Trypanosoma cruzi reinfections provoke synergistic effect and cardiac beta-adrenergic receptors' dysfunction in the acute phase of experimental Chagas' disease

Cardiac beta-adrenergic receptors' function was studied in the acute phase of Chagas' disease in mice reinfected with Trypanosoma cruzi Tulahuen strain (Tul) and with parasites isolated from an infected patient (SGO-Z12). Genetic characterization of SGO-Z12 isolates demonstrated that it belongs to the zimodeme Z12, one of the prevalent ones in humans in Argentina. Electrocardiography, heart histopathology, parasitemias, and survival in infected and reinfected mice were also analyzed. Reinfected mice reached higher parasitemias, 14% of the infected with SGO-Z12 and 76% of the reinfected groups showed electrocardiographic abnormalities. Similar results were found in mice that were infected and reinfected with Tul. SGO-Z12-Reinfected and Tul-Infected groups exhibited cardiac beta-adrenergic receptors' affinity significantly diminished (p<0.001) and its density significantly increased (p<0.001) than in infected and non-infected groups. Histopathologic alterations in hearts from Tul and SGO-Z12-Reinfected mice were detected. Reinfections with T. cruzi, Tulahuen strain or SGO-Z12 isolate provoked cardiac dysfunctions of different degrees, from the acute phase on.     

Experimental Chagas' disease in dogs.

This paper describes the development of experimental Chagas' disease in 64 out-bred young dogs. Twenty-nine animals were inoculated with the Be-62 and 35 with Be-78 Trypanosoma cruzi strains. Twenty-six were infected with blood trypomastigotes by different inoculation routes and 38 with metacyclic trypomastigotes from the vector via the conjunctival route. Twenty of the 26 dogs infected with blood trypomastigotes were autopsied during the acute phase. Eleven died spontaneously and nine were sacrificed. Six remained alive until they died suddenly (two) or were autopsied. (four). Twelve of the 38 dogs infected with metacyclic trypomastigotes evolved naturally to the chronic phase and remained alive for 24-48 months. The parasitemia, clinical aspects and serology (IgM and IgG) as well as electrocardiogram, hemogram and heart anatomo-histopathologic patterns of acute and chronic cardiac forms of Chagas' disease as seen in human infections, were reproduced. The most important finding is the reproducibility of diffuse fibrosing chronic chagasic cardiopathy in all dogs infected with Be-78 T. cruzi strain autopsied between the 90th and 864th days of infection. Thus, the dog can be considered as a suitable experimental model to study Chagas' disease according to the requisites of the Word Health Organization (1984). Furthermore the animal is easily obtained and easy to handle and maintain in experimental laboratory conditions.     

Cardioprotective effects of phosphoramidon on myocardial structure and function in murine Chagas' disease

Chagas' disease is an important cause of cardiomyopathy. Endothelin-1, a vasoactive peptide has been implicated in the pathogenesis of chagasic cardiomyopathy. C57BL/6 x 129sv and CD1 mice were thus, infected with trypomastigotes of Trypanosoma cruzi (Brazil strain) and these infected mice were compared with infected mice treated with phosphoramidon. This compound inhibits endothelin-converting enzyme and neutral endopeptidases and does not affect the growth of the parasite in culture. Phosphoramidon was given in a dose of 10mg/kg for the initial 15 days post-infection None of the C57Bl/6 x 129sv mice died as a result of infection. However, there was marked myocardial inflammation and fibrosis in infected, untreated mice. The hearts of the infected, phosphoramidon-treated mice showed significantly less pathology. Cardiac magnetic resonance imaging of infected mice revealed right ventricular dilation that was less severe in those treated with phosphoramidon. Phosphoramidon-treated CD1 mice survived the acute infection. Transthoracic echocardiography demonstrated left ventricular dilation and reduced percent fractional shortening and relative wall thickness. These alterations were also attenuated as a result of phosphoramidon treatment. These data suggest that endothelin-1 contributes to the pathogenesis of chagasic cardiomyopathy and interventions that inhibit the synthesis of endothelin-1 and/or neutral endopeptidase might have a protective effect on myocardial structure and function in murine Chagas' disease.     

