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.

An in vitro system of autologous lymphocytes culture that allows the study of homeostatic proliferation mechanisms in human naive CD4 T-cells.

The size of peripheral T-cell pool is kept constant throughout life. However, a decline in lymphocyte numbers is a feature of several human disorders, in which fast and slow homeostatic proliferation play a crucial role. Several in vitro and in vivo models have been developed to study such processes. Nevertheless, self- and commensal- antigens, well-known triggers of homeostatic proliferation, have not been examined in these models. We have designed an in vitro culture of human T-cells exposed to rIL7 and autologous antigen-presenting cells (aAPC) that allows the simultaneous characterization of the different types of homeostatic proliferation. Using our model, we first confirmed that both rIL7 and aAPC are survival signals ultimately leading to homeostatic proliferation. In addition, we explored the modulation of different anti-apoptotic, proliferative, activation and homing markers during fast and slow homeostatic proliferation. Finally, different subsets of Treg were generated during homeostatic proliferation in our model. In summary, our in vitro system is able to simultaneously reproduce both types of homeostatic proliferation of human naive CD4 T-cells, and allows the characterization of these processes. Our in vitro system is a useful tool to explore specific features of human homeostatic proliferation in different human lymphopenia-related disorders and could be used as a cell therapy approach.

Effects of recreational activities on Patagonian rocky shores.

Recreational activities can be an important source of anthropogenic disturbance in intertidal benthic assemblages. On rocky shores, activities such as trampling, snorkeling and the handling of organisms may have a negative effect on benthic communities by modifying the abundance and distribution of key species. Here, we describe and quantify impacts due to recreational activities on benthic communities on a Patagonian rocky shore by investigating their resilience to two types of human disturbance: vehicle traffic and human trampling. To evaluate the effects of these activities, we carried out an observational study and assessed post-disturbance assemblage recovery. The rocky shores is most intensively visited during summer, and marked differences in the distribution and abundance of benthic species among disturbed and control plots were found after this season. The benthic community on the high intertidal was weakly impacted by disturbance generated due to vehicle traffic in summer (one vehicle on a single occasion, pulse disturbance); which did not affect the cover of dominant species. This suggests that the high intertidal community would be resistant to the passage of one vehicle on a single occasion. The effects of continuous trampling (press disturbance) were drastic and the community of the mid intertidal level did not recover before the next recreational season. Mid intertidal communities exposed to press disturbances require more than one tourist season of human inactivity to recover from anthropogenic effects, suggesting that resilience mechanisms in this community operate at broad timescales. Our findings highlight the need to establish and implement management actions that contemplate the nature of the disturbance and intertidal level to minimize habitat degradation due to human recreational activities.

Effect of recreational diving on Patagonian rocky reefs.

Tourism has grown considerably in the last decades, promoting activities such as recreational SCUBA diving that may affect marine benthic communities. In Puerto Madryn, Patagonia Argentina, sub-aquatic tourism areas (STA) receive about 7,000 divers per year. Diving is concentrated on a few small rocky reefs and 50% of the dives occur in summer. In this work, we evaluated the effect of recreational diving activities on benthic communities and determined whether diving causes a press (long-term) or a pulse (short-term) response. We quantified the percentage cover of benthic organisms and compared benthic assemblage structure and composition between two sites with contrasting usage by divers, 'highly disturbed' and 'moderately disturbed' sites, and two 'control' sites with similar physical characteristics but no diving activity, twice before and after the diving peak in summer. We found differences in benthic assemblage structure (identity and relative abundance of taxa) and composition (identity only) among diving sites and controls. These differences were consistent before and after the peak of diving in summer, suggesting that recreational diving may produce a press impact on overall benthic assemblage structure and composition in these STA. At the moderately disturbed site, however, covers of specific taxa, such as some key habitat-forming or highly abundant species, usually differed from those in controls only immediately after summer, after which they begun to resemble controls, suggesting a pulse impact. Thus, STA in Golfo Nuevo seem to respond differently to disturbances of diving depending on the usage of the sites. This information is necessary to develop sound management strategies in order to preserve local biodiversity.

Feasibility of Cervical Cancer Screening Utilizing Self-sample Human Papillomavirus Testing Among Mexican Immigrant Women in Harris County, Texas: A Pilot Study.

Here we evaluate the acceptability and feasibility of self-sample human papillomavirus (HPV) testing (self-HPV) among a sample of predominantly Mexican immigrant women who never or sporadically attend for Pap testing. Immigrant women who had not had a Pap test in the past 3 years (n = 100) were recruited. Participants self-collected a cervical sample for HPV testing. High-risk (HR) HPV-positive women were referred for clinical follow-up. Acceptability of self-HPV was high, with 99% reporting that they would be willing to use self-HPV regularly. Nineteen women (19%) tested positive for HR-HPV. Of these, 50% obtained clinical follow-up within 3 months. For those who did not, the primary barrier to follow-up was difficulty obtaining healthcare coverage. In conclusion, self-HPV is highly acceptable to Mexican immigrant women who otherwise do not attend for Pap testing. While the approach addresses critical barriers to primary screening, questions remain as to whether uninsured HR-HPV positive women are able to attend for clinical follow-up.