Release of nitric oxide during the experimental infection with Trypanosoma cruzi

We analysed the production of nitric oxide (NO) intermediates by cells from BALB/c mice infected with either virulent (Tulahuén or RA) or avirulent (CA-1) strains of Trypanosoma cruzi. Peritoneal or spleen cells from mice infected with T. cruzi released NO when incubated without further stimuli. Cells from mice during the acute stage of infection accumulated higher levels of inducible NO synthase mRNA and produced both, before and after lypopolysaccharide stimulation, higher amounts of NO than cells from mice chronically infected with T. cruzi. NO synthesis showed similar kinetics in connection with all three strains ofT. cruzi, but cells from mice inbred with the Tulahuén or RA strains released higher levels of IFN-γ, an activator of the NO pathways, than cells from mice infected with the CA-1 strain. In vivo administration of L-Ng-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase, increased the susceptibility of mice to T. cruzi. We conclude that infection with T. cruzi induces NO production, and suggest that NO plays a role in the resistance against the parasite.     

Immunoregulatory actions of melatonin and zinc during chronic Trypanosoma cruzi infection

After one century of the discovery of Chagas' disease and the development of an efficient drug with amplitude of actions both in the acute and chronic phase is still a challenge. Alternative immune modulators have been exhaustively used. For that purpose, melatonin and zinc were administered during chronic Trypanosoma cruzi‐infected Wistar rats and several endpoints were assessed. Melatonin has a remarkable functional versatility, being associated with important antioxidant, anti‐inflammatory, and anti‐apoptotic effects. The cross‐talk between zinc and the immune system includes its ability to influence the production and signaling of numerous inflammatory cytokines in a variety of cell types. Our study showed that zinc triggered a decrease in the generation of IFN‐γ for TCD4⁺ cells. Reduced percentage of CD4⁺T cells producing TNF‐α was observed in control melatonin or zinc‐and‐melatonin‐treated animals as compared with untreated rats. On the other hand, a significant increase in the percentage of IL‐4 from CD4⁺ and CD8⁺ T lymphocytes producers was observed 60 days after infection, for all zinc‐treated animals, whether infected or not. Melatonin and zinc therapies increased the percentages of CD4⁺ and CD8⁺ T lymphocytes IL‐10 producers. CD4⁺CD25^highFoxp3⁺ T cells were also elevated in zinc‐ and melatonin‐treated animals. The modulation of the immune system influenced by these molecules affected cytokine production and the inflammatory process during chronic T. cruzi infection. Elucidation of the interplay between cytokine balance and the pathogenesis of Chagas’ disease is extremely relevant not only for the comprehension of the immune mechanisms and clinical forms but, most importantly, also for the implementation of efficient and adequate therapies.     

Interleukin‐17, oxidative stress,and inflammation: role of melatonin during Trypanosoma cruzi infection

Although the exact etiology of Chagas' disease remains unknown, the inflammatory process and oxidative stress are believed to be the main contributors to the dysfunction and pathogenesis during chronic Trypanosoma cruzi infection. Our hypothesis is that melatonin administered for 2 months daily could modulate the oxidative stress and the inflammatory response during the chronic infection. Flow cytometric analysis of macrophages and antigen‐presenting cells (APC), expression of RT1B as well as LFA‐1 and MCP‐1 in CD4⁺ and CD8⁺T cells and levels of interleukin‐17A were assessed. The oxidative stress was evaluated through lipid peroxidation (LPO) analysis on the plasma of thiobarbituric acid‐reactive substances (TBARS) and nitric oxide production. Decreased concentrations of nitrite and TBARS were found in infected and melatonin‐treated animals, as well as a rising trend in the production of IL‐17A as compared to infected and untreated counterparts. A significant decrease was found in the percentages of CD4⁺ and CD8⁺T lymphocytes MCP‐1 producers for infected and melatonin‐treated rats. Reduced percentage of CD8⁺T cells producing LFA‐1 was observed in control and melatonin‐treated animals as compared to untreated rats. The cellular response of peritoneal APC cells and macrophages significantly dropped in infected and treated animals. As an endpoint, the use of antioxidant compounds such as melatonin emerges as a new and promising approach to control the oxidative stress during the chronic Chagas' disease partially mediated through the abrogation of LPO and the prevention of the inflammatory response and can be used for further investigation on treatment trials for other infectious diseases.     

