Differential tissue distribution of discrete typing units after drug combination therapy in experimental Trypanosoma cruzi mixed infection.

The aim of the present work was to evaluate the distribution of the different clones of the parasite prevailing after treatment with benznidazole (BZ) and clomipramine (CLO), in mice infected with Trypanosoma cruzi, Casibla isolate which consists of a mixture of two discrete typing units (DTUs). Albino Swiss mice were infected and treated with high and low concentrations of BZ (100 or 6.25 mg/kg), CLO (5 or 1.25 mg/kg), or the combination of both low doses (BZ6.25 + CLO1.25), during the acute phase of experimental infection. Treatment efficacy was evaluated by comparing parasitaemia, survival and tissular parasite presence. For DTUs genotyping, blood, skeletal and cardiac muscle samples were analysed by multiplex quantitative polymerase chain reaction. The combined treatment had similar outcomes to BZ6.25; BZ100 was the most effective treatment, but it failed to reach parasite clearance and produced greater histological alterations. Non-treated mice and the ones treated with monotherapies showed both DTUs while BZ6.25 + CLO1.25 treated mice showed only TcVI parasites in all the tissues studied. These findings suggest that the treatment may modify the distribution of infecting DTUs in host tissues. Coinfection with T. cruzi clones belonging to different DTUs reveals a complex scenario for the treatment of Chagas disease and search for new therapies.

Analysis of mitochondrial enzymatic activity in blood lymphomonocyte fractions during infection with different Trypanosoma cruzi strains.

Proinflammatory and inflammatory mediators induced by Trypanosoma cruzi infection increase the oxidative stress, generating toxicity for cells targeting mitochondria of different tissues. We studied the activity of citrate synthase and complexes I-IV of respiratory chain in mitochondria of blood lymphomonocyte fraction, from albino Swiss mice infected with different isolates of T. cruzi , during Chagas disease evolution. Complexes I-IV were modified in infected groups (p<0.05) in all the stages, and an inflammatory process of different magnitudes was detected in the heart and skeletal muscle according to the isolate. The citrate synthase activity presented modifications in the SGO Z12 and the Tulahuen group (p<0.05). Hearts showed fiber fragmentation and fibrosis; skeletal muscle presented inflammatory infiltrates and in the Tulahuen infected group, there were also amastigote nests. The inflammatory processes produced an oxidative stress that induced different alterations of mitochondrial enzymes activities in the lymphomonocyte fraction that can be detected by a simple blood extraction, suggesting that they could be used as disease markers, especially in the indeterminate phase of Chagas disease.

Differential tissue distribution of Trypanosoma cruzi during acute experimental infection: Further evidence using natural isolates.

In the present work, we evaluated the effect of mixed Trypanosoma cruzi infections, studying the biological distribution of the different parasites in blood, heart and skeletal muscle during the acute phase. Albino Swiss mice were infected with different parasite strain/isolates or with a combination of them. The parasites in the different tissues were typified through specific PCR, population variability was analyzed through RFLP studies and parasitological and histopathological parameters were evaluated. We found a predominance of TcII and TcVI in all tissues samples respect to TcV and different parasite populations were found in circulation and in the tissues from the same host. These results verify the distribution of parasites in host tissues from early stages of infection and show biological interactions among different genotypes and populations of T. cruzi.

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.

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.

Functional and structural alterations of cardiac and skeletal muscle mitochondria in heart failure patients.

BACKGROUND AND AIMS: The fundamental mechanisms involved in the genesis and progression of heart failure are not clearly understood. The present study was conducted to analyze the cardiac mitochondrial involvement in heart failure, the possible parallelism between cardiac and skeletal muscle and if there is a link between clinical symptoms and mitochondrial damage. METHODS: Left ventricle and pectoral biopsies were obtained from patients with heart failure (n: 21) and patients with inter-auricular communication as the unique diagnosis for surgery (n: 6). Mitochondria were isolated from these tissues and studied through electron microscopy, spectrophotometry to measure the activity of respiratory complex III and immunohistochemistry to determine the presence of reactive oxygen species. RESULTS: More than 90% of cardiac and skeletal muscle mitochondria presented structural and functional alterations in relation to an increment in the reactive oxygen species production, even in patients without the presence of any clinical Framingham criteria. CONCLUSIONS: We demonstrated some parallelism between cardiac and skeletal muscle mitochondrial alterations in patients with heart failure and that these alterations begin before the major clinical Framingham criteria are installed, pointing to mitochondria as one of the possibly responsible factors for the evolution of cardiac disease.

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.