Vertical transmission of Trypanosoma cruzi in Wistar rats during the acute phase of infection

Research on this form of transmission was carried out on female rats intradermally injected, before mating, with 1 x 10(4) metacyclic trypomastigotes of T. cruzi strains from dog (Pr) and human (YBM). The infected rats, as well as their offspring, were given parasitological, immunological and histopathological examinations during and after gestation. Healthy gestating rats were used as controls. Rats infected with T. cruzi strains showed clear signs of infection between 18 and 45 days post-inoculation (pi). Of 44 offspring from mothers infected with Pr, 4 males (9.1%) showed high parasitemia (432 and 240 tryps./mm3 of blood) at 30 and 40 days after birth, while direct blood examination, hemoculture and xenodiagnosis showed no infection in the other 40, or in the 52 offspring of rats infected with YBM. Anti-T. cruzi antibodies were found in appreciable quantities in infected mothers and in 44 out of 92 (47.8%) of the offspring, with titers that fluctuated between 1:32 and 1:2048 respectively. Histopathological studies of rats sacrificed at the end of gestation showed acute myocarditis and myositis of varying intensity and extent, characterized by abundant inflammatory infiltrate, in some cases associated with nests of amastigotes. The placentas showed moderate cellular infiltrate without parasites in the vascular stroma and amniotic fluid. The offspring of mothers infected with Chagas' disease were reinoculated and showed an acute phase characterized by low parasitemia (p < 0.05); after 60 days, the beginnings of chronic myocarditis and myositis could be observed, of a similar intensity to that observed in offspring born to infected mothers that were subsequently infected. These results confirm that T. cruzi can be transmitted vertically in Wistar rats; that a small number of offspring contract Chagasic infection congenitally; that anti-T. cruzi antibodies can pass from the mother and that these can modify the immune response in the offspring; that the pathogenicity of the strains of T. cruzi plays an important role in congenital transmission independently of origin or geographical location.     

Modulation of thymocyte subsets during acute and chronic phases of experimental Trypanosoma cruzi infection.

Several observations have demonstrated the importance of T-cell-mediated mechanisms in experimental Chagas' disease. In previous studies, we have shown that mice acutely infected by Trypanosoma cruzi develop a progressive thymic atrophy with severe alterations in the lymphoid compartment. In this report we performed a kinetic analysis of the murine thymic lymphocytes comparing acute and chronic phases of infection. At the chronic phase, we observed that total thymocyte numbers returned to age-matched control values. Additionally, the decrease in the percentage of CD4+CD8+, in parallel with an increase of CD4+CD8-, CD4-CD8+, CD3high, TcR alpha beta and TcR gamma delta cells detected in the acute infection, was also restored in chronically infected mice. This thymocyte recovering is probably linked to the increase in the percentage of thymocyte precursors, such as CD4lowCD8- and CD4-CD8low cells, together with the increase in the number of IL-2R+ and cycling cells, appearing in the late stages of acute infection.     

Impairment of monocytic function during Trypanosoma cruzi infection.

During acute infection, Trypanosoma cruzi, the etiologic agent of Chagas' disease, causes immunosuppression by mechanisms that are not fully delineated. Since mononuclear phagocytes are major target cells in trypanosomiasis, we investigated monocytic function during acute T. cruzi infection. A series of human monocyte and macrophage hybridomas, which represent clonal expansions of subpopulations of human macrophages and possess many normal monocytic functions, were successfully infected with T. cruzi. Clones 63 and 53, chosen for stability in long-term culture, were studied extensively after infection with T. cruzi. Following infection of clone 63, the trypomastigote did not transform into the amastigote multiplicative form, suggesting that clone 63 did not support the entire T. cruzi life cycle. The typical life cycle was completed in clone 53, and thus, clone 53 was used in subsequent studies. Following infection, clone 53 lost expression of class II antigens compared with uninfected cells (DR of 2.2% versus 29.3% and mean channel fluorescence intensity [mean channel] of 4.1 versus 30.5, DQ of 2.3% versus 15.6% and mean channel of 5.4 versus 11.4, and DP of 6.3% versus 27.2% and mean channel of 10.3 versus 33.4). The expression of Class I antigens (87.9% versus 82.8%; mean channel, 20 versus 120) and the adhesion molecules LFA-1 (72.9% versus 28.7%; mean channel, 50.7 versus 23.7) and LFA-3 (10.8% versus 0.7%; mean channel, 20.7 versus 15.1) was increased in infected cells compared with that in uninfected cells. Production of interleukin-1 alpha was decreased and interleukin-6 production was increased in infected clone 53 compared with those in the uninfected cells, while production of tumor necrosis factor alpha was increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Splenocyte membrane changes and immunosuppression during infection and reinfection with Trypanosoma cruzi

Antisera to epi- and trypomastigote forms of Trypanosoma cruzi were used to detect trypanosome antigens on the surface of lymphocytes from infected mice. Only the anti-trypomastigote serum could recognize antigens expressed transiently on the splenocyte membranes from infected animals. The number or structural configuration of Concanavalin A receptors was similarly affected and a clear correlation was seen between these two types of membrane changes and the immunosuppression to mitogens and SRBC presented by the infected mice. Reinfected animals did not show evidences of trypanosome proliferation in blood or tissues nor trypomastigote antigens on splenocytes, but presented a less intense, transient immunosuppression as measured by responsiveness to mitogens and SRBC, suggesting that the primed immune system can eliminate the new parasite inoculum before the host is immunosuppressed and also that the liberation of strong immunosuppressor trypomastigote antigens induce the new state of suppression.     

