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.     

Trypanosoma cruzi: effects of repetitive stress during the development of experimental infection

Activation of the hypothalamus-pituitary-adrenal axis plays a major role in the suppression of the immune system. We have investigated the effects of repetitive stress on Wistar rats infected with the Y strain of Trypanosoma cruzi and a control group that underwent stressor stimuli by exposure to ether vapor for one minute twice a day. Repetitive stress resulted in an elevated number of circulating parasites accompanies by deep tissue disorganization, and cardiac histopathological alterations. The infected and stressed group displayed a decrease in body weight, and an increased parasite burden in heart tissue, and adrenal glands. Histological analysis of the heart also showed a moderate to severe diffused mononuclear inflammatory process. These results suggest that repetitive stress could be considered an important factor during development of experimental Chagas' disease, enhancing pathogenesis through disturbance of the host's immune system.     

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.     

Bioluminescent imaging of Trypanosoma cruzi infection

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.     

Trypanosoma cruzi: effect of the infection on the 20S proteasome in non-immune cells

Human infection with the protozoan Trypanosoma cruzi leads to Chagas disease. After 10-20 years of the normal acute phase, this disease develops to a chronic phase characterized mainly by dilated congestive cardiomyopathy. The mechanisms involved in the chronic phase are poorly understood, and it has been suggested that the parasite evades immune surveillance by down regulating the MHC class I antigen processing pathway. Here we analyzed whether composition or expression of the 20S proteasome, the major proteinase responsible for the generation of MHC class I ligands, were altered upon infection of HeLa cells by T. cruzi. Two-dimensional gel electrophoresis and RT-PCR experiments comparing non-infected and infected cells did not show differences between the composition of 20S proteasome or expression of its subunits. However, the proteasome’s trypsin- and chymotrypsin-like activities were 2.5 and 3.6 times higher in infected cells than in non-infected cells. Our results suggest that in vitroT. cruzi infection of human or rat cells do not alter the expression of 20S proteasomal subunits or particle composition, and fails to induce the formation of immunoproteasome. However, a significant increase in the trypsin- and chymotrypsin-like activities of the host proteasome was observed.     

Trypanosoma cruzi: influence of predominant bacteria from indigenous digestive microbiota on experimental infection in mice

To verify the influence of some predominant components from indigenous microbiota on systemic immunological responses during experimental Chagas disease, germ-free NIH Swiss mice were mono-associated with Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp. and then infected with the Y strain of Trypanosoma cruzi. All the mono-associations predominantly induced a Th1 type of specific immune response to the infection by T. cruzi. A direct correlation was observed between a higher survival rate and increased IFN-gamma and TNF-alpha production (P<0.05) in E. faecalis-, B. vulgatus-, and Peptostreptococcus-associated mice. Moreover, higher levels of anti-T. cruzi IgG1 and anti-T. cruzi IgG2a were also found in mono-associated animals after infection. On the other hand, with the exception of E. faecalis-associated mice, mono-association induced a lower IL-10 production after infection (P<0.05) when compared with germ-free animals. Interestingly, spleen cell cultures from non-infected germ-free and mono-associated mice spontaneously produced higher levels (P<0.05) of IL-10 than cultures from infected mono-associated mice, except again for E. faecalis-associated animals. In conclusion, the presence of the components of the indigenous microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in gnotobiotic mice. However, the degree of increase in production of cytokines depends on each bacterial component.     

Trypanosoma cruzi: plasma corticosterone after repetitive stress during the acute phase of infection

An increased level of plasma corticosterone is one manifestation of severe environmental or physiologic stress. The stress response mediated by the hypothalamic-pituitary-adrenal axis is already known to suppress immunoglobulin production and to impair immune function, but there are few studies relating stress and plasma corticosterone to the outcome of Trypanosoma cruzi infection. In this study, male Wistar rats were infected with the Y strain of T. cruzi and then subjected to repetitive stress by exposure to ether vapor for 1min twice a day during the acute phase of infection. Stressed animals showed decreased lytic antibody activity and lowered levels of peritoneal macrophages. Despite an increase in the weight of the spleen, histological analyses demonstrated tissue alterations, the presence of amastigote nests, and a complete absence of activated lymphoid follicles. These results suggest that stress-induced increases in plasma corticosterone can suppress the immune response and worsen tissue injury during the acute phase of T. cruzi infection.     

