In vitro and in vivo activity of amphotericin B-lipid formulations against experimental Trypanosoma cruzi infections.

The activities of four amphotericin B formulations, Fungizone, AmBisome, Amphocil, and Abelcet, were compared in vitro and in vivo against Trypanosoma cruzi infections. In vitro, Fungizone and Amphocil were highly active against T. cruzi Y strain amastigotes in macrophages with 50% effective dose (ED50) values of 0.027-0.028 microg/ml, which were 7-fold and 42-fold more active than AmBisome and Abelcet, respectively. In vitro activities of all formulations against T. cruzi amastigotes in Vero cells were similar, with ED50 values in the range of 2.0-4.2 microg/ml. Acute infections of the T. cruzi Y strain in BALB/c mice were suppressed in all animals by a single 25 mg/kg dose of the liposomal formulation AmBisome. At the same dose, the two other lipid formulations, Amphocil and Abelcet, increased the survival rate but did not suppress infection in all animals.     

Further studies on the cell surface charge of Trypanosoma cruzi

Trypanosoma cruzi has a negative surface which varies according to the ionic strength and the pH of the solution in which the cells are suspended. At low pH there is a decrease in the negative surface charge with an isoelectric point at pH 2.6 and 3.0 for epimastigote and trypomastigote forms, respectively. Below these pH values the cells have a positive surface charge. At higher pH there is an increase in the surface charge. Glutaraldehyde fixation did not interfere with the electrophoretic mobility (EPM) of the cells. Epimastigote and trypomastigote forms of T. cruzi have a characteristic EPM independent of the medium in which the cells were grown, the origin of the trypomastigotes or the strain of the parasite. Trypomastigotes have a higher negative surface charge than epimastigotes. Based on the change in the EPM of the cells treated with neuraminidase, is concluded that sialic acid is present on the cell surface of T. cruzi and that it is the main component responsible for the high negative surface charge of the trypomastigote form. Trypsin treatment also reduces the EPM of T. cruzi. Neuraminidase or trypsin-treated parasites recovered their normal EPM when incubated for 4 h in fresh culture medium. This process involves synthesis of protein since it is inhibited by puromycin.     

Ketoconazole inhibition of intracellular multiplication of Trypanosoma cruzi and protection of mice against lethal infection with the organism

The effects of ketoconazole against infection with Trypanosoma cruzi both in vivo and in vitro were examined. In vivo, ketoconazole significantly protected mice infected with lethal inocula of the Y strain of T. cruzi even when treatment was initiated seven days after infection; protection was also demonstrated for three other strains. Although mice had demonstrable parasitemia after completion of therapy, tissue sections of treated mice revealed a complete absence of organisms. Concentrations of ketoconazole as low as 0.001 microgram/ml inhibited in vitro replication of intracellular organisms, whereas concentrations of ketoconazole that prevented replication of intracellular amastigotes had no effect on extracellular organisms. This finding and the observation that inhibition of replication of amastigotes occurred in macrophages exposed to ketoconazole before infection suggests that the inhibitory effect depends on interaction of the drug with host cells. Thus ketoconazole should be tested as a therapeutic agent for Chagas' disease in humans.     

Anti-Trypanosoma cruzi activity of nicotinamide

Inhibition of Trypanosoma brucei and Leishmania spp. sirtuins has shown promising antiparasitic activity, indicating that these enzymes may be used as targets for drug discovery against trypanosomatid infections. In the present work we carried out a virtual screening focused on the C pocket of Sir2 from Trypanosoma cruzi. Using this approach, the best ligand found was nicotinamide. In vitro tests confirmed the anti-T. cruzi activity of nicotinamide on epimastigote and trypomastigote forms. Moreover, treatment of T. cruzi-infected macrophages with nicotinamide caused a significant reduction in the number of amastigotes. In addition, alterations in the mitochondria and an increase in the vacuolization in the cytoplasm were observed in epimastigotes treated with nicotinamide. Analysis of the complex of Sir2 and nicotinamide revealed the details of the possible ligand-target interaction. Our data reveal a potential use of TcSir2 as a target for anti-T. cruzi drug discovery.     

