Mechanism of entry and development of Trypanosoma dionisii in non-phagocytic cells.

Cultures of 'buffalo' (bison) lung (BL) cells were infected with epimastigotes or trypomastigotes of Trypanosoma (Schizotrypanum) dionisii derived from cultures in vitro, fixed after various periods of incubation at 37 degrees C and examined by light or electron microscopy. Few if any epimastigotes entered the BL cells, but many trypomastigotes did so; they adhered to the cell surface within 2 h and then appeared to sink into furrows on the cell surface until engulfed in parasitophorous vacuoles. Cytochalasin D (5-10 micrograms ml-1) completely, but reversibly, inhibited entry of trypomastigotes without affecting parasite motility. It was concluded that entry depended on the interaction of stage-specific components on the trypomastigote's surface with receptors on the BL cells, and that this interaction induced active uptake of the protozoa by a phagocytic process not involving pseudopod formation. Soon after entry of the trypomastigotes into BL cells, the membranes of the parasitophorous vacuoles disintegrated and the parasites, which were now lying free in the cytoplasm of the host cell, transformed into amastigotes (micromastigotes) during the next 24-48 h. Replication then occurred, followed by transformation, beginning after 3 days, through a transitional promastigote phase to small intracellular trypomastigotes at 7 days. The promastigotes had a characteristic curved protrusion extending from the lip of the flagellar pocket (or reservoir) into the host cell's cytoplasm. Trypomastigotes, released into the supernatant medium by rupture of the plasma membranes of the BL cells after 8 days, could re-invade other cells.

Trypanosoma (Schizotrypanum) dionisii as a model for chemotherapeutic studies related to Chagas's disease.

Cultivated trypomastigotes of Trypanosoma (Schizotrypanum) dionisii were added to cultures of buffalo lung (BL) cells at 37 degrees C in a ratio of 500 parasites per cell. More than 60% of the cells became infected with amastigotes and the system was used to test trypanosomicidal activity and cytotoxicity of possible chemotherapeutic agents for use in Chagas's disease. Three nitroheterocyclic compounds known to be active against T. cruzi in cell cultures (nifurtimox, benznidazole and SQ 18506) were active at similar levels against intracellular T. dionisii (1, 10 and 0.1 microgram ml-1 respectively); concentrations of 10, 10 and 1 microgram ml-1 respectively were cytotoxic. Other substances, not regarded as active against T. cruzi (metronidazole, pentamidine and pentostam, had little or no trypanosomicidal effect even at 100 microgram ml-1. A recently developed nitroheterocyclic compound (MK 436) was active, and not cytotoxic, at 10 microgram ml-1 and possibly warrants further investigation Tetraethylthiuram disulphide, an inhibitor of threonine dehydrogenase which prevents growth of extracellular T. cruzi and T. dionisii in vitro at 1 microgram ml-1, was only slightly trypanosomicidal to intracellular T. dionisii at this dose; it was also somewhat cytotoxic.

Trypanosoma (Schizotrypanum) dionisii: influence of mouse peritoneal macrophages and calf sera on extracellular growth in vitro at 37 degrees C.

Macrophages and certain batches of sera were essential for extracellular multiplication of Trypanosoma dionisii in vitro in medium 199 with 20% (v/v) calf serum at 37 degrees C. In mixtures of 'good' and 'bad' batches of sera, multiplication increased as the proportion of the former was increased. Mixtures of 'bad' and 'intermediate' sera permitted virtually no growth. Phagocytosis of parasites by macrophages was unaffected by different batches of sera, though reduced in medium 199 alone. Replacement of supernatant medium by medium containing 'bad' serum reduced the macrophage infection rate no more than did transfer to medium containing 'good' serum. Daily addition of medium conditioned by prior contact with macrophages in vitro to cultures of trypanosomes without macrophages resulted in growth at least as good as that in the presence of macrophages. Extracellular replication in medium 199 at 37 degrees C apparently required at least two factors: 'M factor' provided by macrophages, but not required at 28 degrees C; and 'S+ factor' present in some batches of calf serum, essential also at 28 degrees C but not required by intracellular parasites. Some sera appeared to contain an inhibitory 'S- factor'.