In vitro and in vivo drug combination for the treatment of Trypanosoma cruzi infection: A multivariate approach.

Combination therapies based on the available drugs have been proposed as promising therapeutic alternatives for many diseases. Clomipramine (CLO) has been found to modify the evolution of the experimental infection. The objective of this study was to evaluate the combined effect of benznidazole (BZ) and clomipramine (CLO) against different life-stages of Trypanosoma cruzi in vitro and their efficacy in a murine model. Life-stages of T. cruzi, BZ-partially-resistant (Y) strain, were incubated with BZ and CLO and isobolograms and combination index (CI) were obtained. Swiss mice were infected with trypomastigotes and different treatment schedules were performed, each of which consisted of 30 consecutive daily doses. Treatment efficacy was evaluated by comparing parasitemia, qPCR, survival and histological analysis. These results were analyzed using multivariate analysis to determine the combined effect of the drugs in vivo. CLO + BZ showed synergistic activity in vitro against the clinically relevant life-stages of T. cruzi. The most susceptible forms were the intracellular amastigotes (CI: 0.20), followed by trypomastigotes (CI: 0.60), with no toxicity upon mammalian cells. The combination of both drugs CLO (1.25 mg/kg) and BZ (6.25 mg/kg), in vivo, significantly diminished the parasitic load in blood and the mortality rate. CLO + BZ presented a similar inflammatory response in cardiac and skeletal muscle (amount of inflammatory cells) to BZ (6.25 mg/kg). Finally, the results from the principal component analysis reaffirmed that both drugs administered in combination presented higher activity compared with the individual administration in the acute experimental model.

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.

Chronic indeterminate phase of Chagas' disease: mitochondrial involvement in infection with two strains.

Chagasic cardiopathy has become one of the most frequent causes of heart failure and sudden death, as well as one of the most common causes of cardio-embolic stroke in Latin America. The myocyte response to oxidative stress involves the progression of cellular changes, primarily targeting the mitochondria and modifying therefore the energy supply. In this paper we analysed the effect of the infection of mice with 2 different strains of Trypanosoma cruzi (Tulahuen and SGO Z12) in the chronic indeterminate stage (75 days post-infection), upon the structure and function of cardiac mitochondria. The structural results showed that 83% of the mitochondria from the Tulahuen-infected mice presented an increase in their matrix and 91% of the mitochondria from the SGO Z12-infected group showed a reduction in their diameter (P < 0.05). When the Krebs cycle and mitochondrial respiratory chain functionality was analysed through the measurement of the citrate synthase and complexes I to IV activity, it showed that their activity was altered in all cases in a similar manner in both infected groups. In this paper we have demonstrated that the chronic indeterminate phase is not 'silent' and that cardiac mitochondria are clearly involved in the genesis and progression to the chronic chagasic cardiopathy when different factors alter the host-parasite equilibrium.

Búsqueda de marcadores de fisiopatogenia y evolución de la miocardiopatía por enfermedad de Chagas / Search markers pathophysiology and evolution of Chagas disease cardiomyopathy

Las mitocondrias son una de las organelas más importantes del miocardiocito, ya que aportan el 90% de la energía utilizada por el miocardio y la fibra muscular. La infección por T. cruzi induce la producción de mediadores proinflamatorios e inflamatorios que juegan un rol importante en modular la resistencia del parásito; estos mediadores producen aumento del estrés oxidativo generando toxicidad para los componentes celulares y para la misma organela, esto está asociado con una actividad modificada de los complejos respiratorios, lo que hace indispensable identificar marcadores de inicio y de evolución de lesiones en miocardio y en el tejido muscular, como así también en sangre, que nos permitiría con una simple muestra conocer el estado mitocondrial y predecir la evolución de la miocardiopatía, para prevenir la instalación o disminuír la gravedad de la enfermedad. Es por ello que en el presente trabajo hemos reproducido en modelos experimentales el inicio de la infección por T. cruzi y la evolución de la enfermedad en miocardio, músculo esquelético y sangre para comprobar si existen alteraciones a nivel histopatológico y ultraestructural de las mitocondrias, lo que implicaría una menor producción de energía y como consecuencia una disminución en la capacidad contráctil. Se analizaron los efectos de la infección de ratones con dos aislamientos (SGO Z12 y Lucky) y una cepa (Tulahuen) de T.cruzi. El estudio se llevó a cabo en los estadios agudo (35 días post-infección), crónico indeterminado (75 días post-infección) y crónico cardiaco (365 días post-infección) de la infección, analizando la histopatología de los tejidos, la ultraestructura mitocondrial y su función a través de la actividad enzimática de la citrato sintasa (enzima del ciclo de Krebs) y de los complejos (I-IV) de la cadena respiratoria...

Mitochondria are important organelles for myocardiocyte as provide 90% of the energy used by the myocardium and muscle fiber. T. cruzi infection induces the production of proinflammatory and inflammatory mediators that play an important role in modulating parasite resistance; these mediators cause increased oxidative stress causing cellular toxicity for the same components and organelles, this is associated with a modified activity of the respiratory complexes, which is essential to identify markers of evolution starting and myocardial injury and tissue muscle, as well as in blood, which would allow us to know a single sample mitochondrial state and predict the evolution of cardiomyopathy, to prevent installation or diminish the severity of the disease. That is why in this paper we have reproduced in experimental models starting T. cruzi infection and disease progression in myocardium, skeletal muscle and blood to check for histopathological and ultrastructural alterations of mitochondria level, would imply a lower energy output and consequently a decrease in contractility. The effects of infection of mice with two isolates (SGO Z12 and Lucky) and a strain (Tulahuen) of T. cruzi were analyzed. The study was carried out in the acute stage (35 days post-infection), indeterminate chronic (75 days post-infection) and chronic cardiac (365 days post-infection) of infection, analyzing tissue histopathology, ultrastructure mitochondrial and function through the enzymatic activity of citrate synthase (Krebs cycle) and the complexes (I-IV) of the respiratory chain...