Specific glycoprotein antigens on the surface of insect and mammalian stages of Trypanosoma cruzi.

Two major surface antigens on Trypanosoma cruzi, the causative agent of Chagas disease, have been described [Nogueira, N., Chaplan, S., Tydings, J., Unkeless, J. & Cohn, Z. (1981) J. Exp. Med. 153, 629-639]. One, a Mr 75,000 glycoprotein (GP), is specific for the culture forms (insect-host stages) of the organisms--epimastigotes and metacyclic trypomastigotes. The other, a Mr 90,000 GP, was found in vertebrate-host stages of the organisms--bloodstream-form trypomastigotes. We now report that these two major surface antigens of T. cruzi seem to be unrelated proteins, as judged by tryptic and chymotryptic peptide analysis. Antibodies were raised in rabbits against epimastigote or trypomastigote proteins which had been immunoprecipitated with human antisera. These trypomastigote and epimastigote protein antisera reacted only with the homologous immunogen, as determined by immunoprecipitation of surface-labeled organisms and by immunofluorescence. The Mr 75,000 GP is detected only in cultured (insect stage) epimastigotes and metacyclic trypomastigotes. The Mr 90,000 GP is only present in bloodstream-form trypomastigotes, amastigotes, and trypomastigotes obtained from infected muscle cells in vitro. Therefore, the insect and vertebrate stages of this species display distinctive surface GPs that can be identified by surface labeling and immunoprecipitation techniques in six strains of T. cruzi (Y, CL, Peru, Colombiana, SF-12, and SF-21) isolated from widely different areas of South America.     

Phagocytosis of the three developmental forms of Trypanosoma cruzi: effect of specific sera

Studies were carried out aiming at comparing the uptake of the three evolutionary stages of Trypanosoma cruzi by mouse peritoneal macrophages, influenced by specific immunosera. Incorporation of T. cruzi by macrophages was time dependent. In absence of antibody, trypomastigotes are forms more effectively incorporated by macrophages. Pre-incubation of macrophages with specific sera against each of the T. cruzi morphological stages was followed by an increase in the uptake of amastigotes and trypomastigotes but not of epimastigotes. Our results show that amastigotes, in comparison with the other T. cruzi forms are more actively phagocytized in presence of specific serum.     

Chagas'disease: IgA, IgM and IgG antibodies to T. cruzi amastigote, trypomastigote and epimastigote antigens in acute and in different chronic forms of the disease

In an attempt to find a better T. cruzi antigen and possible immunological markers for the diagnosis of different clinical forms of Chagas'disease, amastigote and trypomastigote antigens obtained from immunosuppressed mice infected with T. cruzi (Y strain) were assessed in comparison with conventional epimastigote antigens. A total of 506 serum samples from patients with acute and with chronic (indeterminate, cardiac and digestive) forms, from nonchagasic infections, and from healthy individuals were assayed in immunofluorescence (IF) tests, to search for IgG, IgM and IgA antibodies. Amastigote proved to be the most convenient antigen for our purposes, providing higher relative efficiency indexes of 0.946, 0.871 and 0.914 for IgG, IgM and IgA IF tests, respectively. Anti-amastigote antibodies presented higher geometric mean titers (GMT) than anti-trypomastigote and anti-epimastigote. Anti-amastigote IgG antibodies were found in all forms of Chagas'disease, and predominantly IgA antibodies, in chronic digestive and in acute forms, as well as IgM antibodies, in latter forms. Thus, tests with amastigote antigen could be helpful for screening chagasic infections in blood banks. Practical and economical aspects in obtaining amastigotes as here described speak in favour of its use in developing countries, since those from other sources require more complex system of substruction, specialized personnel or equipment.     

