Culture supernatants from V. cholerae O1 ElTor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity / Cepas de V. cholerae O1 biotipo ElTor aisladas de diferente origen geográfico inducen vacuolización celular y citotoxicidad

OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

OBJETIVO: Analizar el efecto vacuolizante de cepas de V. cholerae O1 ElTor aisladas de diferente origen geográfico, incluyendo México. MATERIAL Y MÉTODOS: Se realizaron pruebas de hemolisis, vacuolización y citotoxicidad en células Vero, así como PCR, análisis por RFLP y clonación molecular. RESULTADOS: Todas las cepas indujeron el efecto vacuolizante. Las cepas del ribotipo 2, aisladas de las costas del Golfo en Estados Unidos, presentaron títulos altos de vacuolización. El gen hlyA fue amplificado en las nueve cepas mediante PCR, aunque sólo ocho fueron hemolíticas. Se clonó el gen hlyA de una cepa toxigénica (2514-88, ctxAB+) y de una cepa no toxigénica aislada en México (CM 91-3, ctxAB-). El sobrenadante de las clonas recombinantes indujo hemólisis, efecto vacuolizante y citotoxicidad. El RFLP mostró alta similitud del gen hlyA de las cepas estudiadas. CONCLUSIÓN: El efecto vacuolizante es un fenotipo ampliamente distribuido en cepas epidémicas de V. cholerae O1 biotipo ElTor.

The use of and ELISPOT assay to evaluate intestinal and systemic antibody responses to locally administered Entamoeba histolytica antigen in mice

The local induction of humoral immune response to Entamoeba histolytica trophozoites in the gut has not been studied. This work reports some of our recent studied. This work reports some of our recent studies aimed to induce optimal immune responses against E. histolytica in mice and to describe a novel approach for monitoring mucosal immune responses induced in the gastrointestinal tract and expressed locally and systemically. We have compared the kinetics of both mucosal and systemic primary antibody responses to E. histolytica in the Peyer's patches (PP) and the spleen in Balb/c mice after a single dose of glutaraldehyde fixed amebas (GFA) by intragastric (IG), rectal (R), and intraperitoneal (IP) routes. The number of antibody-secreting cells directed to E. histolytica was assessed by the technique of ELISPOT on mitrocellulose filters. The antibody response to E. histolytica was detected in both PP and spleen with the three routes, indicating that either mucosal or systemic stimulation by GFA generates both types of response. We also determined the total antibody response in entestinal fluids and the antibody secretions from spleen and PP cells in vitro and found differences between male and female mice