Efficacy of novel benznidazole solutions during the experimental infection with Trypanosoma cruzi

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. About 8 million people throughout Latin America are infected causing approximately 10,000 deaths annually. Benznidazole, available as unique 100 mg tablets in many of the endemic countries, is currently the drug of choice for the specific treatment of this condition. Despite of the large number of pediatric patients infected, there are no commercial liquid dosage forms available to treat this trypanosomiasis. This work showed that novel benznidazole–water–polyethylene glycol 400 solutions are active against T. cruzi in a murine model of Chagas' disease. Present results constitute the first demonstration on the usefulness of benznidazole solutions in infected mice.     

Trypanosoma cruzi: acute and long-term infection in the vertebrate host can modify the response to benznidazole

We analyzed the influence of Trypanosoma cruzi maintenance in different hosts (dog and mouse) on its susceptibility to benznidazole treatment. Five T. cruzi stocks were isolated from dogs inoculated with Be-62 or Be-78 strain (both sensitive to benznidazole) 2-10 years ago, and the benznidazole sensitivity was then determined using the mouse as experimental model. The different T. cruzi stocks obtained from long-term infected dogs showed 50-90% drug resistance right after isolation. However, maintenance of these T. cruzi stocks in mice, by successive blood passages (2.5 years), led to either a decrease or stability of the drug resistance pattern and an increase in parasite virulence. We also demonstrated the effectiveness of the induction of parasitemia reactivation by cyclophosphamide immunosuppression in the evaluation of the response to the specific drug treatment.     

Combined treatment of heterocyclic analogues and benznidazole upon Trypanosoma cruzi in vivo

Chagas disease caused by Trypanosoma cruzi is an important cause of mortality and morbidity in Latin America but no vaccines or safe chemotherapeutic agents are available. Combined therapy is envisioned as an ideal approach since it may enhance efficacy by acting upon different cellular targets, may reduce toxicity and minimize the risk of drug resistance. Therefore, we investigated the activity of benznidazole (Bz) in combination with the diamidine prodrug DB289 and in combination with the arylimidamide DB766 upon T. cruzi infection in vivo. The oral treatment of T.cruzi-infected mice with DB289 and Benznidazole (Bz) alone reduced the number of circulating parasites compared with untreated mice by about 70% and 90%, respectively. However, the combination of these two compounds decreased the parasitemia by 99% and protected against animal mortality by 100%, but without providing a parasitological cure. When Bz (p.o) was combined with DB766 (via i.p. route), at least a 99.5% decrease in parasitemia levels was observed. DB766+Bz also provided 100% protection against mice mortality while Bz alone provided about 87% protection. This combined therapy also reduced the tissular lesions induced by T. cruzi infection: Bz alone reduced GPT and CK plasma levels by about 12% and 78% compared to untreated mice group, the combination of Bz with DB766 resulted in a reduction of GPT and CK plasma levels of 56% and 91%. Cure assessment through hemocultive and PCR approaches showed that Bz did not provide a parasitological cure, however, DB766 alone or associated with Bz cured ≥13% of surviving animals.     

Trypanosoma cruzi: effects of adrenalectomy during the acute phase of experimental infection

Glucocorticoid hormones have been implicated as an important modulator of Trypanosoma cruzi pathogenesis. Since adrenal steroid hormones play a fundamental role in modulating the immune response, we hypothesized that adrenalectomy affect the course of the experimental T. cruzi infection. This study was undertaken to determine the effects of adrenalectomy during the acute phase of T. cruzi infection. Blood and tissue parasitism, macrophages, nitric oxide (NO) production and IFN-γ were evaluated in male Wistar rats infected with the Y strain of T. cruzi. Our results show that adrenalectomized rats displayed increased number of blood and heart parasites accompanied by decreases in the total number of peritoneal macrophages and IFN-γ when compared to controls. Adrenalectomy also reduced the levels of NO released from peritoneal macrophages of infected animals. These results suggest that adrenal corticosteroid insufficiency due to adrenalectomy could be considered an important factor during development of acute phases of experimental Chagas’ disease, enhancing pathogenesis through disturbance of the host’s immune system.     