Identification and synthesis of a major conserved antigenic epitope of Trypanosoma cruzi.

A gene sequence encoding an immunodominant protein with a repetitive epitope from the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, was cloned and expressed. The identified 10-amino acid repeat is present within a high-molecular-weight trypomastigote antigen that appears specific to and conserved among T. cruzi isolates. More importantly, greater than 95% of T. cruzi infection sera, including both chronic and acute Chagas disease, contained elevated levels of antibody to a 15-amino acid synthetic peptide bearing the repetitive B-cell epitope. Considering the wide diversity of T. cruzi parasites, as well as the broad spectrum of clinical manifestations of Chagas disease, such a prevalent immune response among patients is significant and applicable to the control of Chagas disease through the diagnosis of T. cruzi infection.     

Effect of L-leucine methyl ester on growth and ultrastructure of Trypanosoma cruzi

L-Amino acid methyl esters, such as L-leucine methyl ester (Leu-OMe), have been identified as agents targeting the lysosomal system of Leishmania amazonensis amastigotes, by a mechanism that involves ester hydrolysis by parasite enzymes located inside megasomes. We have here analyzed the effect of Leu-OMe on all three evolutive forms of Trypanosoma cruzi, in a search for potential targets of the compound in this protozoan. Treatment of epimastigote forms resulted in dose-dependent growth inhibition, with IC50/1 day = 0.55 +/- 0.21 mM. Incubation with 4-8mM/1 day led to 100% cell death. Treatment of bloodstream trypomastigotes resulted in cell lysis, with an IC50/1 day = 1.46 +/- 0.16 mM. Furthermore, infected macrophages treated with 0.125-1mM Leu-OMe showed a dose- and time-dependent decrease in the percent of amastigote infection. Morphological changes in macrophages were observed only at concentrations above 8mM, at the third day of treatment. Analysis of treated parasites by transmission electron microscopy demonstrated severe morphological alterations in cell shape, mitochondrion and nucleus, while kinetoplast and reservosomes (pre-lysosomal compartments) appeared to be not affected. Lysis of bloodstream trypomastigotes and intracellular amastigotes indicated that lysosomes of T. cruzi are the main target for the drug, since reservosomes occur only in epimastigote forms. The presence of lysosomes in T. cruzi epimastigotes was demonstrated by using ultrastructural cytochemistry.     

Melatonin and dehydroepiandrosterone combination: does this treatment exert a synergistic effect during experimental Trypanosoma cruzi infection?

Abstract:  Previous studies showed that melatonin or dehydroepiandrosterone (DHEA) enhances the immune response against parasitic pathogens. The present study investigated the in vitro activity of melatonin combined with DHEA in a period of 24 hr during the course of in vivo T. cruzi infection. The in vitro activity of melatonin or DHEA alone, as well as together, were tested for the trypomastigote forms (doses ranging from 0.5 to 128 μ m ). In vitro, neither melatonin nor DHEA alone had any activity against trypomastigote forms, although when the highest concentration of combined melatonin and DHEA was used, it was active against the trypomastigote forms of the parasite. However, for this concentration, a quite toxicity on peritoneal macrophages was observed. For in vivo evaluation, male Wistar rats were infected with the Y strain of T. cruzi. They were orally treated with 10 mg/kg body weight/day of melatonin and subcutaneously with 40 mg/kg body weight/day of DHEA. Treatment with melatonin, DHEA and the association showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection as compared to untreated animals ( P  < 0.05). A significant increase in the number of macrophages and nitric oxide (NO) concentrations were observed during the peak of parasitaemia with melatonin alone or combined with DHEA. However, with DHEA alone the highest concentration of NO was observed ( P  < 0.05). Moreover, DHEA treatment increased TNF-alpha levels during the infection ( P  < 0.05). These results show that melatonin, DHEA or the combination of both reduces parasitemia during the acute phase of infection. The combined action of both molecules did not exert a synergic action on the host's ability to fight infection, and it seems that among all treatments DHEA induces a more efficient immune response.     