Markers of oxidative stress in adipose tissue during Trypanosoma cruzi infection

The protozoan parasite Trypanosoma cruzi causes Chagas disease. Cardiac and adipose tissues are among the early targets of infection and are sites of persistent infection. In the heart and adipose tissue, T. cruzi infection results in an upregulation of pro-inflammatory mediators. In the heart, infection is associated with an increase in the markers of oxidative stress. To date, markers of oxidative stress have not been evaluated in adipose tissue in this infection. Brown and white adipose tissues were obtained from CD-1 mice infected with the Brazil strain of T. cruzi for 15, 30, and 130 days post infection. Protein carbonylation and lipid peroxidation assays were performed on these samples. There was an upregulation of these markers of oxidative stress at all time-points in both white and brown adipose tissue. Determinants of anti-oxidative stress were downregulated at similar time-points. This increase in oxidative stress during T. cruzi infection most likely has a deleterious effect on host metabolism and on the heart.     

Gamma interferon suppresses acute and chronic Trypanosoma cruzi infection in cyclosporin-treated mice.

To determine if exogenous gamma interferon is effective in immunosuppressed mice infected with Trypanosoma cruzi, recombinant murine gamma interferon was administered to cyclosporin-treated mice with either acute or chronic T. cruzi infection. Gamma interferon significantly decreased parasitemia and prevented death in acutely infected mice. Parasitemias and mortality of mice treated with both gamma interferon and cyclosporin were similar to those of immunocompetent controls. In chronically infected mice, cyclosporin treatment produced significantly more organ explant cultures positive for T. cruzi. Fewer positive cultures, particularly for spleen and heart, were obtained from cyclosporin-treated mice when they also received gamma interferon. Ketoconazole treatment of mice resulted in no positive cultures. Cyclosporin treatment did not prevent activation of peritoneal macrophages by parenteral gamma interferon, nor did it have a consistent effect on serum titers of alpha/beta or gamma interferon in response to a second challenge inoculum of T. cruzi. These data indicate that exogenous gamma interferon suppresses acute and chronic T. cruzi infection in cyclosporin-treated mice but that gamma interferon is not as effective as the relatively specific antimicrobial ketoconazole. Gamma interferon activates macrophages despite cyclosporin treatment, and its effects appear to be tissue specific.     

Fas ligand-dependent inflammatory regulation in acute myocarditis induced by Trypanosoma cruzi infection

Fas/Fas ligand (Fas-L) engagement, a potent inducer of apoptosis, is also important for cellular activation, regulation of effector and chemotactic activity, and secretion of chemokines and cytokines. We evaluated the relevance of Fas/Fas-L in the regulation of myocarditis induced by Trypanosoma cruzi infection and observed that in Fas-L(-/-) mice (gld/gld), cardiac infiltration was significantly reduced, accordingly showing less cardiomyocyte destruction. Fluorescence-activated cell sorting analysis of cardiac inflammatory cells showed higher numbers of CD8(+) T cells in BALB/c compared with gld/gld mice but similar levels of lymphocyte function-associated antigen-1, intercellular adhesion molecule, CD2, and CD69 expression; MAC-1(+) myeloid cells and mast cells were increased in BALB/c mice, whereas gld/gld mice exhibited an enrichment of CD4(+/low) T cells. Intracellular labeling of cytokines revealed no clear cardiac skewing of Th1 or Th2 responses, but we found a higher number of interleukin-10(+) cells in gld/gld mice and a deficient expression of vascular cell adhesion molecule-1 on cardiac endothelial cells in gld/gld mice. Finally, we found a population of CD3(+) but CD4/CD8 double negative cardiac T cells in both groups of infected mice, but down-regulation of some adhesion molecules and surface receptors was only observed in gld/gld mice, indicating a targeted T-cell population mostly affected by the lack of Fas-L engagement. These results point to a role for myocarditis regulation by Fas/Fas-L beyond its possible direct relevance in cellular death.     