Trypanosoma cruzi: alteration in the lymphoid compartments following interruption of infection by early acute benznidazole therapy in mice

Disability and mortality as consequence of Chagas disease is enormous in South America. Recently, the success of the trypanocidal treatment with benznidazole, the only available drug, has been associated with the host immune response. In the current study, the impact of benznidazole administration immediately after the experimental infection with Trypanosoma cruzi was evaluated in the main lymphocyte populations in lymphoid organs. Untreated mice displayed enlargement of spleen and lymph node related to the increased frequency of T and B lymphocytes, respectively. An intense thymus involution with the depletion of CD4(+)CD8(+) double-positive thymocytes also occurred. Benznidazole treatment led to a partial reversion of the spleen and lymph node enlargement related to changes in the frequency of lymphocyte subsets due to infection. Prevention of thymus involution was achieved, with the profile of thymocyte subsets similar to that of non-infected mice. The parasitic load at the onset of T. cruzi infection seems critical to trigger immune system activation.     

Cell-mediated immunity in experimental Trypanosoma cruzi infection

Infection of humans with the protozoan Trypanosoma cruzi leads to Chagas disease, or American trypanosomiasis, a disease that affects nearly 20 million people, and constitutes one of the largest socioeconomic burdens in Latin America. Much of the present knowledge on pathogenic mechanisms underlying T. cruzi infection comes from experimental murine models. Here, George A. DosReis reviews recent findings about the features of host cell-mediated immunity against the parasite and possible mechanisms leading to chronic infection.     

Role of dystrophin in acute Trypanosoma cruzi infection

Previous studies have demonstrated loss/reduction of dystrophin in cardiomyocytes in both acute and chronic stages of experimental Trypanosoma cruzi (T. cruzi) infection in mice. The mechanisms responsible for dystrophin disruption in the hearts of mice acutely infected with T. cruzi are not completely understood. The present in vivo and in vitro studies were undertaken to evaluate the role of inflammation in dystrophin disruption and its correlation with the high mortality rate during acute infection. C57BL/6 mice were infected with T. cruzi and killed 14, 20 and 26 days post infection (dpi). The intensity of inflammation, cardiac expression of dystrophin, calpain-1, NF-κB, TNF-α, and sarcolemmal permeability were evaluated. Cultured neonatal murine cardiomyocytes were incubated with serum, collected at the peak of cytokine production and free of parasites, from T. cruzi-infected mice and dystrophin, calpain-1, and NF-κB expression analyzed. Dystrophin disruption occurs at the peak of mortality and inflammation and is associated with increased expression of calpain-1, TNF-α, NF-κB, and increased sarcolemmal permeability in the heart of T. cruzi-infected mice at 20 dpi confirmed by in vitro studies. The peak of mortality occurred only when significant loss of dystrophin in the hearts of infected animals occurred, highlighting the correlation between inflammation, dystrophin loss and mortality.     

Trypanosoma cruzi: allopurinol in the treatment of mice with experimental acute Chagas disease

The therapeutic effect of allopurinol was studied in an experimental Trypanosoma cruzi infection (Chagas disease) in outbred IVIC-NMRI and inbred C57B1/6J mice intraperitoneally inoculated with the parasites 2–6 days before drug treatment. Allopurinol protected against T. cruzi infection. This effect was evidenced by highly significant reductions in both parasitemias and mortality rates and increased survival time in allopurinol-treated animals compared with untreated infected mice. Allopurinol protected effectively when administered in 10 daily doses of 32–64 mg/kg body wt/day injected intraperitoneally. Using direct methods, parasitemia remained undetectable for at least 310 days. An indirect method, subinoculation to susceptible mice, showed a few circulating trypanosomes which decreased greatly in number after a second schedule of allopurinol treatment; finally no trypanosomes were detectable 275 days after treatment initiation. Allopurinol also induced a strong trypanostatic effect when tested in vitro on five different Trypanosoma cruzi strains (optimal inhibitory concentration: 3 μg/ml). These results suggest that allopurinol protects mice with acute Chagas infection by a direct trypanostatic effect. The low toxicity of this drug suggests its use in more chronic experimental Chagas infections.     