Effects of melatonin on thymic and oxidative stress dysfunctions during Trypanosoma cruzi infection

Although the exact etiology of Chagas disease is not completely elucidated, thymic atrophy and oxidative stress are believed to be important contributors to the pathogenesis during acute Trypanosoma cruzi (T. cruzi ) infection. We hypothesized that exogenous melatonin, administered by gavage (5 mg/kg, p.o., gavage) to young (5 weeks old) and middle‐aged (18 months old) male Wistar rats, would modulate thymic oxidative damage and reverse the age‐related thymus regression during T. cruzi acute infection. Increased levels of superoxide anion (O₂^?) were detected in the thymus of infected animals, and treatment with melatonin reverted this response. We found reduced TBARS levels as well as a significant increase in superoxide dismutase (SOD ) activity in the thymus of all middle‐aged melatonin‐treated animals, infected or not with T. cruzi . Furthermore, melatonin increased the thymic expression of SOD 1 and SOD 2 in middle‐aged control animals. Nox2 expression was not affected by melatonin treatment in young or middle‐aged animals. Melatonin reverted the age‐related thymic regression as revealed by the increase in thymus weight, total number of thymocytes, and reduction in age‐related accumulation of double‐negative thymocytes. This is the first report to directly examine the effects of melatonin treatment on the thymic antioxidant/oxidant status and thymic changes during T. cruzi infection. Our results revealed new antioxidant features that turn melatonin a potentially useful compound for the treatment of Chagas disease, a condition in which an excessive oxidative damage occurs.     

Immune response and Trypanosoma cruzi infection in Trypanosoma rangeli-immunized mice.

The capacity of Trypanosoma rangeli antigens to induce immune response in mice was analyzed and the course of the infection was studied in immunized animals challenged with virulent forms of T. cruzi. BALB/c mice were immunized with supernatant of disrupted epimastigotes of T. rangeli and with epimastigotes (EPI) of T. rangeli fixed with glutaraldehyde. Both of the antigens were emulsified with incomplete Freund's adjuvant (IFrAdj). All of the animals received T. cruzi Tulahuen antigens in the footpad and the skin reactivity was later studied. The mice that received EPI with or without IFrAdj showed significantly higher skin reactivity than controls, both in Arthus (3 hr) and delayed type hypersensitivity (24 hr) reactions. Furthermore, the mice immunized with T. rangeli developed antibodies against T. cruzi detectable through hemagglutination and immunofluorescence tests. When the animals were challenged with trypomastigotes of T. cruzi, only the groups immunized with EPI-IFrAdj had significantly lower parasitemias and greater survival against infection than controls. These results suggest that T. rangeli can induce humoral and cellular immune response against T. cruzi and attenuate the acute period of the infection produced by this parasite. This is the first time that partial resistance to T. cruzi in T. rangeli-immunized mice is reported. These findings may provide a useful tool for future studies directed at the immunoprevention of Chagas' disease.     

Endogenous glucocorticoids cause thymus atrophy but are protective during acute Trypanosoma cruzi infection

The cytokine-mediated stimulation of the hypothalamus-pituitary-adrenal (HPA) axis is relevant for survival during bacterial endotoxemia and certain viral infections. However, only limited information is available regarding the effects of endogenous glucocorticoids on parasite diseases. We have studied this issue using, as a model, C57Bl/6 and Balb/c mice infected with Trypanosoma cruzi, the causal agent of Chagas' disease. These two mouse strains differ in the susceptibility to infection with the parasite. An intense stimulation of the HPA-axis was observed 3 weeks after infection in both strains, but glucocorticoid levels were already increased two- to threefold in the less susceptible Balb/c strain during the first week. Blockade of glucocorticoid receptors with the glucocorticoid antagonist RU486, starting on day 10 after infection, partially reversed the thymic atrophy and decreased the number of CD4(+)CD8(+) thymocytes without affecting parasitemia and the number of inflammatory foci in the heart. However, tumor necrosis factor-alpha blood levels were increased in infected mice of both strains treated with RU486. Furthermore, the blockade of glucocorticoid receptors accelerated death in C57Bl/6J mice and increased lethality to 100% in Balb/c mice. The results obtained represent the first evidence that an endocrine host response that is coupled to the immune process can strongly affect the course of a parasite infection.     

Antibody response and antigen recognition in human infection with Trypanosoma cruzi

Sequential serum samples of an individual accidentally infected with Trypanosoma cruzi were examined to study the evolution of the antibody response, particularly of those with the capacity to lyse trypanosomes, and to determine the antigens of each of the 3 stages of the life cycle of T. cruzi, recognized by antibodies formed as the infection progresses. T. cruzi specific IgM and subclasses of IgG antibodies were detected and reached peak levels at the same period of time. Lytic antibodies were detected 2 weeks before demonstration of parasitemia and of antibodies reacting in the ELISA and IFA tests. Western blots used to examine antigen recognition revealed a complex array of antigens of epimastigotes and amastigotes, but not of blood trypomastigotes, recognized by antibodies in the various serum samples. Most of the antigens recognized by antibodies were common to all 3 stages of T. cruzi, although a few were specific for each of the stages. Certain antigens were only recognized by antibodies in serum collected at distinct periods of time during the course of the infection. The pattern noted after immunoprecipitation of 125I labeled cell membrane surface antigens was simple. Antigens of molecular weight of 90, 72, 50, and 30 K were immunoprecipitated in higher quantities. Antibodies in serum collected early and late in the infection recognized similar antigens in epimastigotes of the infecting strain and in epimastigotes of 3 other strains of T. cruzi from widely separated geographic areas.     

