A quantitative method for experimentally separating the adhesion and interiorization phases of the interaction of Trypanosoma cruzi with mammalian cells was developed. Incubation of confluent monolayers of mammalian cells with epimastigotes or trypomastigotes at 4 degrees C allowed the evaluation of the number of adhered parasites that do not become interiorized at this temperature. Quantification of interiorized parasites at 34 degrees C was achieved by employing hypotonic lysis to eliminate the extracellularly adhered trypomastigotes. Both adhesion and interiorization were found to be proportional to the time of exposure of cells to parasites and to the multiplicity of infection. These phenomena occur normally for trypomastigotes in the absence of serum with LLC-MK2 cells, HeLa cells, and 3T3 fibroblasts. Moreover, it was possible to obtain trypomastigotes that presented the same infectivity to LLC-MK2 cells as did parasites obtained in the presence of 2% fetal calf serum after 10 serial passages in a medium devoid of serum. Inhibition of adhesion (of epimastigotes and trypomastigotes) and of interiorization (of trypomastigotes) was obtained with inactivated normal serum from several sources, a saturation effect being observed at a final concentration of 20%. Bovine serum albumin, at the concentrations present in the sera, had no inhibitory effect. Trypomastigotes that have been pre-incubated with 40% FCS (45 min at 4 degrees C) showed decreased adhesion and interiorization indices, effects that can be reversed by trypsinization of the parasites prior to exposure of the cells. A progressive internalization of previously attached trypomastigotes was observed on raising the temperature from 4 degrees C to 34 degrees C; no spontaneous detachment of parasites was detected up to 120 min. Approximately 75% of the adhered parasites were found inside the cells after 45 min at 34 degrees C. The presence of normal inactivated calf serum during incubation at 34 degrees C resulted in a certain degree of detachment and in a lower interiorization index.
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