Trypanosoma cruzi upregulates nitric oxide release by IFN-γ-preactivated macrophages, limiting cell infection independently of the respiratory burst

The relationship between nitric oxide (N = O) produced by mouse peritoneal macrophages (MPM) and Trypanosoma cruzi infection is still poorly understood. The conditions of MPM activation by gamma-interferon (IFN-γ) to trigger a N = O-dependent trypanocidal activity, as well as the effect of parasite infection or of reactive oxygen species (ROS) inhibitors on the N = O release were studied. T. cruzi infection occurring after a previous 24 h MPM activation induced an enhancement of nitrite levels (the stable degradation product of N = O) in cell supernatants; both the percentage of infected MPM and the number of amastigotes per infected cell were decreased in comparison to infected but non-activated MPM. Addition of superoxide dismutase or catalase to non-infected but activated MPM increased the nitrite levels; these were not detectable when L-arginine inhibitors were added together with ROS inhibitors. The latter had no effect on infection nor on nitrite levels when infection occurred after pre-activation, and induced only a weak nitrite release when infection took place before MPM activation. Altogether, these results support the involvement of N = O in the inhibition of T. cruzi infection by IFN-γ-preactivated macrophages, together with the upregulation of N = O release by T. cruzi infection independently of the respiratory burst.     

A survey of congenital Chagas' disease, carried out at three health institutions in São Paulo City, Brazil

The congenital transmission of Chagas' disease was evaluated in 57 pregnant women with Chagas' disease and their 58 offspring. The patients were selected from three Health Institutions in S?o Paulo City. The maternal clinical forms of Chagas' disease were: indeterminate (47.4%), cardiac (43.8%) and digestive (8.8%); 55 were born in endemic areas and two in S?o Paulo City. The transmission of Chagas' disease at fetal level was confirmed in three (5.17%) of the 58 cases studied and one probably case of congenital Chagas' disease. Two infected infants were born to chagasic women with HIV infection and were diagnosed by parasitological assays (microhematocrit, quantitative buffy coat-QBC or artificial xenodiagnosis). In both cases the placenta revealed T. cruzi and HIV p24 antigens detected by immunohistochemistry. In one case, a 14-week old abortus, the diagnosis of congenital T. cruzi infection was confirmed by immunohistochemistry. The other probable infection, a 30-week old stillborn, the parasites were found in the placenta and umbilical cord. The Western blot method using trypomastigote excreted/secreted antigens of T. cruzi (TESA) was positive for IgG antibodies in 54/55 newborns and for IgM in 1/55 newborns. One of the two newborns with circulating parasites had no detectable IgG or IgM antibodies. The assessment of IgG antibodies in the sera of pregnant women and their newborns was performed by ELISA using two different T. cruzi antigens: an alkaline extract of epimastigotes (EAE) and trypomastigote excreted/secreted antigens (TESA). The analysis showed a linear correlation between maternal and newborn IgG antibody titers at birth.     

Arginine decarboxylase inhibitors reduce the capacity of Trypanosoma cruzi to infect and multiply in mammalian host cells.

