Influence of organ extracts of Triatoma infestans on differentiation of Trypanosoma cruzi.

The influence that extracts of different T. infestans organs possess to induce Trypanosoma cruzi differentiation from epimastigotes to metacyclic forms in Grace's and modified Grace's media was analyzed. Growth of epimastigotes was adequate in Grace's medium when homogenates of stomach, testes, and ovaries from recently fed insects were added. Growth of epimastigotes was adequate when testes or ovaries obtained from recently fed triatomes were added to modified Grace's medium. Similar results were obtained in Grace's or modified Grace's media supplemented with stomach or intestinal homogenates obtained from "starved" triatomes. None of the above culture conditions induced differentiation of epimastigotes to the metacyclic stage. When extracts of intestine or stomach obtained from triatomes fed 24 to 48 hr previously were used to supplement the modified Grace's medium, growth and differentiation to the metacyclic stage was induced in a high percentage of the population. When extracts of intestine obtained from recently fed insects were used to supplement Grace's medium, differentiation and growth was an in modified Grace's medium. No important differences were observed if cultures were prepared at pH 6.4 or 7.0. The triatome food source did not seem to be important for the differentiation. The parasite population after differentiation to metacyclic type forms showed a higher degree of infectivity than the original population constituted mostly by epimastigotes. These studies suggest the importance of factors from the insect vector in differentiation of T. cruzi.

Isolation by Percoll gradient centrifugation and radiolabeling of bloodstream forms of Trypanosoma cruzi.

A method to isolate T. cruzi bloodstream forms was designed taking advantage of Percoll self-generated gradients and isopycnic centrifugation, resulting in a good resolution between parasites and host cells. Purified parasites incorporated 3H-uridine giving values of 23% precipitable by TCA. Viability of the parasites was totally preserved as evidenced by the "in vivo" infectivity assays.

Isolation by Percoll gradient centrifugation and radiolabeling of bloodstream forms of Trypanosoma cruzi

Se diseñó un método para purificar las formas sanguíneas de T. cruzi utilizando un gradiente de Percoll contínuo de 6 cm de longitud (Percoll 60% en "HSA-buffer", preformado por centrifugación a 20.000 g durante 90 min a 4-C en un rotor Sorvall SS 34). El mismo se sembró con 6 x 10**7 elementos (parásitos semipurificados de glóbulos rojos por centrifugación diferencial a partir de sangre desfibrinada) y se centrifugó a 800 g, 15 min. Los parásitos se ubicaron en 1,063 a 1,064 g/ml, existiendo una buena resolución entre ellos y los elementos figurados de la sangre. Los parásitos purificados incorporaron H-uridina y dieron valores del 23% precipitables por TCA. La viabilidad de los mismos fue totalmente preservada según lo evidenciado en los ensayos de infectividad "in vivo"

Fagocitosis de las formas epi y tripomastigote de Trypanosoma cruzi por leucocitos humanos perifericos. / Phagocytosis of the epi and trypomastigote forms of Trypanosoma cruzi by peripheral human leukocytes

Las formas de cultivo (epimastigotes) y sanguineas (tripomastigotes) de Trypanosoma cruzi son fagocitadas por leucocitos humanos perifericos normales (monocitos y neutrofilos) en presencia de antisuero de conejo anti-epimistigote y de suero de conejo o de pacientes cronicamente infectados con T. cruzi. Los neutrofilos mostraron niveles bajos de fagocitosis en ausencia de antisuero. El porcentaje de fagocitosis se determino morfologicamente en base al numero de celulas con parasitas intracelulares. Tanto en el caso de los monocitos como los neutrofilos, la fagocitosis de los epimastigotes es considerablemente mayor que la de los tripomastigotes.El pretratamiento de tripomastigotes con tripsina aumenta su fagocitosis por monocitos y neutrofilos tanto en presencia como en ausencia de antisuero. La presencia de epi o tripomastigotes sensibilizados incrementa el consumo de O2 de los leucocitos polimorfonucleares