Protective role of ETA endothelin receptors during the acute phase of Trypanosoma cruzi infection in rats

Chagas' disease, caused by Trypanosoma cruzi, has an acute phase characterized by blood-circulating trypomastigotes and amastigote proliferation in several cell types, especially muscle cells. In the chronic phase, around 70% of infected people are asymptomatic (latent form). The remainder develop chagasic cardiomyopathy and/or digestive syndromes. There is evidence for aggravation of the chronic cardiac pathology by endothelin-mediated vasoconstriction. Holtzman rats have proven to be a good model for Chagas' disease acute phase and latent chronic phase. Now, we investigate the effects of prolonged treatment with an endothelin ET(A) receptor antagonist, BSF 461314, during the acute phase on parasitemia, coronary flow, tissue parasitism and the inflammatory process. Using isolated heart in Langendorff's preparation, endothelial dysfunction was observed only in non-treated infected animals. Histoquantitative analyses carried out in heart and diaphragm showed higher tissue parasitism and/or inflammatory process in BSF 461314-treated animals. Our data indicate that endothelin ET(A) receptors contribute to the initial mechanisms of parasite control. Impairment of the endothelium-dependent vasodilatation favors hazardous effects. However, blocking endothelin ET(A) receptors can prevent the latter.     

Different infective forms trigger distinct immune response in experimental Chagas disease

Although metacyclic and blood trypomastigotes are completely functional in relation to parasite-host interaction and/or target cell invasion, they differ in the molecules present on the surface. Thus, aspects related to the variability that the forms of T. cruzi interacts with host cells may lead to fundamental implications on the immune response against this parasite and, consequently, the clinical evolution of Chagas disease. We have shown that BT infected mice presented higher levels of parasitemia during all the acute phase of infection. Moreover, the infection with either MT or BT forms resulted in increased levels of total leukocytes, monocytes and lymphocytes, specifically later for MT and earlier for BT. The infection with BT forms presented earlier production of proinflammatory cytokine TNF-α and later of IFN-γ by both T cells subpopulations. This event was accompanied by an early cardiac inflammation with an exacerbation of this process at the end of the acute phase. On the other hand, infection with MT forms result in an early production of IFN-γ, with subsequent control in the production of this cytokine by IL-10, which provided to these animals an immunomodulatory profile in the end of the acute phase. These results are in agreement with what was found for cardiac inflammation where animals infected with MT forms showed intense cardiac inflammation later at infection, with a decrease in the same at the end of this phase. In summary, our findings emphasize the importance of taking into account the inoculums source of T. cruzi, since vectorial or transfusional routes of T. cruzi infection may trigger distinct parasite-host interactions during the acute phase that may influence relevant biological aspects of chronic Chagas disease.     

Evaluation of the rabbit as a model for Chagas' disease. I. Parasitological studies

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, 72 animals have been inoculated by intraperitoneal and conjunctival route with bloodstream forms, vector-derived metacyclic trypomastigotes and tissue culture trypomastigotes of Trypanosoma cruzi strains Y, CL and Ernane. In 95.6% of the animals trypomastigotes had been detected at the early stages of infection by fresh blood examination. The course of parasitemia at the acute phase was strongly influenced by the parasite strain and route of inoculation. At the chronic phase parasites had been recovered by xenodiagnosis and/or hemoculture in 40% of the examined animals. The xenodiagnosis studies have shown selective interactions between the T. cruzi strains and the four species of vectors used, inducing significant variability in the results. The data herein present are consistent with the parasitological requirements established for a suitable model for chronic Chagas' disease.     

