Tania C. de Araújo-Jorge

Biological aspects of the DM28C clone of Trypanosoma cruzi after metacylogenesis in chemically defined media

The biological characterization of the Trypanosoma cruzi clone Dm 28c in terms of its growth in LIT medium, cell-cycle, infectivity to mice and interaction with professional and non-professional phagocytic cells shows that it behaves as a bona fide T. cruzi representant. The biological properties of this myotropic clone do not change according to the origin of the trypomastigote forms (i. e., from triatomines, infected mice, cell-culture or from the chemically defined TAUP and TAU3AAG media). In addition Dm 28c metacyclic trypomastigotes from TAU3AAG medium display a high infectivity level to fibroblasts and muscle cells. Experiments on binding of cationized ferritin to trypomastigotes surface show the existence of cap-like structures of ferritin in regions near the kinetoplast, however the nature and role of these anionic sites remain to be determined. The results indicate that metacyclic trypomastigotes from the Dm 28c clone obtained under chemically defined conditions reproduce the biological behaviour of T. cruzi, rendering this system very suitable for the study of cell-parasite interactions and for the isolation of trypanosome relevant macromolecules.

Towards a Paradigm Shift in the Treatment of Chronic Chagas Disease

ABSTRACT Treatment for Chagas disease with currently available medications is recommended universally only for acute cases (all ages) and for children up to 14 years old. The World Health Organization, however, also recommends specific antiparasite treatment for all chronic-phase Trypanosoma cruzi-infected individuals, even though in current medical practice this remains controversial, and most physicians only prescribe palliative treatment for adult Chagas patients with dilated cardiomyopathy. The present opinion, prepared by members of the NHEPACHA network (Nuevas Herramientas para el Diagnóstico y la Evaluación del Paciente con Enfermedad de Chagas/New Tools for the Diagnosis and Evaluation of Chagas Disease Patients), reviews the paradigm shift based on clinical and immunological evidence and argues in favor of antiparasitic treatment for all chronic patients. We review the tools needed to monitor therapeutic efficacy and the potential criteria for evaluation of treatment efficacy beyond parasitological cure. Etiological treatment should now be mandatory for all adult chronic Chagas disease patients.

INCREASED SUSCEPTIBILITY OF FAS LIGAND-DEFICIENT GLD MICE TO TRYPANOSOMA CRUZI INFECTION DUE TO A TH2-BIASED HOST IMMUNE RESPONSE

Infection of BALB / c mice with Trypanosoma cruzi resulted in up‐regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4+ T cells, activation‐induced CD4+ T cell death (AICD), and in Fas  :  FasL‐mediated cytotoxicity. When CD4+ T cells from infected mice were co‐cultured with T. cruzi‐infected macrophages, onset of AICD exacerbated parasite replication. CD4+ T cells from T. cruzi‐infected FasL‐deficient BALB gld / gld mice had no detectable AICD in vitro and their activation with anti‐TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld / gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild‐type mice. Secretion of Th2 cytokines IL‐10 and IL‐4 by CD4+ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti‐IL‐4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild‐type mice. These results indicate that, besides controlling CD4+ T cell AICD and parasite replication in vitro, an intact Fas  :  FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2‐type immune response to the parasite.

Pivotal role for TGF-β in infectious heart disease : The case of Trypanosoma cruzi infection and consequent Chagasic myocardiopathy

This paper summarizes recent data from the literature suggesting that transforming growth factor-beta (TGF-beta) participates at least in four different processes influencing development of myocardiopathy in Chagas disease, a major parasitic illness caused by Trypanosoma cruzi infection: (a) invasion of cardiac fibroblasts and myocytes; (b) intracellular parasite cycle; (c) regulation of inflammation and immune response; (d) fibrosis and heart remodeling during acute and chronic disease. All these effects point to an important role of TGF-beta in Chagas disease myocardiopathy and suggest that monitoring the circulating levels of this cytokine could be of help in clinical prognosis and management of patients. Moreover, TGF-beta-interfering therapies appear as interesting adjuvant interventions during acute and chronic phases of T. cruzi infection.

