Basic surface properties of mononuclear cells from Didelphis marsupialis.

The electrostatic surface charge and surface tension of mononuclear cells/monocytes obtained from young and adult marsupials (Didelphis marsupialis) were investigated by using cationized ferritin and colloidal iron hydroxyde, whole cell electrophoresis, and measurements of contact angles. Anionic sites were found distributed throughout the entire investigated cell surfaces. The results revealed that the anionic character of the cells is given by electrostatic charges corresponding to -18.8 mV (cells from young animals) and -29.3 mV (cells from adult animals). The surface electrostatic charge decreased from 10 to 65.2% after treatment of the cells with each one of trypsin, neuraminidase and phospholipase C. The hydrophobic nature of the mononuclear cell surfaces studied by using the contact angle method revealed that both young and adult cells possess cell surfaces of high hidrofilicity since the angles formed with drops of saline water were 42.5 degrees and 40.8 degrees, respectively. Treatment of the cells with trypsin or neuraminidase rendered their surfaces more hydrophobic, suggesting that sialic acid-containing glycoproteins are responsible for most of the hydrophilicity observed in the mononuclear cell surfaces from D. marsupialis.

Heart muscle cells share common neutral glycosphingolipids with Trypanosoma cruzi.

Neutral glycosphingolipids were isolated from mouse heart muscle cells and their structures were analyzed. The molecular compositions of these glycosphingolipids were examined using column chromatography, HPTLC, GC-MS and fast atom bombardment-mass spectrometry (FAB-MS). Monohexosylceramides are a mixture of glucosyl- and galactosylceramides in a ratio of 1:1, sphingosine as the long chain base and as fatty acyl groups mainly C16, C18 saturated and C22 and C24 hydroxy fatty acids. Dihexosylceramide, identified as lactosylceramide contains C18 sphingosine and C18, C20 and C22 were the major fatty acids. No evidence for the occurrence of hydroxylated fatty acids in this glycolipid could be obtained from the GC-MS data. Our results clearly demonstrated that Trypanosoma cruzi and heart muscle cells have similar glycosphingolipid structures. In addition, heart muscle cells neutral glycosphingolipids have been shown to be immunoreactive. Antibodies reactive with each of the immunogenic glycolipids from heart cells or T. cruzi epimastigotes were present in the sera of human patients with Chagas disease as detected by ELISA. These cross-reactive antigens could be involved in the Chagasic autoimmunity.

Alterations in the surface charge of heart muscle cells during interaction with Trypanosoma cruzi.

The surface charge of heart muscle cells (HMC) and Trypanosoma cruzi trypomastigotes was estimated during their interaction by means of zeta potential (ZP). Metacyclic and bloodstream trypomastigote, but not amastigote forms, are able to decrease the surface charge of HMC as well as other nonphagocytic cells. However, no alteration could be detected on T. cruzi-infected macrophage cell line. Trypomastigote forms collected from the supernatant after 20 h of contact with HMC also have their ZP value decreased. The analysis of the surface components of both the parasite and HMC involved in such interaction was also carried out. Assays concerning the kinetics of the cell-parasite interaction demonstrated the influence of parasite surface anionogenicity during its interaction with HMC. The binding of bloodstream forms to HMC was enhanced after their incubation with cationized ferritin (CF), whereas phospholipase C and neuraminidase treatments improved and trypsin treatment inhibited parasite uptake in HMC. Conversely, the incubation of HMC with phospholipase C impaired, and with trypsin enhanced, the interiorization of the parasites. These results suggest that trypomastigote forms of T. cruzi may process the surface of HMC and its own surface either by removing molecules or by exposing ligands for their internalization.

Trypanosoma cruzi: effect of phenothiazines on the parasite and its interaction with host cells.

Phenothiazines were observed to have a direct effect on Trypanosoma cruzi and on its in vitro interaction with host cells. They caused lysis of trypomastigotes (50 uM/24 h) and, in axenic medium, dose-dependent inhibition of amastigote and, to a lesser extent, epimastigote proliferation. Treatment of infected peritoneal macrophages with 12.5 uM chlorpromazine or triflupromazine inhibited the infection; this effect was found to be partially reversible if the drugs were removed after 24 h of treatment. At 60 uM, the drugs caused damage to amastigotes interiorized in heart muscle cells. However, the narrow margin of toxicity between antitrypanosomal activity and damage to host cells mitigates against in vivo investigation at the present time. Possible hypotheses for the mechanism of action of phenothiazines are discussed.

Ultrastructural detection in vitro of WGA-, RCA I-, and Con A-binding sites involved in the invasion of heart muscle cells by Trypanosoma cruzi.

The presence of carbohydrate residues in the plasma membrane of normal and Trypanosoma cruzi-infected heart muscle cells was investigated cytochemically using ruthenium red, lanthanum nitrate, periodic acid-Schiff/thiocarbohydrazide/silver, and gold- and ferritin-lectin complexes. The study combined conventional electron microscopy with the new analytical technique of electron spectroscopic imaging (ESI). Galactosyl, mannosyl, and sialyl residues were detected in regions of host-cell plasma membrane that undergo interiorization together with the parasite. Lectin-binding sites were sometimes found to show a punctate or patchy distribution in the endocytic vacuole membrane. These findings suggest the that glycoconjugates cytochemically detected in the host-cell plasma membrane participate in the invasion of heart muscle cells by T. cruzi.

