The in vivo trypanocidal effect of the diterpene 5-epi-icetexone obtained from Salvia gilliesii.

The search for new compounds with trypanocidal activity is crucial for the treatment of Chagas' disease. Previous in vitro studies have shown that the diterpene 5-epi-icetexone (ICTX) is active against Trypanosoma cruzi. The aim of this work was to evaluate the effect of ICTX on the parasites in infected mice, in an experimental model that mimics the acute phase of the disease. Swiss albino mice were infected with T. cruzi and treated daily with 10mg/kg/day ICTX (i.p.). Infected mice and mice injected with either saline or the vehicle DMSO were used as controls. Animals' survival and parasitemia were monitored once a week and histological studies were made at necropsy by the 5th week after infection. It was observed that the administration of ICTX increased the survival of mice infected, and induced a significant decrease in the parasitemia, as compared to controls. A similar protective effect was observed when animals were treated orally with benznidazole (BZN, used as a control of antiparasitic effect). By the 5th week post-infection, the presence of amastigote nests was observed within the fibers of the cardiac and skeletal muscle in controls, but not in animals treated with either ICTX or BZN. In addition, inflammatory infiltrates were observed in the tissues of controls, but not in animals treated with the drugs. We conclude that ICTX has an antiparasitic effect against T. cruzi, thus constituting an interesting option for the treatment of Chagas' disease, alone or combined with other drugs.

Involvement of the beta-adrenergic system in the cardiac chronic form of experimental Trypanosoma cruzi infection.

Changes in the cardiac beta-adrenergic system in early stages of Trypanosoma cruzi infection have been described. Here, we studied an early (135 days post-infection-p.i.) and a late stage (365 days p.i.) of the cardiac chronic form of the experimental infection (Tulahuen or SGO-Z12 strains), determining plasma epinephrine and norepinephrine levels, beta-receptor density, affinity and function, cardiac cAMP concentration and phosphodiesterase activity, cardiac contractility, and the presence of beta-receptor autoantibodies. Tulahuen-infected mice presented lower epinephrine and norepinephrine levels; lower beta-receptor affinity and density; a diminished norepinephrine response and higher cAMP levels in the early stage, and a basal contractility similar to non-infected controls in the early and augmented in the late stage. The Tulahuen strain induced autoantibodies with weak beta-receptor interaction. SGO-Z12-infected mice presented lower norepinephrine levels and epinephrine levels that diminished with the evolution of the infection; lower beta-receptor affinity and an increased density; unchanged epinephrine and norepinephrine response in the early and a diminished response in the late stage; higher cAMP levels and unchanged basal contractility. The SGO-Z12 isolate induced beta-receptor autoantibodies with strong interaction with the beta-receptors. None of the antibodies, however, acted a as beta-receptor agonist. The present results demonstrate that this system is seriously compromised in the cardiac chronic stage of T. cruzi infection.

Trypanosoma cruzi: Cardiac mitochondrial alterations produced by different strains in the acute phase of the infection.

The parasite Trypanosoma cruzi is the causative agent of Chagas disease. T. cruzi invasion and replication in cardiomyocytes induce cellular injuries and cytotoxic reactions, with the production of inflammatory cytokines and nitric oxide, both source of reactive oxygen species. The myocyte response to oxidative stress involves the progression of cellular changes primarily targeting mitochondria. We studied the cardiac mitochondrial structure and the enzymatic activity of citrate synthase and respiratory chain CI-CIV complexes, in Albino Swiss mice infected with T. cruzi, Tulahuen strain and SGO Z12 isolate, in two periods of the acute infection. Changes in the mitochondrial structure were detected in both infected groups, reaching values of 71% for Tulahuen and 88% for SGO Z12 infected mice, 30 days post infection. The citrate synthase activity was different according to the evolution of the infection and the parasite strain, but the respiratory chain alterations were similar with either strain.

Trypanosoma cruzi: chemotherapeutic effects of clomipramine in mice infected with an isolate obtained from an endemic area.

The susceptibility of Trypanosoma cruzi strains to nifurtimox and benznidazole has been investigated and resistant strains have been described. Some tricyclic drugs are lethal for trypomastigote and epimastigote forms of T. cruzi (Tulahuen strain) and prevent the disease in mice. We investigated whether clomipramine, a tricyclic antidepressant drug with anti-trypanothione reductase and anti-calmodulin effects, could be effective in treating Albino Swiss mice infected with trypomastigotes of a new T. cruzi isolate from a chronic patient from an endemic area of Argentina in two different treatment schedules. Both treatment schedules were effective in reducing electrocardiographic changes and preventing myocardial structural damage. The cardiac beta-receptors low affinity was compensated for by an increment in their density. This probably maintained cardiac function since 70% of the mice survived for more than 2 years even though anti-cruzipain titers remained high. These results demonstrate that clomipramine, clinically used as a neuroleptic, could be a promising trypanocidal agent for the treatment of Chagas' disease.

