Alejandra Baez

Trypanothione reductase inhibitors: Overview of the action of thioridazine in different stages of Chagas disease.

Thioridazine (TDZ) is a phenothiazine that has been shown to be one of the most potent phenothiazines to inhibit trypanothione reductase irreversibly. Trypanothione reductase is an essential enzyme for the survival of Trypanosoma cruzi in the host. Here, we reviewed the use of this drug for the treatment of T. cruzi experimental infection. In our laboratory, we have studied the effect of TDZ for the treatment of mice infected with different strains of T. cruzi and treated in the acute or in the chronic phases of the experimental infection, using two different schedules: TDZ at a dose of 80 mg/kg/day, for 3 days starting 1h after infection (acute phase), or TDZ 80 mg/kg/day for 12 days starting 180 days post infection (d.p.i.) (chronic phase). In our experience, the treatment of infected mice, in the acute or in the chronic phases of the infection, with TDZ led to a large reduction in the mortality rates and in the cardiac histological and electrocardiographical abnormalities, and modified the natural evolution of the experimental infection. These analyses reinforce the importance of treatment in the chronic phase to decrease, retard or stop the evolution to chagasic myocardiopathy. Other evidence leading to the use of this drug as a potential chemotherapeutic agent for Chagas disease treatment is also revised.

Clomipramine and benznidazole association for the treatment of acute experimental Trypanosoma cruzi infection.

Alternative strategies are being designed to identify candidates among drugs already available on the market that could be used in combination to improve the efficacy of Chagas disease treatment. This work evaluates the effect of the association of clomipramine (CLO) with benznidazole (BZN) for the treatment of experimental Chagas disease in the acute stage, in Swiss albino mice infected with Trypanosoma cruzi Tulahuen strain. Infected mice were treated with CLO 5mg/kg/day and BZN 50 and 100mg/kg/day, each separately or together. Efficacy of the treatment was evaluated through parasitemia, survival, electrocardiography, histopathological studies, serological and PCR assays at 90 days post-infection (dpi). All treatments significantly (P<0.05) reduced mortality and decreased parasitemia. Histopathological analysis of liver and kidneys of mice treated with CLO and the drug combination showed less injury than mice treated only with BZN. The lower dose of BZN (50mg/kg/day) combined with CLO showed the same efficacy as the habitual dose of BZN (100mg/kg/day) combined with CLO. The therapeutic results from the combination of BZN with CLO presented lesser side effects than the treatment with BZN.

Use of clomipramine as chemotherapy of the chronic phase of Chagas disease

Chagas infection is a major endemic disease affecting Latin American countries. The persistence of Trypanosoma cruzi generates a chronic inflammatory reactivity that induces an immune response directed to the hosts tissues. The effectiveness of the treatment in the chronic phase is still unsatisfactory due, amongst other reasons, to the collateral effects of the drugs used. We investigated the effect of clomipramine, a tricyclic antidepressant that, when used as a treatment of T. cruzi-chronically infected mice, inhibits trypanothione reductase, an exclusive and vital enzyme of T. cruzi. Clomipramine improved survival (P<0.05) by diminishing the parasite intensity as demonstrated by PCR studies in the heart and skeletal muscle, and significantly prevented the evolution to fibrosis of the inflammatory infiltrates. Clomipramine could be a good candidate for the treatment of chronic Chagas disease.

Association of clomipramine and allopurinol for the treatment of the experimental infection with Trypanosoma cruzi

We have previously shown that clomipramine and allopurinol used separately are effective in preventing chronic chagasic cardiomyopathy. The aim of the present study was to evaluate the effect of the association of clomipramine (Clo—5 mg/kg/day/90 days) and allopurinol (Allo—5, 10, or 15 mg/kg/day/90 days) for the treatment of experimental Chagas disease in the acute stage. Treatment effectiveness was evaluated through parasitemia, survival, electrocardiography, serology, and cardiac histopathology. Groups treated showed no electrocardiographic abnormalities, in contrast to those untreated which presented 25% of mice with conduction alterations. The myocardium of treated mice (Clo, Allo10+Clo, and Allo15+Clo) presented no structural alterations. Cardiac b-receptor affinity was preserved in mice treated with Clo or Clo+Allo at the different doses; receptor density of the Clo and Allo15+Clo groups did not differ from the non-infected group. Anti-cruzipain antibody levels were similar in treated and untreated groups. Survival was significantly increased in the treated groups (p < 0.05), with Clo and all the Clo+Allo groups presenting the highest rates. These results show that the association of clomipramine + allopurinol is effective for Chagas disease treatment and has the same effect as clomipramine alone.

Mitochondrial involvement in chronic chagasic cardiomyopathy

The pathogenesis of chronic chagasic cardiopathy is still under discussion; there is considerable evidence that inflammatory infiltrates and their mediators have a direct effect on cardiac cells. Here we studied the structure and function of cardiac mitochondria in chronic chagasic myocardiopathy. Cardiac mitochondrial structure and enzyme activity of citrate synthase and complexes I to IV of the respiratory chain were studied in albino Swiss mice infected with Trypanosoma cruzi (Tulahuen strain or SGO Z12 isolate) on 365 days post-infection (dpi). The presence of parasites in cardiac and skeletal muscle was also investigated. The activity of complexes I to IV was altered in different ways, according to the strain employed (P<0.0001), in relation to the cristae disorganisation and the parasite persistence found in the Tulahuen group, and the chronic inflammatory process described in both groups; citrate synthase activity also increased in both infected groups. Changes in mitochondrial structure were detected in 89% of Tulahuen- and 58% of SGO Z12-infected mice. In this paper we demonstrate that parasite persistence and inflammation are likely to be involved in the structural and functional alterations in cardiac mitochondria from chronically T. cruzi-infected mice, demonstrating that the parasite strain determines different mitochondrial changes in chagasic cardiopathy.