Trypanosoma cruzi: in vivo evaluation of iron in skin employing X-ray fluorescence (XRF) in mouse strains that differ in their susceptibility to infection

Trypanosoma cruzi, the causative agent of Chagas' disease (CD), is a substantial public health concern in Latin America. Laboratory mice inoculated with T. cruzi have served as important animal models of acute CD. Host hypoferremic responses occur during T. cruzi infection; therefore, it has been hypothesized that T. cruzi requires iron for optimal growth in host cells and, unlike extracellular pathogens, may benefit from host hypoferremic responses. Recent technological improvements of X-ray fluorescence are useful for diagnostics or monitoring in biomedical applications. The goal of our study was to determine whether the iron availabilities in Swiss and C57BL/6 mice differ during the acute phase of T. cruzi infection and whether the availability correlates with oxidative stress in the susceptible and resistant phenotypes identified in these mice. Our results showed that the decrease in iron levels in the skin of resistant infected mice correlated with the increase in oxidative stress associated with anemia and the reduction in parasite burden.     

Trypanosoma cruzi: preliminary investigation of NADH-positive and somatostatin-immunoreactive neurons in the myenteric plexus of the mouse colon during the infection

In this paper, the distribution of NADH-positive and somatostatin (SOM) immunoreactive neurons in the myenteric plexus of the colon of mice infected with Trypanosoma cruzi was studied. Ten young, male, BALB/c mice were inoculated with the Y strain of T. cruzi, 60 days previously (chronic phase of the infection). Another 10 mice were uninfected controls. Distal and proximal colonic neurons from five chronically infected mice and their controls were stained using the NADH-diaphorase method. Quantitative results showed a significant decrease of 39% in the number of neurons in the proximal colon of infected mice and 58% in the distal colon (p<0.05). SOM was localized in five animals from each group by light microscopy, using an indirect immunofluorescence technique. It was observed that there were far fewer nerve cells and fibres and less intensely stained neuron bodies and varicose SOM-positive nerve fibres in both, control and chronic infected mice. These findings could be related to the disturbances in intestinal motility observed in patients in the chronic phase of Chagas' disease.     

Aspirin treatment of mice infected with Trypanosoma cruzi and implications for the pathogenesis of Chagas disease

Chagas disease, caused by infection with Trypanosoma cruzi, is an important cause of cardiovascular disease. It is increasingly clear that parasite-derived prostaglandins potently modulate host response and disease progression. Here, we report that treatment of experimental T. cruzi infection (Brazil strain) beginning 5 days post infection (dpi) with aspirin (ASA) increased mortality (2-fold) and parasitemia (12-fold). However, there were no differences regarding histopathology or cardiac structure or function. Delayed treatment with ASA (20 mg/kg) beginning 60 dpi did not increase parasitemia or mortality but improved ejection fraction. ASA treatment diminished the profile of parasite- and host-derived circulating prostaglandins in infected mice. To distinguish the effects of ASA on the parasite and host bio-synthetic pathways we infected cyclooxygenase-1 (COX-1) null mice with the Brazil-strain of T. cruzi. Infected COX-1 null mice displayed a reduction in circulating levels of thromboxane (TX)A(2) and prostaglandin (PG)F(2α). Parasitemia was increased in COX-1 null mice compared with parasitemia and mortality in ASA-treated infected mice indicating the effects of ASA on mortality potentially had little to do with inhibition of prostaglandin metabolism. Expression of SOCS-2 was enhanced, and TRAF6 and TNFα reduced, in the spleens of infected ASA-treated mice. Ablation of the initial innate response to infection may cause the increased mortality in ASA-treated mice as the host likely succumbs more quickly without the initiation of the "cytokine storm" during acute infection. We conclude that ASA, through both COX inhibition and other "off-target" effects, modulates the progression of acute and chronic Chagas disease. Thus, eicosanoids present during acute infection may act as immunomodulators aiding the transition to and maintenance of the chronic phase of the disease. A deeper understanding of the mechanism of ASA action may provide clues to the differences between host response in the acute and chronic T. cruzi infection.     