Inducible cyclooxygenase released prostaglandin mediates immunosuppression in acute phase of experimental Trypanosoma cruzi infection

We investigated the possible role of prostaglandins produced by COX-2 in the immunosuppression observed during Trypanosoma cruzi infection. Con-A-stimulated splenocytes isolated from mice on days 5, 10, and 15 of infection released large amounts of PGE2 and this release was inhibited by the treatment of animals with sodium salicylate or meloxicam. The treatment of the animals with these drugs enhanced the release of IL-2 by splenocytes from T. cruzi-infected animals and significantly reduced the blood parasitemia and delayed the mortality of the infected mice. Furthermore, the release of TNF-alpha, IFN-gamma, IL-4, and IL-10 by Con-A-stimulated splenocytes obtained from infected mice on days 5, 10, and 15 of the infection was significantly inhibited by treatment of the animals with salicylate or meloxicam. In conclusion, the results suggest that the prostaglandins produced mainly by COX-2 mediate the immunosuppression observed in the acute phase of T. cruzi infection.     

CD8low T cells expanded following acute Trypanosoma cruzi infection and benznidazole treatment are a relevant subset of IFN-γ producers

CD8 T cells are regarded as pivotal players in both immunoprotection and immunopathology following Trypanosoma cruzi infection. Previously, we demonstrated the expansion of CD8⁺ T lymphocytes in the spleen of T. cruzi-infected mice under treatment with benznidazole (N-benzyl-2-nitroimidazole acetamide; Bz), a drug available for clinical therapy. This finding underlies the concept that the beneficial effects of Bz on controlling acute T. cruzi infection are related to a synergistic process between intrinsic trypanocidal effect and indirect triggering of the active immune response. In the present study, we particularly investigated the effect of Bz treatment on the CD8⁺ T cell subset following T. cruzi infection. Herein we demonstrated that, during acute T. cruzi infection, Bz treatment reduces and abbreviates the parasitemia, but maintains elevated expansion of CD8⁺ T cells. Within this subset, a remarkable group of CD8^low cells was found in both Bz-treated and non-treated infected mice. In Bz-treated mice, early pathogen control paralleled the lower frequency of recently activated CD8^low cells, as ascertained by CD69 expression. However, the CD8^low subset sustains significant levels of CD44^highCD62L^low and CD62L^lowT-bet^high effector memory T cells, in both Bz-treated and non-treated infected mice. These CD8^low cells also comprise the main group of spontaneous interferon (IFN)-γ-producing CD8⁺ T cells. Interestingly, following in vitro anti-CD3/CD28 stimulation, CD8⁺ T cells from Bz-treated T. cruzi-infected mice exhibited higher frequency of IFN-γ⁺ cells, which bear mostly a CD8^low phenotype. Altogether, our results point to the marked presence of CD8^low T cells that arise during acute T. cruzi infection, with Bz treatment promoting their significant expansion along with a potential effector program for high IFN-γ production.     

Trypanosoma cruzi: Immunological predictors of benznidazole efficacy during experimental infection

C3H/HeN male mice were infected with a lethal population of Trypanosoma cruzi and treated with benznidazole (Bz). Parasitemia, body weight and survival rate were registered during the therapy with significant improvement for T. cruzi-infected Bz-treated animals. Besides, flow cytometry resulted a useful method to discriminate between cured animals from those not cured by monitoring IgG₁ bound to live trypomastigotes levels. At the end of Bz therapy, the LT splenocyte compartment was studied for activation/memory cell surface markers ( and ). Cytofluorometric analysis showed that T. cruzi-infected untreated mice increased their activated LT numbers and this effect was completely abolished only in cured mice at the end of Bz administration. The same behavior was observed for the memory LT subpopulation correlating to an effector memory () displayed by T. cruzi infection. Bz treatment was able to modulate the immunological response by reducing the deleterious effect of the acute phase in all T. cruzi-infected mice.     

