Trypanosoma cruzi: isolation and characterization of membrane and flagellar fractions.

A cell fractionation procedure for obtaining membrane and flagellar fractions was developed using Trypanosoma cruzi epimastigote forms. The cells, swollen in an hypotonic medium, were disrupted in the presence of a nonionic detergent, and fractions were isolated by differential centrifugation. The flagellar fraction, pelleted in 10 min at 10,000g, was further purified on a sucrose gradient. The membrane fraction was obtained by centrifugation of the supernatant at 27,000g for 30 min. Electron microscopy of the isolated fractions demonstrated a high degree of purity of each fraction. The membrane fraction showed homogeneous vesicles with low ribosome content. In frozen-etched preparations, the distribution of intramembranous particles on the vesicles was similar to that of the plasma membrane of intact cells. Enzymatic assays indicated that the membrane and flagellar fractions had low contamination with mitochondria and lysosomes. 5′-Nucleotidase activity was not detected in the membrane fraction; Mg²⁺-dependent ATPase activity was slightly enhanced, although, the enzyme was not sensitive to Na⁺, K⁺, and Ca²⁺ ions. The membrane fraction showed about five times the adenylyl cyclase activity of the whole homogenate. Gel immunodiffusion revealed the whole antigen of T. cruzi extracted by formamide to be identical to the membrane fraction when both were tested against rabbit anti- T. cruzi (epimastigote) immune serum.     

Trypanosoma cruzi: location of a specific antigen on the surface of bloodstream trypomastigote and culture epimastigote forms.

The nature of surface antigens of culture epimastigote and bloodstream trypomastigote forms of Trypanosoma cruzi was investigated by light and electron microscopy using indirect immunofluorescence and peroxidase labeling techniques and antisera against unique, common, and contaminant antigens. A specific antigen, identified by monospecific rabbit antiserum (anti-component 5 antiserum), is the major constituent of the cell surface and flagellar membrane of both the culture epimastigote and bloodstream trypomastigote forms. Antigens of heterologous stercorarian trypanosomes (Trypanosoma rangeli) and of culture medium proteins could not be detected on the cell surface of culture epimastigote forms and bloodstream trypomastigote forms.     

Leishmania species: Comparative ultrastructure of experimental nodules and diffuse human cutaneous lesions in American leishmaniases

Experimental nodules of American leishmaniases were obtained by inoculating 0.1–1 × 10⁵ amastigotes into the dorsum of the hindpaws of golden hamsters and of C57B1/6J mice. The amastigotes were obtained by biopsy of lesions in six human cases of cutaneous leishmaniases and were serially maintained in golden hamsters and in a fetal calf serum-containing medium. Human nodules were obtained by biopsy from several patients with diffuse cutaneous leishmaniases, always prior to treatment. Within the same host species, no ultrastructural differences were seen in the tissue response to isolates of Leishmania mexicana, L. brasiliensis, or L. garnhami, nor were there differences between the host species in response to a particular isolate of the genus Leishmania. The typical inflammatory response was a macrophage granuloma with abundant polymorphonuclear neutrophils, some eosinophils, and plasma cells. Simple human cutaneous leishmanial lesions, as well as experimental nodules in regression, show many fibroblasts, much collagen fiber, but very few parasites. In typical lesions, parasites occurred within macrophage phagolysosomes, within distended lacunar cells, and in the intercellular spaces. Leishmaniae strongly adhered to parasitophorous vacuoles by a site of their plasma membrane directly opposite the flagellum, and the host cell cytoplasm close to the adherence site became highly vacuolated. In most cases the intra- and extracellular parasites show normal morphology, which suggest the inability of phagocytic cells to attack them.