Augmentation of suppressed antibody responses in mice during experimental Chagas' disease by T helper cells activated in a time-dependent mode of immunization

Mice infected with the protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas' disease, develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC), hapten-conjugated SRBC (TNP-SRBC), and horse erythrocytes (TNP-HRBC). Studies in vivo demonstrated that anti-SRBC responses were best enhanced when T. cruzi-infected mice were injected with primed T cells derived from normal or infected mice immunized four days previously. The presence of enhancing capacities for DPFC responses by T cells from T. cruzi-infected mice were also supported by experiments examining the hapten-carrier effect. Preimmunization of infected mice with SRBC or HRBC four days before injection of hapten-homologous (TNP-SRBC or TNP-HRBC) carrier resulted in markedly augmented anti-hapten antibody responses. These results show that functional help provided by T cells activated during priming and exposed to a challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in T. cruzi-infected mice.     

Follow-up of patent and subpatent parasitemias and development of muscular lesions in mice inoculated with very small numbers of Trypanosoma cruzi.

A sequential analysis of patent and subpatent parasitemias, mortality, and histopathology during acute Chagas' disease experimentally produced by inoculation of 10 or 100 bloodstream forms of Trypanosoma cruzi Y strain in susceptible mice was carried out. Parasites were searched for comparatively using three different methods: direct counting, Ficoll-MI density flotation, and hemoculture. Ficoll-MI density flotation promptly discriminated with high reproducibility subpatent parasitemic states not detected in the blood samples analyzed by direct counting. Despite the high proportion of supposedly uninfected animals and depending on the postinfection time, the majority of the mice had bloodstream parasites at the subpatent level detected by Ficoll-MI, and all of them had muscular lesions during the acute phase. All Ficoll-MI-negative blood samples from infected mice were also negative by hemoculture. Normal mouse blood purposely contaminated with parasite quantities ranging from 200 to 2000/ml was tested comparatively by density flotation and hemoculture and showed frequencies of reisolation varying from 25 to 100%. Overall, these data showed that inoculum as low as 10 infective forms of Y strain is able to induce acute Chagas' disease in susceptible mice and that a subpatent parasitemic state of 600-1000 forms/ml is a common finding. The use of Ficoll-MI to detect subpatent parasitemia is discussed.     

Immunological consequences of infection and vaccination in South American trypanosomiasis

Trypanosoma cruzi infection provokes a vigorous immune response that terminates the parasitaemia associated with the acute stage within two to three months of initial infection. Even so, a variable proportion of patients may develop severe Chagas' disease, often decades after initial infection. Recent experimental findings suggest that trypomastigotes of T. cruzi possess a surface bound neuraminidase and sugar binding protein by means of which they invade host cells--a mechanism very reminiscent of influenza virus. Studies of the antibody response to trypomastigotes in patients or murine models have identified a series of antibodies able to mediate lysis of live parasites in a complement mediated lysis (c.m.l.) assay. These antibodies have also been linked to resistance to infection in vivo and disappear following successful parasitological 'cure' in drug-treated animals and human patients. Immunochemical studies have shown that sera from infected patients or mice lacking this c.m.l. activity also lack those antibodies able to bind trypomastigote surface components of 85 and 160 kDa relative molecular mass. The availability of rabbit and mouse models of Chagas' disease have produced data that suggest that chronic stage pathology may have an immunological basis dependent on the known cross reactivity between host and parasite cells. Delivery of the lethal hit leading to host cell destruction is probably facilitated by the ability of parasite antigens to bind to host cells thus exposing them to the host's own anti-parasite immune response. If Chagas' disease does indeed have an immunological basis, then this might be controlled in turn by immunoregulation, in a manner analogous to that achieved in experimental allergic encephalomyelitis.     

Exacerbation of experimental Trypanosoma cruzi infection in mice by concomitant malaria.