Trypanosoma cruzi: the effects of zinc supplementation during experimental infection

It is well recognized that zinc is an essential trace element, influencing growth and affecting the development and integrity of the immune system. The use of oligoelements as zinc can be considered a tool in modulating the effectiveness of the immune response. In this work zinc was daily and orally supplied in male Wistar rats infected with the Y strain of Trypanosoma cruzi. Parasitemia was evaluated and a significant reduction on blood parasites was observed. In order to check some immunological parameters peritoneal macrophages were counted revealing higher percentages for zinc supplied group. Consequently enhanced concentrations of IFN-gamma was found and for the first time NO was evaluated in T. cruzi infected animals under the influence of zinc therapy, revealing enhanced concentrations when compared to unsupplied counterparts. We conclude that zinc is able to up-regulate the host's immune response against parasite replication.     

Absence of Bim sensitizes mice to experimental Trypanosoma cruzi infection

Chagas disease is a life-threatening disorder caused by the protozoan parasite Trypanosoma cruzi. Parasite-specific antibodies, CD8⁺ T cells, as well as IFN-γ and nitric oxide (NO) are key elements of the adaptive and innate immunity against the extracellular and intracellular forms of the parasite. Bim is a potent pro-apoptotic member of the Bcl-2 family implicated in different aspects of the immune regulation, such as negative selection of self-reactive thymocytes and elimination of antigen-specific T cells at the end of an immune response. Interestingly, the role of Bim during infections remains largely unidentified. To explore the role of Bim in Chagas disease, we infected WT, Bim^+/−, Bim^−/− mice with trypomastigotes forms of the Y strain of T. cruzi. Strikingly, our data revealed that Bim^−/− mice exhibit a delay in the development of parasitemia followed by a deficiency in the control of parasite load in the bloodstream and a decreased survival compared to WT and Bim^+/− mice. At the peak of parasitemia, peritoneal macrophages of Bim^−/− mice exhibit decreased NO production, which correlated with a decrease in the pro-inflammatory Small Peritoneal Macrophage (SPM) subset. A similar reduction in NO secretion, as well as in the pro-inflammatory cytokines IFN-γ and IL-6, was also observed in Bim^−/− splenocytes. Moreover, an impaired anti-T. cruzi CD8⁺ T-cell response was found in Bim^−/− mice at this time point. Taken together, our results suggest that these alterations may contribute to the establishment of a delayed yet enlarged parasitic load observed at day 9 after infection of Bim^−/− mice and place Bim as an important protein in the control of T. cruzi infections.Subject terms: Cell death and immune response, Infectious diseases     

Isolation of cardiac mast cells in experimental Trypanosoma cruzi infection

Mast cells (MC) secrete diverse pre-stored chemical mediators that are pivotal in inflammatory and fibrotic etiologies, such as Trypanosoma cruzi-induced myocardiopathy. However, due to reduced number of cardiac MC, in situ and in vitro identification, and difficult tissue isolation, these cells are rarely addressed. In this work we optimized the identification of cardiac and peritoneal MC and developed an enzymatic method for MC isolation using control and T. cruzi-infected mice. MC were identified by: toluidine blue (TB); alcian blue (AB)/safranin (S); AB or a mixed solution composed by AB/S/TB. Previous evaluations of cardiac MC in T. cruzi infection were based on TB staining and our results using AB/S/TB solution showed an increase in, at least, five times the detection of MC. This mixed solution may improve the identification of MC populations also from skin, mucosa and tissues that are infected by other pathogens or under the influence of chronic inflammation, leading to more precise results. Furthermore, the appropriate combination of samples (frozen/unfixed/thick slices) and staining protocols can assure the best evaluation of MC. We have also isolated cardiac MC using collagenase and developed a highly efficient 60%/70% Percoll-graded protocol that enriched in, at least, 95% the population of cardiac MC.     