The capacity of blood (trypomastigote) forms of Trypanosoma cruzi to infect mouse peritoneal macrophages or rat heart myoblasts in vitro was inhibited by treatment of the trypomastigotes with DL-alpha-difluoromethylarginine (F2Me Arg), monofluoromethylagmatine, or (E)-alpha-monofluoromethyl-3-4-dehydroarginine--all irreversible inhibitors of arginine decarboxylase. Similar results were obtained when F2MeArg-treated parasites were incubated with rat heart myoblasts. The inhibitory effects were characterized by marked reductions in both the proportion of infected cells and the number of parasites per 100 host cells. The concentrations of the arginine decarboxylase inhibitors that affected infectivity had no detectable effect on either the concentration or motility of the parasite and, therefore, could not have affected the collision frequency. F2MeArg appeared to inhibit the ability of T. cruzi to penetrate the host cells since the drug had no significant effect on the extent of parasite binding to the surface of the host cells. The inhibitory effect of F2MeArg was markedly reduced or abrogated in the presence of either agmatine or putrescine, as would have been expected if F2MeArg acted by inhibiting arginine decarboxylase. Addition of F2MeArg to macrophage or myoblast cultures immediately after infection or at a time when virtually all of the intracellular parasites had transformed into the multiplicative amastigote form, resulted in a markedly reduced parasite growth rate. This effect was also prevented by exogenous agmatine. These results indicate the importance of polyamines and polyamine biosynthesis in the following two important functions of T. cruzi: invasion of host cells and intracellular multiplication. Furthermore, concentrations of the inhibitors tested that affected the parasite did not alter the viability of the host cells, the cellular density of the cultures, or the ability of uninfected myoblasts to grow. Thus, arginine decarboxylase inhibitors may have a potential application in chemotherapy against T. cruzi infection.     

Congenital Trypanosoma cruzi infection in a laboratory-born squirrel monkey, Saimiri sciureus

A Colombian Phenotype, laboratory-born squirrel monkey, Saimiri sciureus, was found to be congenitally infected with biologically proven Trypanosoma cruzi. The parasite was observed in blood smears and by xenodiagnoses of the mother and the offspring, and the isolates produced infection in mice and amastigotes in VERO tissue culture cells. The finding was accidental; both animals were healthy. Tissues of the mother did not show macro-microscopic evidence of T. cruzi infection and the electrocardiograph of the offspring was normal. This seems to be the first report of congenital T. cruzi transmission in an otherwise healthy non-human primate.     

Modification of the n-6/n-3 fatty acid ratio in the phospholipids of rat ventricular myocytes in culture by the use of synthetic media: functional and biochemical consequences in normoxic and hypoxic conditions

The respective roles of exogenous polyunsaturated fatty acids on the lipid composition, physiological properties and enzyme release was investigated on isolated cardiac muscle cells in normoxia and hypoxia. Rat neonatal ventricular myocytes were grown for 5 days in conventional serum-supplemented medium. Cells were then incubated for 24 h in fully chemically-defined media featuring a balanced fatty acid composition containing either linoleic acid (18:2 n-6) or linolenic acid (18:3 n-3) as sole polyunsaturated fatty acid source. Transmembrane potentials were monitored with microelectrodes and contractions with a photoelectric device. The radio of n-6 to n-3 phospholipid fatty acids increased from 6.3 in control cells to 20.2 in cells exposed to n-6 fatty acids (SM6) and decreased to 1.4 in those exposed to n-3 fatty acids (SM3). These modifications had no influence on the electrical and mechanical activities and on automaticity in normoxic conditions. The action potential depression under hypoxia was less severe in SM6 cells, whereas there was a better electrophysiological recovery upon reoxygenation in SM3 cells. However, the loss of lactate dehydrogenase during sustained hypoxic treatment was not affected by changes in phospholipid fatty acid pattern. These results suggest that the effect of the polyunsaturated fatty acid balance depends on the cellular function under study and on the environmental conditions.     

Enalapril prevents cardiac immune‐mediated damage and exerts anti‐Trypanosoma cruzi activity during acute phase of experimental Chagas disease

