S. Castanys

Purification of metacyclic forms ofTrypanosoma cruzi by Percoll discontinuous gradient centrifugation

A method for the purification of metacyclic forms ofTrypanosoma cruzi has been developed. Metacyclic forms obtained in modified Grace medium were separated from the epimastigote forms by Percoll discontinuous density gradient centrifugation. Four different osmotic pressures were applied: 160±10, 260±10, 310±10 and 510±10 mosmol/kg H2O. At 160 mosmol/kg H2O, 100% of the metacyclic forms with a 21.3% yield were found in the interphase 1.120/1.125 g/ml, while 92.7% of the metacyclic forms with a 73.7% yield were found in the interphase 1.115/1.120 g/ml. At 310 mosmol/kg H2O, 100% of the metacyclic forms in the interphase 1.135/1.140 g/ml with a 36.8% yield were obtained. Metacyclic forms purified in this way do not show alterations in their capacity to infect cultures of HeLa cells.

Some factors affecting the in vitro invasion of HeLa cells by Trypanosoma cruzi

Abstract The penetration of metacyclic forms of Trypanosoma cruzi into HeLa cells after different treatments was studied. When cell development was synchronized by two different processes, maximum rates of parasitization occurred during the S phase of cell cycle (29.48 and 24.3%). However, when cells were treated with trypsin (0.1%), parasitization rates appeared to be lower than controls, reaching values similar to controls 14 h after the beginning of the treatment. Infection values remained unaltered after treatment with colcemid (0.6 μg ml−1). Cell treatment either with valinomycin (1 μg ml−1) or with actinomycin D (250 μg ml−1) caused a marked decrease in the percentage of parasitization. When cells were treated and infected in the presence of tunicamycin (100 ng ml−1), parasitization rates were increased (14.7%) compared to control cells (6%). On the other hand, no differences in parasitization rates were observed when cells were treated with cycloheximide (100 μg ml−1). Infection in a low redox medium (−100 mV) resulted in considerable increase in parasitization.

Trypanosoma cruzi: Calcium ion movement during internalization in host HeLa cells

The role of cytosolic Ca2+ and cytoplasmic calcium movement during the parasitization of HeLa cells by T. cruzi were studied. The level of calcium in parasitized cells increased compared to the control cells. Our experiments demonstrate that this cytosolic calcium originates from the release of the intracellular calcium deposits, especially from the mitochondria of the host cell. The parasitization rates decreased after the cells were treated with drugs to increase the cytosolic Ca2+ levels to inhibit the host-cell calmodulin.

Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages

Abstract Osuna A. , Gamarro F. , Castanys S. and Ruiz-Perez L.M. 1986. Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages. International Journal for Parasitology16: 629–632. Prelabelling of lysosomes with acridine orange has been performed in order to verify whether metacyclic forms of Trypanosoma cruzi are capable of inhibiting lysosomal fusion during the first moments of interiorization in non-sensitized mouse peritoneal macrophages. Thus, the degree of degranulation (lysosomal fusion) in metacyclic forms is low while epimastigote forms present higher levels. When epimastigote forms are made to interact with the macrophages in the presence of various concentrations of the medium used for transformations of epimastigotes to metacyclic forms or when interaction was performed in the presence of NH4Cl, the degree of degranulation was similar to that obtained when interaction was carried out with metacyclic forms. The present results suggest that during the first moments of the interaction of T. cruzi, only the infective forms may increase the cytoplasmic pH value of the host phagocytic cell, avoiding lysosomal fusion and the subsequent destruction of the parasite.

Isolation and purification of amastigotes ofTrypanosoma cruzi from cultured Vero cells

A method is described for the isolation and purification of the intracellular amastigotes ofTrypanosoma cruzi from cultured Vero cells. Host cells were infected with metacyclic forms obtained in Graces medium. Six days after infection, the cells were subjected to treatment with trypsin to obtain the intracellular forms. The parasites were collected and purified by Percoll discontinuous gradient centrifugation.

A protein secreted by Trypanosoma cruzi capable of inducing the entry of inert particles into HeLa cells

Trypanosoma cruzi requires an intracellular environment to multiply within its mammalian host. We describe the purification and some properties of a protein secreted exclusively by the metacyclic (infective) forms of the parasite. This permeabilizing protein (relative molecular mass 64,000) was secreted under our experimental conditions only when the parasites interacted with HeLa cells, HeLa membranes, or wheat-germ lectin. The protein is thermostable, and its biological activity is inhibited by formaldehyde but not by ethanol or acetone. At low concentrations and over short treatment times, this protein acts as a permeabilizer and induces endocytosis. No significant protease or neuraminidase activity was found. When adsorbed onto bentonite particles and incubated in the presence of non-phagocytic cells the protein facilitated the penetration of the particles into the cells. Immune serum directed against the protein neutralized its cytotoxic action and reduced the rate of penetration of metacyclic forms into both macrophages and non-phagocytic cells. Our results suggest that the protein secreted by the parasite plays a key role in the penetration of its infective form into the host cell.

New antiparasitic agents. III. Comparison between trypanocidal activities of some acridine derivatives against Trypanosoma cruzi in vitro.

Some acridine compounds referred to as 9-imino, 9-oxo and 9-thio derivatives were screened for activity against Trypanosoma cruzi in vitro. The results are discussed here with reference to the structure of the compounds tested. Attempts to elucidate the mode of action of active acridines are also included. The most active compounds that were tested were 9-thioacridanones and 9-thio-1,2,3,4-tetrahydroacridanones Added to this, the dialkylaminoalkylthio group seems to be the most convenient molecular moiety for trypanocidal activity in the 9-substituted acridine series.

Purification of a glycoprotein excreted by Trypanosoma cruzi to increase the permeability of the host-cell membrane.

During invasion of the prospective host cell, metacyclic forms of Trypanosoma cruzi render the membrane of HeLa cells permeable to the alpha-sarcin toxin, by excreting a glycoprotein with N-acetyl-D-glucosamine residues. The molecular weight of the glycoprotein is 64,000 dalton and its isoelectric point is 4.8.

Antiamebic Activity of New Acridinic Derivatives against Naegleria and Acanthamoeba Species in vitro

In vitro antiamebic activity of selected acridine derivatives has been investigated against Naegleria and Acanthamoeba species. The most active compounds belong to the 9-thioacridanone and the 1,2,3,4-tetrahydro-9-thioacridanone series. In addition, some structure-activity relationships are proposed.

Effect of interferon on the infectivity of Trypanosoma cruzi in cultured HeLa cells.

Abstract Results are presented on the effects of human lymphoblastoid interferon (HUIFN-α-ly) on the infectivity of metacyclic culture forms of Trypanosoma cruzi in HeLa cells. When cells were pretreated with interferon the parasitisation ratios of the cultures increased with respect to controls. This phenomenon also occurs when interferon was added during the period of parasite-cell interaction. When parasites were pretreated with interferon but cells were not, no increase in the parasitisation ratios was observed.