations. Bothrops colombiensis venom had no effect on these two types of cell populations. As it is known, lymphocytes are cells of enormous flexibility and can operate in diverse aspects, warranting that the correct immune response persists controlled. Conclusions: These results suggested that these different toxins can modulate lymphocyte functional activation toward an inhibitory or stimulatory state.