Weekly electrocardiographic pattern in mice infected with two different Trypanosoma cruzi strains.

BACKGROUND: Chagas' disease, which is caused by Trypanosoma cruzi, affects 20 million people. The electrocardiographic alterations are usually the first evidence of disease progression. In this work, we evaluated if two different T. cruzi strains presented electrocardiographic and heart histopathological alterations that could be characteristic and only achieved to the parasite strain. The moment when the electric alterations began was also studied. METHODS: Albino mice (n=100) were inoculated with 50 (n=50) and 500 (n=50) trypomastigotes of T. cruzi, for Tulahuen strain and SGO-Z12 isolate, respectively. Electrocardiograms were obtained before infection and once a week from 7 to 147 days post infection (d.p.i). Dipolar and unipolar leads were analyzed. Hearts were removed by necropsy on 14, 90 and 147 d.p.i. Each heart was cut horizontally into 5-mum sections and they were stained with Hematoxilin-Eosine. RESULTS: At 147 d.p.i., 30% of Tul-infected mice were found alive, while in the SGO-Z12 infected group, 75% were alive at the same moment. The Tul-infected group showed more intraventricular blockage alterations than the other groups from 49 to 70 d.p.i, (p<0.01). No structural cardiac alterations were detected in SGO-Z12-infected mice at 7 d.p.i., while the Tul-infected group showed mononuclear cell infiltrates. At 147 d.p.i., fiber disorganization and cell infiltration were observed in the SGO-Z12 and Tul-infected groups. CONCLUSIONS: We demonstrated that T. cruzi Tulahuen strain and SGO-Z12 isolate determined different electrocardiographic alterations which were characteristic for each stage of the experimental Chagas' disease. These results highlight the importance of the T. cruzi strain in the severity of the cardiopathy.

Thioridazine treatment prevents cardiopathy in Trypanosoma cruzi infected mice.

Trypanosoma cruzi trypanothione reductase is an enzyme that has been identified as a potential target for chemotherapy. Thioridazine inhibits it and prevented cardiopathy in mice infected with T. cruzi Tulahuen strain. As not all T. cruzi strains respond to treatment in the same way, an isolate from a chronic patient (SGO Z12) was used; parasitaemias were studied along with, survival, serology, electrocardiography, histology and cardiac beta receptor function. Parasitaemia in thioridazine (80 mg/(kg day) for 3 days) treated mice was less and lasted for a shorter period (P < 0.01), there were reduced electrocardiographic and histological alterations and significantly improved survival (80% of non-treated died). Treated mice had lower receptor affinity and higher density as a compensatory mechanism, modifying the course of T. cruzi infection (SGO Z12 isolate) and preventing the consequent cardiopathy.

Electrocardiographic characterization in Trypanosoma cruzi reinfected mice.

Chagas' disease, caused by Trypanosoma cruzi, affects approximately 20 million people. There are 3 stages in the disease: acute, intermediate and chronic, the diversity and severity of the symptoms range from a mild electrocardiographic alteration to sudden death. We have previously demonstrated that when reinfections were carried out in the acute phase they produce greater cardiac damage. The aim of the present work was to investigate whether T. cruzi reinfected mice present electrocardiographic abnormalities that could be characteristic and only achieved after reinfections. Of the mice reinfected during the acute phase 100% showed abnormalities from days 90 post-infection, with a predominance of auricle ventricle blocks (67-71%). All the mice reinfected during the chronic infection showed electrocardiographic alteration after 30 days post-first reinfection. Of the mice infected, without reinfection, 60% exhibited electrocardiographic dysfunction at 90 days post-infection. Our results demonstrated that when the host was reinfected in the acute phase, more serious electrocardiographic alterations were developed than when the reinfections were carried out in the chronic stage. Sudden death described in some chagasic patients, might be related to some of the findings described here.

Trypanosoma cruzi trypanothione reductase inhibitors: phenothiazines and related compounds modify experimental Chagas' disease evolution.