Implication of Transforming Growth Factor–β1 in Chagas Disease Myocardiopathy

Cardiac dysfunction with progressive fibrosis is a hallmark of Chagas disease. To evaluate the involvement of transforming growth factor (TGF)-beta1 in this disease, TGF-beta1 levels in patients were measured at 3 stages: asymptomatic indeterminate (IND), cardiac with no or slight heart dysfunction (Card 1), and cardiac with moderate or severe heart dysfunction (Card 2). All patients had significantly higher circulating levels of TGF-beta1 than did healthy persons, and 27% of patients in the Card 1 group had higher TGF-beta1 levels than did patients in the IND group. Immunohistochemical analysis of cardiac biopsy specimens showed strong fibronectin staining in the extracellular matrix and staining for phosphorylated Smad 2 (activation of the TGF-beta1 signaling pathway) in cell nuclei. The higher levels of latent TGF-beta1 observed in patients with myocardiopathy, together with intracellular activation of the TGF-beta1 pathway and tissue fibrosis, suggest that TGF-beta1 plays an important role in Chagas disease. TGF-beta1 may represent a new target for preventive and curative treatments of Chagas disease.

A comparative study of posaconazole and benznidazole in the prevention of heart damage and promotion of trypanocidal immune response in a murine model of Chagas disease

A comparative study was performed between the trypanocidal efficacy of and associated immune response to benznidazole and posaconazole in a murine model of Chagas disease. Both drugs led to 100% survival, suppression of parasitaemia and reduction of specific anti-Trypanosoma cruzi antibodies following chronic infection. All posaconazole-treated animals had negative haemocultures at 54 days post infection, whilst 50% of those treated with benznidazole had positive results. Although both drugs were effective in reducing parasitism and inflammation in the heart, posaconazole-treated animals had plasma enzymatic levels of cardiac lesion that were indistinguishable from those of uninfected mice, whilst for benznidazole the enzyme levels were significantly higher than those of uninfected controls 31 days after the start of treatment. Posaconazole was more effective than benznidazole in controlling spleen enlargement and unspecific splenocyte proliferation in the early acute phase, but allowed higher levels of activation of CD4(+) and CD8(+) T-cells in the late acute phase when the adaptive immune response takes control of the infection. These results support the notion that posaconazole could be superior to benznidazole for the treatment of T. cruzi infection in humans.

Increased Trypanosoma cruzi Invasion and Heart Fibrosis Associated with High Transforming Growth Factor β Levels in Mice Deficient in α2-Macroglobulin

ABSTRACT Trypanosoma cruzi proteinases are involved in host cell invasion in human patients and in mouse models. In mice, murine α2-macroglobulin (MAM) and murinoglobulin are circulating plasma proteinase inhibitors that also have important roles in inflammation and immune modulation. To define their role in experimental Chagas disease, we investigated the susceptibility to T. cruzi infection of mice that are deficient only in α2-macroglobulins (AM-KO) or in both MAM and monomeric murinoglobulin-1 (MM-KO), relative to the wild type (WT). Despite the high parasite load, parasitemia was lower in AM-KO and MM-KO mice than in WT mice. Nevertheless, we observed a significantly higher parasite load in the hearts of AM-KO and MM-KO mice, i.e., more amastigote nests and inflammatory infiltrates than in WT mice. This result demonstrates a protective role for MAM in the acute phase of murine T. cruzi infection. We further demonstrated in vitro that human α2-macroglobulins altered the trypomastigote morphology and motility in a dose-dependent way, and that also impaired T. cruzi invasion in cardiomyocytes. Finally, we demonstrated that the levels of transforming growth factor β in AM-KO mice increased significantly in the third week postinfection, concomitant with high amastigote burden and important fibrosis. Combined, these in vivo and in vitro findings demonstrate that the MAM contribute to the resistance of mice to acute myocarditis induced by experimental T. cruzi infection.