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 in the immune response during the course of experimental disease

Zinc is an essential nutritional component required for normal development and maintenance of immune functions. The possible effects of zinc in upregulating the host immune response during the acute and chronic phases of experimental Chagas’ disease were evaluated. In young, infected and Zn-supplemented animals, higher concentrations of IFN-γ and NO were observed. During the chronic phase, decreased concentrations of NO and IFN-γ were found for older infected animals that received Zn supplementation. For young animals, hearts from Zn-supplemented groups displayed reduced inflammatory infiltrate, heart weight and number of amastigote burdens. For older, infected and Zn-supplemented animals amastigote nests were absent with reduced inflammatory cell infiltrate. This study identifies a potentially novel therapeutic approach that could control the parasite load during acute phase of disease, consequently preventing the deleterious, parasite-elicited responses observed during chronic phase.     

Trypanosoma cruzi: effects of social stress in Calomys callosus a natural reservoir of infection

Social environment can represent a major source of stress affecting cortisol and/or corticosterone levels, thereby altering the immune response. We have investigated the effects of social isolation on the development of Trypanosoma cruzi infection in female Calomys callosus, a natural reservoir of this protozoan parasite. Animals were divided in groups of five animals each. The animals of one group were kept together in a single cage. In a second group, four females were kept together in a cage with one male. In the final group, five individuals were kept isolated in private cages. The isolated animals showed body weight reduction, decreased numbers of peritoneal macrophages, lower global leucocytes counts, smaller lytic antibody percentage and a significantly higher level of blood parasites compared to the other animals. Their behavior was also altered. They were more aggressive than grouped females, or females exposed to the presence of a male. These results suggest that isolation creates a distinct social behavior in which immunity is impaired and pathogenesis is enhanced.     

Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.     

Platelet-activating factor-like activity isolated from Trypanosoma cruzi

Platelet-activating factor is a phospholipid mediator that exhibits a wide variety of physiological and pathophysiological effects, including induction of inflammatory response, chemotaxis and cellular differentiation. Trypanosoma cruzi, the etiological agent of Chagas' disease, is transmitted by triatomine insects and while in the triatomine midgut the parasite differentiates from a non-infective epimastigote stage into the pathogenic trypomastigote metacyclic form. We have previously demonstrated that platelet activating factor triggers in vitro cell differentiation of T. cruzi. Here we show a platelet activating factor-like activity isolated from lipid extract of T. cruzi epimastigotes incubated in the presence of [14C]acetate. Trypanosoma cruzi-platelet activating factor-like lipid induced the aggregation of rabbit platelets, which was prevented by platelet activating factor-acetylhydrolase. Mouse macrophage infection by T. cruzi was stimulated when epimastigotes were kept for 5 days in the presence of T. cruzi-platelet activating factor, before interacting with the macrophages. The differentiation of epimastigotes into metacyclic trypomastigotes was also triggered by T. cruzi-platelet activating factor. These effects were abrogated by a platelet activating factor antagonist, WEB 2086. Polyclonal antibody raised against mouse platelet activating factor receptor showed labelling for T. cruzi epimastigotes using immunoblotting and immunofluorescence assays. These data suggest that T. cruzi contain the components of an autocrine platelet activating factor-like ligand-receptor system that modulates cell differentiation towards the infectious stage.     

Trypanosoma cruzi: sensitivity of the polymerase chain reaction for detecting the parasite in the blood of mice infected with different clonal genotypes

The polymerase chain reaction showed high sensitivity for detecting Trypanosoma cruzi in the blood of mice, independent of clonal genotype (19, 20-T. cruzi I; 32, 39-T. cruzi II) or phase of the infection (acute or chronic).     

Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole

Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor β and PGE₂ activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect.Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively.The observed effect of aspirin upon T. cruzi infection was not related with the PGE₂ synthesis inhibition. Nevertheless, NO levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement.Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages.     

Trypanosoma cruzi: orchiectomy and dehydroepiandrosterone therapy in infected rats

The ability of gonadal hormones to influence and induce diverse immunological functions during the course of a number of parasitic infections has been extensively studied in the latest decades. Dehydroepiandrosterone and its sulfate are the most abundant steroid hormones secreted by the human adrenal cortex and are considered potent immune-activators. The effects of orchiectomy on the course of Trypanosoma cruzi infection in rats, treated and untreated with DHEA were examined, by comparing blood and cardiac parasitism, macrophage numbers, nitric oxide and IFN-γ levels. Orchiectomy enhanced resistance against infection with elevated numbers of macrophages, enhanced concentrations of NO and IFN-γ and reduced amastigote burdens in heart when compared to control animals. DHEA replacement exerted a synergistic effect, up-modulating the immune response. Male sex steroids appear to play fundamental role in determining the outcome of disease, through the regulation and modulation of the activity of the immune response.     

TcRho1 of Trypanosoma cruzi: role in metacyclogenesis and cellular localization

Here we have investigated the function of TcRho1, a Rho family orthologue from the parasite Trypanosoma cruzi. We have selected parasites overexpressing wild-type TcRho1 and a truncated form of TcRho1 (TcRho1-DeltaCaaX) which is unable to undergo farnesylation and supposed to interfere with recruitment of Rho effectors to membranes. TcRho1 protein was localized at the anterior region of wild-type and TcRho1 overexpressing epimastigotes, suggesting association with the Golgi apparatus. Accordingly, parasites overexpressing TcRho1-DeltaCaaX presented cytoplasmic fluorescence. To address the function of TcRho1 during differentiation, from epimastigotes to trypomastigotes, we submitted parasites overexpressing the above-cited lineages to metacyclogenesis assays. Parasites overexpressing TcRho1-DeltaCaaX generated a discrete number of metacyclic trypomastigotes when compared with other lineages. Strikingly, TcRho1-DeltaCaaX cells died synchronously during the process of metacyclogenesis.     

Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids

Trypanosoma cruzi, the causative agent of Chagas disease, has at least two principal intraspecific subdivisions, T. cruzi I (TCI) and T. cruzi II (TCII), the latter containing up to five subgroups (a-e). Whilst it is known that TCI predominates from the Amazon basin northwards and TCII to the South, where the disease is considered to be clinically more severe, the precise clinical and evolutionary significance of these divisions remains enigmatic. Here, we present compelling evidence of an association between TCI and opossums (Didelphis), and TCII and armadillos, on the basis of key new findings from the Paraguayan Chaco region, together with a comprehensive analysis of historical data. We suggest that the distinct arboreal and terrestrial ecologies, respectively, of these mammal hosts provide a persuasive explanation for the extant T. cruzi intraspecific diversity in South America, and for separate origins of Chagas disease in northern South America and in the southern cone countries.     

Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles

The comparisons of 170 sequences of kinetoplast DNA minicircle hypervariable region obtained from 19 stocks of Trypanosoma cruzi and 2 stocks of Trypanosoma cruzi marenkellei showed that only 56% exhibited a significant homology one with other sequences. These sequences could be grouped into homology classes showing no significant sequence similarity with any other homology group. The 44% remaining sequences thus corresponded to unique sequences in our data set. In the DTU I ("Discrete Typing Units") 51% of the sequences were unique. In contrast, in the DTU IId, 87.5% of sequences were distributed into three classes. The results obtained for T. cruzi marinkellei, showed that all sequences were unique, without any similarity between them and T. cruzi sequences. Analysis of palindromes in all sequence sets show high frequency of the EcoRI site. Analysis of repetitive sequences suggested a common ancestral origin of the kDNA. The editing mechanism that occurs in kinetoplastidae is discussed.