The effect of malaria on the chronic phase of Chagas' disease was investigated in mice. The animals were given Plasmodium bergheri-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y. CL and Gilmar). in all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas' disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curved of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phas with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Trypanosoma cruzi: cross-reactive anti-heart autoantibodies produced during infection in mice

Preimmunization with attenuated Corpus Christi stain Trypanosoma cruzi provides survival to C3H mice and enhances resistance of C57 mice to Brazil strain infection. C3H(He) and C57 B1/6 mice surviving acute infection of T. cruzi are shown to have heart specific autoantibodies through acute and chronic infection. ELISA assays were performed using nondenatured extract of hearts from normal syngeneic mice as target antigen reacted with sera from immunized and/or infected mice. Surviving C3H mice developed a specific anti-heart response as early as Day 21 of infection and this response continued at a high level to Day 300. The response in C57 mice, both immunized-infected and infected only, increased to Day 100 followed by a decline in intensity. The heart specificity of the response in mice was suggested by negligible reaction of sera with smooth muscle preparations and a reduced autoreactivity with skeletal muscle. Laminin, a suggested target of autoimmunity in Chagas' disease, was shown not to be the target of the responses in these mice. Immunoaffinity-purified heart specific antibodies show strong cross-reactivity with parasite antigen and like purified parasite specific antibodies, reacted with heart antigen.     

Morphological changes and EGF expression in the granular convoluted tubule cells of submandibular glands of Trypanosoma cruzi infected rats

We have previously demonstrated in rats that Chagas' disease affects the salivary glands, by promoting an enlargement of the submandibular gland. In order to further investigate possible functional alterations on infected submandibular glands, the objective of the present study was to analyze epidermal growth factor (EGF) expression on rat submandibular glands during Trypanosoma cruzi infection. Results demonstrated that infected rats presented lower levels of testosterone, and morphological changes in the granular convoluted tubule (GCT) cells of the submandibular glands, along with acinar enlargement and delayed ductal maturation at the developing granular ducts. Immunohistochemistry analysis additionally showed that only few cells immunolabelled with anti-EGF on infected rats during the acute phase of Chagas' disease, while after 64 and 90 days (chronic phase) of infection, EGF expression was similar to non-infected rats. The present findings suggest that at the acute phase of Chagas' disease, lower levels of testosterone may lead to a delayed maturation of GCT, which positively correlates with decreased EGF production by submandibular glands cells.     

Prevalence of anti-R-13 antibodies in human Trypanosoma cruzi infection

Abstract Infection with Trypanosoma cruzi develops in three phases: acute, indeterminate or asymptomatic, and chronic phase (with cardiac or digestive manifestations). Moreover, transmission may occur from infected mothers to newborn, the so-called congenital form. In the present study, humoral responses against T. cruzi total extract and against the 13 amino acid peptide named R-13 derived from the parasite ribosomal P protein, previously described as a possible marker of chronic Chagas heart disease, were determined pateints and in blood bank donors from endemic areas. While in sera from acute phase, only IgM anti- T.cruzi response was observed, both IgM and IgG anti- T. cruzi antibodies were detected in sera from congenitally infected newborns. The percentage of positive response in sera from blood bank donors was relatively high in endemic regions. Antibodies against the R-13 peptide were present in a large proportion of cardiac chagasic patients but were totally lacking in patients with digestive form of Chagas disease. Furthermore, anti-R-13 positive responses were detected in congenitally infected newborns.     