Chagas heart disease (CHD), caused by Trypanosoma cruzi infection, is a significant cause of morbidity and mortality in South and Central America. Enalapril, an angiotensin converting enzyme (ACE) inhibitor, is an important drug used to ameliorate heart functional capacity and its remodelling in individuals presenting CHD. In this study, we evaluated the effects of enalapril on systemic and cardiac immune response during experimental acute CHD. C57BL/6 mice infected with 50 trypomastigote forms of T. cruzi (Colombian strain) were treated daily with enalapril (25 mg/kg) and, after 30 days, a reduction in seric levels of IFN‐gamma, TNF‐alpha, CCL5/RANTES and nitric oxide, but not in that of IL‐10, was detected. This imbalance of cytokines reflects in a reduction of heart mononuclear infiltration and in an increasing of cardiac mast cells. Enalapril also presents a new and interesting in vitro and in vivo anti‐T. cruzi activity probably acting on parasite oxidative pathway via cytochrome‐P450. Our data show that enalapril exerts an important anti‐T. cruzi and anti‐inflammatory activity during acute CHD reducing inflammatory cells and, possibly, preventing fibrotic process in the chronic phase. Nevertheless, further studies are still necessary to clarify the mechanisms by which this drug is acting on the parasites and on the immune pathways.     

Antibody response and antigen recognition in human infection with Trypanosoma cruzi

Sequential serum samples of an individual accidentally infected with Trypanosoma cruzi were examined to study the evolution of the antibody response, particularly of those with the capacity to lyse trypanosomes, and to determine the antigens of each of the 3 stages of the life cycle of T. cruzi, recognized by antibodies formed as the infection progresses. T. cruzi specific IgM and subclasses of IgG antibodies were detected and reached peak levels at the same period of time. Lytic antibodies were detected 2 weeks before demonstration of parasitemia and of antibodies reacting in the ELISA and IFA tests. Western blots used to examine antigen recognition revealed a complex array of antigens of epimastigotes and amastigotes, but not of blood trypomastigotes, recognized by antibodies in the various serum samples. Most of the antigens recognized by antibodies were common to all 3 stages of T. cruzi, although a few were specific for each of the stages. Certain antigens were only recognized by antibodies in serum collected at distinct periods of time during the course of the infection. The pattern noted after immunoprecipitation of 125I labeled cell membrane surface antigens was simple. Antigens of molecular weight of 90, 72, 50, and 30 K were immunoprecipitated in higher quantities. Antibodies in serum collected early and late in the infection recognized similar antigens in epimastigotes of the infecting strain and in epimastigotes of 3 other strains of T. cruzi from widely separated geographic areas.     

In vivo and in vitro effects of cyclosporin A on Trypanosoma cruzi

The in vivo and in vitro effects of cyclosporin A on T. cruzi were examined. Mice receiving 150 or 75 mg/kg/day of cyclosporin A and infected with T. cruzi 48 hr later had significantly higher parasitemias and earlier mortality than controls. Mice receiving cyclosporin A after infection had parasitemias similar to controls. Infections with both the reticulotropic Y and the miotropic CL strains of T. cruzi were enhanced by cyclosporin A. The in vitro replication of epimastigotes, but not the intracellular replication of amastigotes, in mouse macrophages was inhibited by 5 micrograms cyclosporin A/ml. Enhancement of the infection by cyclosporin A was not due to an effect on macrophages since the drug did not prevent development of activated macrophages capable of killing intracellular T. cruzi.     

Arachidonic acid deficiency in streptozotocin-induced diabetes.

Fatty acid compositions of phospholipids of heart, liver, kidney, aorta, and serum from rats having streptozotocin-induced diabetes were determined and compared with those of nondiabetic controls. Linoleic and dihomo-gamma-linolenic acids were increased whereas arachidonic acid was decreased in most tissues, suggesting an impairment of delta 5-desaturase activity. Acids derived from linolenic acid were increased in some diabetic tissues from diabetic animals although the linolenic content was normal, indicating less impairment in the desaturation of the omega 3 series of fatty acids. Diabetes suppressed all polyunsaturated acids in the whole animal, but the competition between omega 3 and omega 6 acids favored the excessive suppression of long-chain omega 6 acids and an increase in the proportion of omega 3 acids in lipids of vital tissues. These changes in fatty acid composition of the phospholipids may have significant effects on cellular functions and vasoregulatory control mechanisms in diabetes.