Trypanosoma cruzi: Induction of benznidazole resistance in vivo and its modulation by in vitro culturing and mice infection

Through a continuous in vivo drug pressure protocol, using mice as experimental model, we induced benznidazole resistance in Trypanosoma cruzi stocks. Full resistance was obtained for four out of five T. cruzi stocks analyzed. However, the number of benznidazole doses (40–180), as well as the time (4–18 months) necessary to induce resistance varied among the different T. cruzi stocks. The resistance phenotype remained stable after T. cruzi stocks has been maintained by 12 passages in mice (six months) and in acellular culture for the same time. However, the maintenance of resistant parasite for 12 months in acellular culture induces a reduction in its level of benznidazole resistance, while no alteration was detected in parasite maintained for the same time in mice. The data showed the stability of the resistance acquired by drug pressure, but suggest the possibility of reversible changes in the resistance levels after maintenance for long time in acellular culture.     

Trypanosoma cruzi: the role of PGE2 in immune response during the acute phase of experimental infection

Chagas disease is characterized by cardiac lesions and a high level of PGE2. Our objective was to investigate the role of PGE2 in cardiac lesions. BALB/c mice were infected with Trypanosoma cruzi (1x10(3) trypomastigote forms) and were treated daily with PBS, meloxicam (0.5 mg/kg) or etoricoxib (0.6 mg/kg). The animals were sacrificed on the 21st day of infection and we collected the cardiac tissue and spleen cells for tissue culture. We observed that treatment with COX-2 inhibitors was able to decrease synthesis of PGE2 by spleen cells. This reduction was accompanied by reduction of the inflammatory infiltrate, parasite nets, cardiac fibrosis and fewer COX-2 positive cells in cardiac tissue obtained from these animals. In conclusion, treatment with COX-2 inhibitors, and consequent inhibition of PGE2 synthesis, was able to reduce the cardiac damage observed during the acute phase of experimental Chagas disease, thus demonstrating the involvement of this mediator in the cardiac lesion.     

Myeloid-derived suppressor cells infiltrate the heart in acute Trypanosoma cruzi infection

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects several million people in Latin America. Myocarditis, observed in the acute and chronic phases of the disease, is characterized by a mononuclear cell inflammatory infiltrate. We previously identified a myeloid cell population in the inflammatory heart infiltrate of infected mice that expressed arginase I. In this study, we purified CD11b(+) myeloid cells from the heart and analyzed their phenotype and function. Those CD11b(+) cells were ～70% Ly6G(-)Ly6C(+) and 25% Ly6G(+)Ly6C(+). Moreover, purified CD11b(+)Ly6G(-) cells, but not Ly6G(+) cells, showed a predominant monocytic phenotype, expressed arginase I and inducible NO synthase, and suppressed anti-CD3/anti-CD28 Ab-induced T cell proliferation in vitro by an NO-dependent mechanism, activity that best defines myeloid-derived suppressor cells (MDSCs). Contrarily, CD11b(+)Ly6G(+) cells, but not CD11b(+)Ly6G(-) cells, expressed S100A8 and S100A9, proteins known to promote recruitment and differentiation of MDSCs. Together, our results suggest that inducible NO synthase/arginase I-expressing CD11b(+)Ly6G(-) myeloid cells in the hearts of T. cruzi-infected mice are MDSCs. Finally, we found plasma l-arginine depletion in the acute phase of infection that was coincident in time with the appearance of MDSCs, suggesting that in vivo arginase I could be contributing to l-arginine depletion and systemic immunosuppression. Notably, l-arginine supplementation decreased heart tissue parasite load, suggesting that sustained arginase expression through the acute infection is detrimental for the host. This is, to our knowledge, the first time that MDSCs have been found in the heart in the context of myocarditis and also in infection by T. cruzi.