Circulating Trypanosoma cruzi populations differ from those found in the tissues of the same host during acute experimental infection.

We evaluated the presence and distribution of two Trypanosoma cruzi natural isolates in blood, heart, skeletal muscle, liver, and spleen tissues in the acute phase of the experimental infection (35 days postinfection) in order to determine if the populations present in blood were different to those found in the tissues of the same host. Thirty mice were infected with 50 forms of each isolate or with a combination of them. Presence and molecular characterization of the parasites in the host tissues were determined by specific PCR. Cardiac and skeletal muscle alterations were analyzed by histological studies. T. cruzi variability in the host tissues was analyzed through RFLP studies. Both isolates used consisted of a mixture of two T. cruzi lineages. Specific PCRs were positive for most of the samples from the 3 groups analyzed. Cardiac and skeletal muscle sections from the groups infected with one isolate presented mild to moderate inflammatory infiltrates; the group infected with both isolates showed severe inflammatory infiltrates and the presence of amastigote nests in both tissues. Different parasite populations were found in circulation and in the tissues from the same host. These results are important for patients with high probability of mixed infections in endemic areas and contribute to the knowledge of parasite/host interactions.

Mitochondrial dysfunction in skeletal muscle during experimental Chagas disease.

Trypanosoma cruzi invasion and replication in cardiomyocytes and other tissues induce cellular injuries and cytotoxic reactions, with the production of inflammatory cytokines and nitric oxide, both sources of reactive oxygen species. The myocyte response to oxidative stress involves the progression of cellular changes primarily targeting mitochondria. Similar alterations could be taking place in mitochondria from the skeletal muscle; if that is the case, a simple skeletal muscle biopsy would give information about the cardiac energetic production that could be used as a predictor of the chagasic cardiopathy evolution. Therefore, in the present paper we studied skeletal muscle mitochondrial structure and the enzymatic activity of citrate synthase and respiratory chain complexes I to IV (CI-CIV), in Albino Swiss mice infected with T. cruzi, Tulahuen strain and SGO Z12 and Lucky isolates, along the infection. Changes in the mitochondrial structure were detected in 100% of the mitochondria analyzed from the infected groups: they all presented at least 1 significant abnormality such as increase in their matrix or disorganization of their cristae, which are probably related to the enzymatic dysfunction. When we studied the Krebs cycle functionality through the measurement of the specific citrate synthase activity, we found it to be significantly diminished during the acute phase of the infection in Tulahuen and SGO Z12 infected groups with respect to the control one; citrate synthase activity from the Lucky group was significantly increased (p<0.05). The activity of this enzyme was reduced in all the infected groups during the chronic asymptomatic phase (p<0.001) and return to normal values (Tulahuen and SGO Z12) or increased its activity (Lucky) by day 365 post-infection (p.i.). When the mitochondrial respiratory chain was analyzed from the acute to the chronic phase of the infection through the measurement of the activity of complexes I to IV, the activity of CI remained similar to control in Tulahuen and Lucky groups, but was significantly augmented in the SGO Z12 one in the acute and chronic phases (p<0.05). CII increased its activity in Tulahuen and Lucky groups by day 75 p.i. and in SGO Z12 by day 365 p.i. (p<0.05). CIII showed a similar behavior in the 3 infected groups, remaining similar to control values in the first two stages of the infection and significantly increasing later on (p<0.0001). CIV showed an increase in its activity in Lucky throughout all stages of infection (p<0.0001) and an increase in Tulahuen by day 365days p.i. (p<0.0001); SGO Z12 on the other hand, showed a decreased CIV activity at the same time. The structural changes in skeletal muscle mitochondria and their altered enzyme activity began in the acute phase of infection, probably modifying the ability of mitochondria to generate energy; these changes were not compensated in the rest of the phases of the infection. Chagas is a systemic disease, which produces not only heart damage but also permanent skeletal muscle alterations.

Quantitative PCR and unconventional serological methods to evaluate clomipramine treatment effectiveness in experimental Trypanosoma cruzi infection

Clomipramine (CLO), a tricyclic antidepressant drug, has been used for the treatment of mice infected with Trypanosoma cruzi. In this work we evaluated the effectiveness of CLO treatment upon T. cruzi-infected mice in the chronic phase of the experimental infection using Quantitative polymerase chain reaction (qPCR) and recombinant ELISA. Sixty Swiss albino mice were inoculated with 50 trypomastigote forms of T. cruzi (Tulahuen strain). CLO treatment consisted of 5mg/kg/day during 60days by intraperitoneal injection, beginning on day 90 post infection (p.i) when the mice presented electrocardiographic (ECG) alterations compatible with the chronic phase of the disease. The evolution of experimental infection and the treatment efficacy were studied through survival, electrocardiography, serology using a mixture and individual (1, 2, 13, 30, 36 and SAPA) recombinant proteins from epimastigotes and trypomastigotes of T. cruzi; and qPCR on days 180 and 270 p.i. CLO treatment in the chronic phase decreased the parasite load, reduced the levels of antibodies against antigen 13 throughout 270days p.i and reversed the ECG abnormalities in the treated animals, from 100% of the mice with alterations at the beginning of the treatment to only 20% of the mice with alterations by day 270 p.i. This study shows that qPCR and the use of recombinant antigens are more sensitive to evaluate the effectiveness of the treatment and proves that clomipramine may be considered as a new chemotherapy for the chronic phase of the disease.