ABSTRACT. Fluorescence microscopy, using dyes which specifically label mitochondria, endoplasmic reticulum and the Golgi complex, and transmission electron microscopy, were used to analyze the changes which occur in the organization of these structures during interaction of Toxoplasma gondii with host cells. In uninfected cells the mitochondria are long filamentous structures which radiate from the nuclear region toward the cell periphery. After parasite penetration they become shorter and tend to concentrate around the parasite-containing vacuole (parasitophorous vacuole) located in the cytoplasm of the host cell. The mitochondria of extracellular parasites, but not of those located within the parasitophorous vacuole, were also stained by rhodamine 123. Labeling with DiOC6, which binds to elements of the endoplasmic reticulum, in association with transmission electron microscopy, revealed a concentration of this structure around the parasitophorous vacuole. The membranelining this vacuole was also stained, suggesting that components of the endoplasmic reticulum are also incorporated into this membrane. The Golgi complex, as revealed by staining with NBD-ceramide and electron microscopy, maintains its perinuclear position throughout the evolution of the intracellular parasitism.

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Conidial forms of Fonsecaeapedrosoi, grown under conditions where melanin was or was not synthesized, were allowed to interact with normal and cytochalasin treated macrophages. Melaninfree conidia were more infective to the macrophages. Treatment of macrophages with either cytochalasin B or D before the interaction decreased, but did not totally prevent their infection by the fungi. This inhibitory effect was higher (approximately 90%) if E pedrosoi was grown under conditions where melanin was not synthesized. When melanin-containing conidia were used, the inhibitory effect of the cytochalasin on the infection was lower (approximately 50%). At least two mechanisms of infection of the host cell were observed: typical phagocytosis and another process in which the fungi played a more active role. Infection by E pedrosoi was also observed in the nonprofessional phagocytic MDCK epithelial cell line. Two types of cytoplasmic vacuoles which contained parasites were seen in thin sections of host cells infected with E pedrosoi: a 'tight' type and a 'loose' type. At least 200 conidia-containing vacuoles were analysed by transmission electron microscopy. The 'tight' type was observed in 75% of the vacuoles of non-treated macrophages, suggesting an association with classical phagocytosis. On the other hand, the 'loose' type vacuole was seen in 75% of the vacuoles present in cytochalasin treated macrophages and seemed to be related to induced phagocytosis or active penetration by the fungi.Fonsecaea pedrosoi, a polymorphic pathogenic fungus, is one of the aetiologic agents of chromoblastomycosis, a chronic disease usually limited to the skin and subcutaneous tissue [25]. Tissue forms comprise hypha! segments and brown, thick-walled sclerotic bodies, while culture forms are mainly filamentous with hyphae and conidia. Chromoblastomycosis is thought to be initiated by traumatic implantation of hyphae or conidia into the skin. The disease progresses slowly and any hyphae or conidia inoculated deeply within the skin of a patient tend to disappear so that only sclerotic forms are observed [9]. F. pedrosoi, like other dematiaceous fungi pathogenic for humans, usually forms a dark pigment, generally referred to as melanin, which is deposited on the fungal cell wall and in cytoplasmic structures [1,12].We have already demonstrated that mouse peritoneal resident macrophages have little or no cytotoxic effect on E pedrosoi [13]. During the phagocytic process, melaninpigmented particles are released by the fungus. These particles can be then seen in the same phagocytic vacuoles that contain ingested fungi, as well in other small cytoplasmic vacuoles [13]. It has been suggested that melanin present in the cell wall of fungi, may protect them against destruction by phagocytes, providing resistance to microbial lysis [24].

sites have developed mechanisms for evading this fate. Toxoplasma gondii [12], Mycobacterium leprae [25], Rickettsiae and Chlamydia [29,31] are ingested by macrophages and block the process of L-P fusion, whereas Trypanosoma cruzi disrupts the 373

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A method is described for the isolation and purification of trypomastigotes and amastigotes of Trypanosoma cruzi from cell cultures. L-A9, a transformed fibroblast cell line, and J774G8, a macrophage-like cell line of tumor origin, were used. Both cell lines were infected with bloodstream trypomastigotes of T. cruzi, which once within host cells transform into dividing amastigotes. After 6--8 days infection the host cells ruptured, spontaneously liberating parasites into the culture medium. L-A9 cells liberated mainly trypomastigotes while J774G8 cells liberated amastigotes. The parasites were collected and purified by centrifugation in a gradient of metrizamide. The purity of the preparation as well as the morphology of the parasites and the host cells were analysed by electron microscopy.

SUMMARYThe infectivity amastigotes of Trypanosoma cruzi, isolated from the supernatant of the J774G8 macrophage-like cell line infected with trypomastigotes to normal macrophages in vitro was tested. After a period of 1 h of T. cruzi-macro¬ phage interaction about 2% of the mouse peritoneal macrophages had ingested amastigotes. In contrast 12% of the macrophages had ingested epimastigotes. Treatment of the amastigotes with trypsin did not interfere with their ingestion by macrophages. Once inside the macrophages the amastigotes divided and after some days transformed into trypomastigotes. When i.p. inoculated into mice the amastigotes were highly infective, inducing high levels of parasitaemia and tissue parasitism. As previously described for trypomastigotes, amastigotes were not lysed when incubated in the presence of fresh guinea-pig serum. Contrasting with what has been described for trypomastigotes, the resistance of amastigotes to complement-mediated lysis persisted after treatment with trypsin.

NADH- or NADPH-oxidase activity was cytochemically detected at the ultrastructural level during the process of interaction between Trypanosoma cruzi and activated mouse peritoneal macrophages. The reaction product, indicative of enzyme activity, was found in the portion of the plasma membrane of the macrophages to which the parasites attached. It was also found in the membrane which surrounds endocytic vacuoles containing ingested parasites.