Trypanosoma cruzi: experimental Chagas' disease in rhesus monkeys. II. Ultrastructural and cytochemical studies of peroxidase and acid phosphatase activities.

Ultrastructural and cytochemical studies of peroxidase and acid phosphatase were performed in skin, lymph node and heart muscle tissue of rhesus monkeys with experimental Chagas' disease. At the site of inoculation there was a proliferative reaction with the presence of immature macrophages revealed by peroxidase technique. At the lymph node a diffuse inflammatory exudate with mononuclear cells, fibroblasts and immature activated macrophages reproduces the human pattern of acute Chagas' disease inflammatory lesions. The heart muscle cells present different degrees of degenerative alterations and a striking increase in the number of lysosomal profiles that exhibit acid hydrolase reaction product. A strong inflammatory reaction was present due to lymphocytic infiltrate or due to eosinophil granulocytes associated to ruptured cells. The present study provides some experimental evidences that the monkey model could be used as a reliable model to characterize histopathological alterations of the human disease.

Trypanosoma cruzi: killing and enhanced uptake by resident peritoneal macrophages treated with alpha-2-macroglobulin.

We report that alpha-2-macroglobulin (A2M), the physiologically important plasma protease inhibitor and suspected immunomodulator, alters the functional ability of murine resident peritoneal macrophages (RM) to ingest and kill the infective trypomastigote stage of Trypanosoma cruzi, the aetiological agent of Chagas' disease. Treatment of RM with 500 micrograms/ml A2M for 30 min enhanced the uptake of trypomastigotes, epimastigotes, and amastigotes by 125%, 46%, and 300%, respectively. The same treatment also increased the phagocytosis of sheep erythrocytes opsonized with complement and IgG as well as of galactosylated asialoerythrocytes. After 60-90 min parasite-cell interaction, epi- and amastigotes were killed by the RM, whereas the infection with trypomastigotes was controlled only after 24 h. Other protease inhibitors, bovine serum albumin, and LPS showed no such effect. The production of hydrogen peroxide was not affected by A2M treatment, but the ultrastructural aspects showed trypomastigote damage and enhancement of macrophage membrane ruffling, indicative of macrophage activation. These results suggest that A2M has the ability to modulate, at least functionally, certain receptor-mediated endocytic pathways that, in concert with an activation of possibly oxygen-independent microbicidal mechanisms, could contribute to resistance against the parasite.

Leishmania mexicana amazonensis: heterogeneity in 5'-nucleotidase and peroxidase activities of mononuclear phagocytes during in vivo and in vitro infection.

The degree of maturation of cells of the Mononuclear Phagocyte System (MPS), during in vivo and in vitro infection by Leishmania mexicana amazonensis, was evaluated in this study. The macrophages' differentiation was assayed by cytochemical characterization at the ultrastructural level, using two well-established markers: 5'-nucleotidase enzyme activity, for revealing the mature cells; and the peroxidase activity present in the cell granules to demonstrate immature mononuclear phagocytes. Only a few macrophages, demonstrating 5'-nucleotidase positive reaction in both the plasma membrane and within their cytoplasmic vesicles, were found scattered in the chronic inflammation at the L. m. amazonensis lesions in albino mice. However, by the peroxidase activity analysis, we were also able to demonstrate the presence of immature MPS cells, which predominate, together with parasitized vacuolated macrophages, in chronic lesions induced in this system by L. m. amazonensis. The implications of these results on the pathogenesis of murine cutaneous leishmaniasis are discussed.

Trypanosoma cruzi: phagolysosomal fusion after invasion into non professional phagocytic cells.

Parasite-containing endocytic vacuoles are formed during the process of in vitro interiorization of the trypomastigote forms of Trypanosoma cruzi by primary culture of mouse fibroblasts, heart and skeletal muscle cells. Fusion of these vacuoles with host cell lysosomes takes place. The process of T. cruzi-muscle cell interaction was analysed by ultrastructural cytochemistry. Two lysosomal enzymes, acid phosphatase and aryl sulphatase and the fusion of peroxidase-labeled secondary lysosomes with the parasitophorus vacuoles were studied. These finding indicate that the basic mechanism of interaction of T. cruzi with the so called non phagocytic cells is similar to that which occurs with phagocytic cells.

Effect of drugs on Trypanosoma cruzi and on its interaction with heart muscle cell "in vitro".

Megazol, nifurtimox, benznidazol and allopurinol were investigated, by light and electron microscopy, for their action on T. cruzi. Both the direct effect upon amastigote and trypomastigote forms and the effect upon the interaction of heart muscle cells (HMC) with bloodstream trypomastigotes were studied. The proliferation of amastigotes in Warren medium was inhibited in a dose-dependent manner by megazol, nifurtimox and benznidazol. Treatment of amastigotes (25-50 microM/24 h) and trypomastigotes (25 microM/24h) led to several ultrastructural alterations in the parasites. These three drugs also had a potent effect on the treatment of infected heart muscle cells when added at the beginning of the interaction or after one or three days of infection. The interiorized parasites showed a similar pattern of ultrastructural alterations as observed by the direct effect on the amastigotes. The primary heart muscle cell culture proved to be a suitable model for the study of drugs on intracellular parasites. Likewise, the amastigote proliferation in axenic medium was shown to be an adequate assay for an initial trial of drugs. These parameters seem very reliable to us for a systematic investigation of the mechanism of action of new drugs.