Chagas' disease affects about 18 million people and 25% of the population of Latin America is at risk of acquiring Chagas' disease. The chemotherapy of Chagas' disease is still an open field and remains as an unsolved problem. Nifurtimox and benznidazole are currently used to treat this disease, however, both drugs have high toxicity and are mutagenic with the result that the patients frequently fail to follow treatment. T. cruzi enzimes such as trypanothione reductase, represent potential drug targets because they play an essential role in the life of this organism. This enzyme has been isolated, purified and studied by X ray crystallography. Phenothiazines and related compounds inhibit trypanothione reductase and a specially favoured fit is a phenothiazine with a 2- substitued with 2- chloro and 2- trifluoromethyl with a remote hydrophobic patch. The essential phenothiazine nucleus can adopt more than one inhibitory orientation in its binding site. Phenothiazines and related compounds are drugs used in psychiatric treatments. These anti-depressants inhibit trypanothione reductase through the peroxidase/ H2O2/ system, and also exert other trypanocidal effects upon epimastigotes and tripomastigotes forms: clomipramine through an anticalmodulin action; trifluopherazine and thioridazine induced disruption of mitochondria and prometazine provoked serious cell membrane disorganization. Clomipramine and thioridazine were also effective in treatment of mice with experimental Chagas' disease, significantly modifying the natural evolution of the infection; cardiac function and survival of infected and treated animals were not different from non infected animals. Phenothiazines and related compounds are promising trypanocidal agents for treatment of Chagas' disease. Other trypanocidal agents as nifurtimox, benznidazol,Allopurinol, cystein protease inhibitors and others, are also discussed.

Effects of clomipramine on Trypanosoma cruzi infection in mice.

Trypanosoma cruzi, widely distributed in Latin American countries, provokes Chagas disease, characterized by cardiomyopathy and mega-viscera. The drugs used currently for treatment of acute Chagas disease are highly toxic; the side-effects are undesirable and patients may abandon treatment. We have previously demonstrated that clomipramine (CLO) exerts trypanocidal effects upon epimastigotes and trypomastigotes in vitro with anticalmodulin activity. The present study analyses the effectiveness of CLO treatment in mice infected with a low number of T. cruzi, an animal model that reproduces acute, indeterminate and chronic phases of this trypanosomiasis. In this work, our results demonstrated that CLO 5 mg/kg daily for 30 days, or 2 doses of CLO 40 mg/kg given intraperitoneally at 1 h and 7 days after infection, was not toxic for the host, but was effective against the parasite in that parasitaemias became negative and only mild heart structural and electrocardiographic alterations were detected in the chronic phase in the group treated with CLO 5 mg/kg. In mice treated with CLO 40 mg/kg, none of these alterations was detected. Cardiac beta receptor density and affinity returned to normal in the chronic stage in both experimental groups. T. cruzi enzymes such as calmodulin and trypanothione reductase represent potential drug targets. It has been reported that both can be inhibited by CLO, a tricyclic drug used in clinical therapeutics. We have shown that CLO strongly decreased the mortality rate and electrocardiographic alterations; in addition cardiac beta receptor density and heart histology returned to, or close to, normality 135 days post infection. These results clearly demonstrated that CLO treatment modified significantly the natural evolution of T. cruzi infection.

Induction of antibodies reactive to cardiac myosin and development of heart alterations in cruzipain-immunized mice and their offspring.

Human and murine infection with Trypanosoma cruzi parasite is usually accompanied by strong humoral and cellular immune response to cruzipain, a parasite immunodominant antigen. In the present study we report that the immunization of mice with cruzipain devoid of enzymatic activity, was able to induce antibodies which bind to a 223-kDa antigen from a mouse heart extract. We identified this protein as the mouse cardiac myosin heavy chain by sequencing analysis. The study of IgG isotype profile revealed the occurrence of all IgG isotypes against cruzipain and myosin. IgG1 showed the strongest reactivity against cruzipain, whereas IgG2a was the main isotype against myosin. Anti-cruzipain antibodies purified by immunoabsorption recognized the cardiac myosin heavy chain, suggesting cross-reactive epitopes between cruzipain and myosin. Autoimmune response in mice immunized with cruzipain was associated to heart conduction disturbances. In addition, ultrastructural findings revealed severe alterations of cardiomyocytes and IgG deposit on heart tissue of immunized mice. We investigated whether antibodies induced by cruzipain transferred from immunized mothers to their offsprings could alter the heart function in the pups. All IgG isotypes against cruzipain derived from transplacental crossing were detected in pups' sera. Electrocardiographic studies performed in the offsprings born to immunized mothers revealed conduction abnormalities. These results provide strong evidence for a pathogenic role of autoimmune response induced by a purified T. cruzi antigen in the development of experimental Chagas' disease.