Ketoconazole protects against infection with Trypanosoma cruzi in a murine model

The oral administration of ketoconazole to mice protected them against death caused by infection with Trypanosoma cruzi. The addition of ketoconazole to cultures of macrophages infected with the organism markedly inhibited the intracellular multiplication of amastigotes. These observations suggest that ketoconazole may be a potent agent against T. cruzi and should be evaluated more extensively as a chemotherapeutic agent for Chagas' disease.     

Modulation of T-cell responsiveness during Trypanosoma cruzi infection: analysis in different lymphoid compartments

Spleen and lymph node cells of Trypanosoma cruzi-infected mice were studied for mitogen-induced responsiveness in terms of proliferation and lymphokine production (IL-2, IFN-gamma). Splenocyte (SP) as well as lymph node cell (LN) proliferation and IL-2 production were depressed during the acute phase of the infection. Proliferative capacity of LN cells recovered completely and that of SP partially during the chronic phase. In contrast to these suppressive effects, the mitogen-induced IFN-gamma response was enhanced. In vitro co-incubation of normal SP or LN cells with trypomastigotes resulted in a reduced mitogen-induced cell proliferation and IL-2 secretion, similar to those seen with cells taken from infected mice. In contrast, trypomastigotes exerted a stimulatory activity on the mitogen-induced IFN-gamma response of both SP and LN cells. Addition of lymph node cells from T. cruzi-infected mice (LN-I) to lymph node cells of control mice (LN-C) suppressed strongly the mitogen-induced responsiveness of such cocultures. A marginal level of suppression was recorded in cocultures of spleen cells from infected mice (SP-I) and control spleen cells (SP-C). The potent suppressive cells within LN-I populations were identified as macrophage-like and such cells were absent in SP-C and peritoneal exudate cells from T. cruzi infected animals.     

Trypanosoma cruzi: the effects of dehydroepiandrosterone (DHEA) treatment during experimental infection

The aim of this study was to evaluate the efficacy of the immunomodulator dehydroepiandrosterone (DHEA) in the treatment of Trypanosoma cruzi infection and the possible biochemistry alterations in male and female Wistar rats. DHEA also known as the steroid of multiple actions has attracted distinct medical areas. Prior studies show that DHEA enhances immune responses against a wide range of viral, bacterial and parasitic pathogens. Furthermore, administration of DHEA seems to protect animals against obesity and diabetes. Male animals subcutaneous treated with 40 mg/kg body weight/day of DHEA displayed a significant reduction in blood parasites during parasitaemia peak, when compared to untreated animals (P<0.001). For female group parasitaemia was also reduced although values are not statistically significant (P>0.05). Sexual dimorphism was also observed, since females displayed lesser parasitaemia levels compared to males group treated (P>0.05) and untreated (P<0.001). Enhanced leucocytes number was observed in control females when compared to control males (P<0.05). DHEA treatment did not triggered any significant alterations in leucocytes levels (P>0.05). DHEA administration induced an enhanced number of macrophages in infected male (P<0.01). DHEA administration causes a decrease in glucose (P<0.001). Cholesterol and tryglicerides levels did not display results statistically significant (P>0.05) during the treatment. These results suggest that DHEA treatment enhances the immune response as evidenced here by reduced levels of parasites. Up-regulation of the immune system by exogenous DHEA may be useful in the treatment of American tripanosomiasis.     

Trypanosoma cruzi parasitemia in US blood donors with serologic evidence of infection

Trypanosoma cruzi infection (which causes Chagas disease) is typically undiagnosed and persists if untreated. We sought to affirm that T. cruzi-seropositive US blood donors have persistent infection with demonstrable parasitemia long after acquisition of infection. Fifty-two previously identified seropositive donors (positive by 2 methods) provided up to 3 blood specimens for testing by polymerase chain reaction (PCR) and hemoculture; most participants (67%) provided only 1 specimen. When evaluated 2 decades after immigration, 33 donors (63%) had PCR evidence of parasitemia; 3 also had culture-confirmed infection. This affirmation that bloodstream parasites are detectable--and potentially transmissible--decades after immigration strengthens the rationale for donor screening.     

Experimental chemotherapy against Trypanosoma cruzi infection: essential role of endogenous interferon-gamma in mediating parasitologic cure

Studies in humans and in experimental models suggest the involvement of the immune system for efficacy of drug treatment against protozoan parasites. This study tested this hypothesis by using various cytokine and inducible nitric oxide synthase (iNOS) knockout (KO) mice infected with Trypanosoma cruzi and treated with benznidazole. In contrast with the 100% parasitologic cure rate achieved in wild-type animals, benznidazole failed to cure 100%, 42%, 35%, and 28% of interferon-gamma, interleukin-12 (protein 40), protein 55-tumor necrosis factor receptor, and iNOS KO mice, respectively. These results suggest that activation of the immune system by the parasite and endogenous interferon-gamma play a major role in the efficacy of benznidazole against infection with T. cruzi.