Enzymatic markers of heart lesion in mice infected with Trypanosoma cruzi and submitted to benznidazole chemotherapy

Abstract Creatine kinase (CK total and CK-MB) were studied as markers of lesion progression induced by Trypanosoma cruzi infection. After 3 weeks mice infected with 104 parasites showed an increase in both enzyme levels and in their frequency distribution. A trend to increase was already detected in the 2nd week. A short duration per os treatment with benznidazole (Bz) prevented the occurrence of tissue lesions, since no changes were observed in enzymes. However, in the 4th week, about 40% of Bz-treated mice showed an increase in CK-MB, as did those that survived until the 8th week. Long-term treatment with Bz in drinking water of mice infected with 102 parasites showed, after 32 weeks, a partial reversion of the occurrence of high CK-MB levels from 85.7% to 50%. We found a positive correlation between inflammatory infiltrates and CK-MB levels, indicating that this marker could be useful to monitor the occurrence of experimental chagasic myocarditis.

Benznidazole Treatment following Acute Trypanosoma cruzi Infection Triggers CD8+ T-Cell Expansion and Promotes Resistance to Reinfection

ABSTRACT Many studies have shed light on the mechanisms underlying both immunoprotection and immune dysregulation arising after Trypanosoma cruzi infection. However, little is known about the impact of benznidazole (N-benzyl-2-nitroimidazole acetamide), the drug available for clinical treatment of the infection, on the immune system in the infected host. In the present study we investigated the effect of benznidazole therapy on the lymphoid compartment during the course of experimental T. cruzi infection. Although amelioration of a variety of clinical and parasitological signs was observed in treated mice, amelioration of splenocyte expansion was not detected. Interestingly, this sustained splenomegaly observed in benznidazole-treated mice showed a preferential expansion of CD8+ T lymphocytes. Moreover, although benznidazole treatment blocked the expansion of recently activated CD4+ and CD8+ T cells seen in infected hosts, benznidazole treatment led to a selective expansion of effector and memory CD8+ T lymphocytes in association with a lower rate of apoptosis. In addition, the surviving treated animals were protected from reinfection. Together, these data suggest that, in addition to its well-known direct role in blocking parasite replication in vivo, benznidazole appears to directly affect immune regulation in T. cruzi-infected hosts.

Evidence for a perforin-mediated mechanism controlling cardiac inflammation in Trypanosoma cruzi infection

Summary.  CD8+ T lymphocytes are considered an important cell population involved in the control of parasitaemia and mortality after Trypanosoma cruzi infection. However, despite recent developments in this field, the mechanism whereby this control is exerted is still not completely understood. Here we have used perforin knockout (–/–) mice infected with Y strain T. cruzi in order to evaluate specifically the participation of the perforin‐based cytotoxic pathway in the destruction of cardiomyocytes, cellular inflammatory infiltration, and control of parasitaemia and mortality. We observed that although parasitaemia was equivalent in perforin (+/+) and (–/–) groups, survival rate and spontaneous physical performance were significantly lower in the perforin deficient mice. The cardiac inflammatory cell infiltration, mostly composed of CD8+ cells, was more evident in perforin (–/–) mice. Ultrastructural and immunofluorescence analysis, as well as plasma creatine kinase activity, revealed cardiomyocyte damage and necrosis, more evident in perforin (–/–) mice. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assays performed in heart samples revealed similar and modest levels of apoptosis in both perforin (+/+) and (–/–) mice. These results indicate that perforin does not play a pivotal role in the control of parasitaemia and direct lysis of cardiomyocytes, but seems to be an important molecule involved in the control of cardiac inflammation and pathology induced by a highly virulent strain of T. cruzi.