Immunization with the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins induces long-term duration cross-reactive antibodies with heart functional and structural alterations in young and aged mice.

The R13 peptide sequence (EEEDDDMGFGLFD) that corresponds to the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins differs from the eukariotic P concensus sequence EESDDDMGFGLFD (H13) only in a nonconservative amino acid substitution. The immunization of BALB/c mice with R13 synthetic peptide coupled to a carrier protein (OVA) induces specific (anti-R13) and autoreactive (anti-H13 and anti-heart) antibodies as well as heart functional alterations. Since aged human and experimental animals are impaired in their responses to most foreign antigens but they produce greater amounts of autoantibodies, in this work we used aged mice as an experimental model able to exaggerate the autoimmune component of the R13-induced response in case it was present. We studied whether these antibodies generated in the absence of the parasite would induce pathological changes in heart tissues. The levels of antibodies against R13 (foreign antigen) and H13 (autoantigen) studied comparatively in 2- and 12-month-old mice 10 days after the third immunization with R13 coupled to OVA were, as we expected for a foreign antigen, higher in almost all sera from 2-month-old mice tested than in sera from 12-month-old mice. Besides, these specific and cross-reactive antibody response remain elevated as long as 150 days post third immunization. In addition, the isotype pattern that recognizes R13 and the self-sequence H13 showed no differences between sera from young and aged mice. Moreover, when ECG traces were obtained from immunized mice, the heart functional alterations observed at 10 days continued at 80 and 150 days after the third immunization, showing an association with the levels of antibodies. In addition, despite the fact that the heart tissue morphology showed no alterations 10 days post third immunization, several abnormalities in the tissue architecture were revealed at 80 and 150 days post third immunization. This report demonstrates the biological relevance of R13-induced cross-reactive antibodies in some of the electrophysiologic and histological changes found in T. cruzi-infected mammalians.

Characterization of autoantibodies generated in mice by immunization with the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins.

The R13 peptide sequence (EEEDDDMGFGLFD) that corresponds to the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins differs from the eukariotic P consensus sequence EESDDDMGFGLFD (H13) only in a nonconservative amino acid substitution. Since the immunization with R13 peptide coupled to a carrier protein like OVA would break the tolerance to a self-sequence and generate autoantibodies, we characterized the antibodies induced in mice by R13 immunization, analyzing by ELISA their capacity to bind to R13 and the self-sequence H13. Besides, we studied the course of these reactivities a long time after immunization. It was found that all R13-immunized mice had antibodies against H13 and this reactivity was always lower than R13 reactivity. The anti-H13 reactivity evaluated by competitive ELISA demonstrated that the H13 peptide is able to inhibit the binding of immune sera to R13 at high doses. When the levels and the avidity of anti-R13 and anti-H13 were evaluated at 10 and 80 days post third immunization, it was observed that anti-R13 levels were higher than anti-H13 levels in all sera from 10 days after the third immunization. However, avidity of both antibodies was high. In sera from 80 days post third immunization, anti-R13 and anti-H13 levels and avidity either remained elevated or showed a rise, whereas anti-OVA levels declined. Moreover, when ECG traces were obtained from immunized mice, the heart functional alterations observed at 10 days continued at 80 days after the third immunization, showing an association with the levels of the antibodies. In addition, the isotype pattern that recognizes R13 and the self-sequence H13 is different. For anti-R13 response, IgG1 reactivity was higher than IgG2; meanwhile, for anti-H13 response IgG2 reactivity was higher than IgG1. These results indicate that sera from R13-immunized mice bind the H13 sequence and this autoreactivity may be self-perpetuating.

Thioridazine treatment modifies the evolution of Trypanosoma cruzi infection in mice.

Thioridazine, a tricyclic drug, is known to have a direct effect on Trypanosoma cruzi, disrupting the parasites' mitochondria and kinetoplasts. In the present study, the drug was used orally, at 80 mg/kg.day for 3 days, to treat mice inoculated with low numbers of T. cruzi. The drug caused no apparent toxicity in the host. It cleared trypomastigotes from the bloodstream, prevented the histological and functional alterations of the heart normally observed in the chronic phase of the experimental disease, and greatly reduced the mortality rate compared with that in untreated, infected controls. When checked 135 days post-infection, the density of cardiac beta receptors and the cardiac histology of the treated mice were indistinguishable from those of uninfected, untreated controls. The drug is already used to treat humans, as a neuroleptic drug. It appears to be able to prevent acute infection with T. cruzi evolving into chronic disease, at least in mice, and may be a useful base from which to design new agents for the treatment of Chagas disease.