Experimental benznidazole treatment of Trypanosoma cruzi II strains isolated from children of the Jequitinhonha Valley,Minas Gerais,Brazil, with Chagas disease

Trypanosoma cruzi strains from distinct geographic areas show differences in drug resistance and association between parasites genetic and treatment response has been observed. Considering that benznidazole (BZ) can reduce the parasite burden and tissues damage, even in not cured animals and individuals, the goal is to assess the drug response to BZ of T. cruzi II strains isolated from children of the Jequitinhonha Valley, state of Minas Gerais, Brazil, before treatment. Mice infected and treated with BZ in both phases of infection were compared with the untreated and evaluated by fresh blood examination, haemoculture, polymerase chain reaction, conventional (ELISA) and non-conventional (FC-ALTA) serologies. In mice treated in the acute phase, a significant decrease in parasitaemia was observed for all strains. Positive parasitological and/or serological tests in animals treated during the acute and chronic (95.1-100%) phases showed that most of the strains were BZ resistant. However, beneficial effect was demonstrated because significant reduction (p < 0.05%) and/or suppression of parasitaemia was observed in mice infected with all strains (acute phase), associated to reduction/elimination of inflammation and fibrosis for two/eight strains. BZ offered some benefit, even in not cured animals, what suggest that BZ use may be recommended at least for recent chronic infection of the studied region.     

Evidence for Trypanosoma cruzi in adipose tissue in human chronic Chagas disease

Trypanosoma cruzi the cause of Chagas disease persists in tissues of infected experimental animals and humans. Here we demonstrate the persistence of the parasite in adipose tissue from of three of 10 elderly seropositive patients with chronic chagasic heart disease. Nine control patients had no parasites in the fat. We also demonstrate that T. cruzi parasitizes primary adipocytes in vitro. Thus, in humans as in mice the parasite may persist in adipose tissue for decades and become a reservoir of infection.     

CTLA-4 blockage increases resistance to infection with the intracellular protozoan Trypanosoma cruzi

Recent studies have revealed an important role for CTLA-4 as a negative regulator of T cell activation. In the present study, we evaluated the importance of CTLA-4 to the immune response against the intracellular protozoan, Trypanosoma cruzi, the causative agent of Chagas' disease. We observed that the expression of CTLA-4 in spleen cells from naive mice cultured in the presence of live trypomastigote forms of T. cruzi increases over time of exposure. Furthermore, spleen cells harvested from recently infected mice showed a significant increase in the expression of CTLA-4 when compared with spleen cells from noninfected mice. Blockage of CTLA-4 in vitro and/or in vivo did not restore the lymphoproliferative response decreased during the acute phase of infection, but it resulted in a significant increase of NO production in vivo and in vitro. Moreover, the production of IFN-gamma in response to parasite Ags was significantly increased in spleen cells from anti-CTLA-4-treated infected mice when compared with the production found in cells from IgG-treated infected mice. CTLA-4 blockade in vivo also resulted in increased resistance to infection with the Y and Colombian strains of T. cruzi. Taken together these results indicate that CTLA-4 engagement is implicated in the modulation of the immune response against T. cruzi by acting in the mechanisms that control IFN-gamma and NO production during the acute phase of the infection.     

Absence of interferon-gamma-inducible gene IGTP does not significantly alter the development of chagasic cardiomyopathy in mice infected with Trypanosoma cruzi (Brazil strain)

Interferon-gamma (IFN-gamma) contributes to host resistance during acute infection with Trypanosoma cruzi, the causative agent of Chagas' disease. Inducibly expressed guanosine triphosphatase (IGTP), a 48-kDa guanosine triphosphatase (GTPase), is a member of a family of GTPase proteins inducibly expressed by IFN-gamma. The expression pattern of IGTP suggests that it may mediate IFN-gamma-induced responses in a variety of cell types. IGTP has been demonstrated to be important for control of Toxoplasma gondii infection but not for resistance against Listeria monocytogenes. We evaluated the role of IGTP in development of chronic chagasic cardiomyopathy in IGTP null mice and C57X129sv (wild type [WT]) mice infected with the Brazil strain for 6 mo. There was no significant difference in parasitemia or cardiac histopathology between null and WT mice. Right ventricular remodeling was observed in infected IGTP null mice, suggesting that IGTP does not significantly alter the course of T. cruzi infection.