Lack of association between NRAMP1 gene polymorphisms and Trypanosoma cruzi infection

Genetic analysis in mice and humans have established the key role of the human natural resistance-associated macrophage protein 1 (NRAMP1) in resistance to intracellular infections. In the present study we investigated whether four NRAMP1 polymorphisms (5'(GT)n, -236 C-->T, D543N, and 3'UTR deletion) were important in determining the susceptibility to Trypanosoma cruzi infections as well as in the development of chagasic cardiac disease. Genotyping for these variants was assessed in 83 seropositive (asymptomatic, n=51, cardiomyopathic, n=32) and 85 seronegative individuals from a Peruvian population where T. cruzi is endemic. No statistically significant differences either between patients and controls or between asymptomatic and cardiomyopathic individuals were observed with respect to NRAMP1 variants. Our data suggest that the NRAMP1 genetic polymorphism analysed do not play a major role in the pathogenesis of T. cruzi infection in this Peruvian sample.     

Trypanosoma cruzi infection in B-cell-deficient rats.

The effect of neonatally initiated injections of anti-mu rabbit antiserum on immunity of rats against Trypanosoma cruzi infection was investigated in vivo. Anti-mu treatment resulted in a loss of immunoglobulin M (IgM) and IgG2a synthesis and, subsequently, of antibody production. These rats so treated were shown to be significantly more susceptible to the acute phase of the infection than the control rats treated with normal rabbit serum, as measured by increased parasitemia and mortality. These results indicate the essential role of antibodies, probably in association with complement or effector cells or both, in immunity to acute Chagas' disease.     

Alterations in production of immunoglobulin classes and subclasses during experimental Trypanosoma cruzi infection

The spleens of mice infected with Trypanosoma cruzi were tested for their contents of cells producing IgM, IgG1, IgG2a, IgG2b, and IgG3 specific for the trinitrophenyl hapten after immunization with trinitrophenyl-Ficoll and trinitrophenyl-bovine serum albumin at various times during the acute and chronic phases of the disease. Reduced splenic contents of all of these cells was the characteristic of the acute period, and a return to normal levels occurred during the chronic stage. However, the density of IgG2a- and IgG2b-producing plaque-forming cells in the spleen was not restored to normality until 130 days postinfection, i.e., long after the chronic phase had been attained, reflecting a diluting effect of splenomegaly on cells that produce immunoglobulin isotypes known to be cytotoxic for the blood form of the parasite.     

Modulation induced by estradiol in the acute phase of Trypanosoma cruzi infection in mice

We investigated the effect of 17beta-estradiol on mice resistant to infection by Trypanosoma cruzi. Infected Balb/C, C3H and C57BL/6 female mice had a longer survival time than males, C57BL/6 showing the highest difference (50% cumulative mortality in females versus 100% in males). This lineage was treated with estradiol (from 0.05 microg to 500 microg/mouse) 1 day before infection. Treatment with 50 microg or 500 microg estradiol/ mouse increased mortality and parasitaemia. Low doses had no effect or tended to reduce both parameters. Given that estradiol presented no in vitro effect on trypomastigotes or epimastigotes, the involvement of a direct hormonal effect on the parasite is improbable. Alterations in the humoral T. cruzi-specific response were also discarded, since the kinetics and concentration of anti-T. cruzi IgG were not affected by the treatment. Females infected during an estradiol-descending phase (meta-oestrus) survived longer than those infected during other phases of the oestrous cycle. We confirmed that estradiol interferes with T. cruzi infection.     

Adoptive transfer of resistance to acute Trypanosoma cruzi infection with T-lymphocyte-enriched spleen cells.

Inbred C57BL/10 mice immunized with live culture forms of Trypanosoma cruzi were resistant to acute infection after challenge with bloodstream forms. Splenic leukocytes or serum from immunized mice were transferred to syngeneic recipients 2 days before of 2 days after challenge. Protection was not observed in recipients of serum, although the serum contained high levels of agglutinating antibody. Unfractionated splenic leukocytes from immunized donors conferred partial protection, and preparations enriched for T lymphocytes were significantly more effective than preparations enriched for B lymphocytes. Recipients of T-lymphocyte-enriched spleen cells had significantly higher survival times and significantly lower parasitemias than did recipients of B-lymphocyte-enriched spleen cells.     

Curcumin treatment provides protection against Trypanosoma cruzi infection

Trypanosoma cruzi, the etiologic agent of Chagas disease, causes an acute myocarditis and chronic cardiomyopathy. The current therapeutic agents for this disease are not always effective and often have severe side effects. Curcumin, a plant polyphenol, has demonstrated a wide range of potential therapeutic effects. In this study, we examined the effect of curcumin on T. cruzi infection in vitro and in vivo. Curcumin pretreatment of fibroblasts inhibited parasite invasion. Treatment reduced the expression of the low density lipoprotein receptor, which is involved in T. cruzi host cell invasion. Curcumin treatment of T. cruzi-infected CD1 mice reduced parasitemia and decreased the parasitism of infected heart tissue. This was associated with a significant reduction in macrophage infiltration and inflammation in both the heart and liver; moreover, curcumin-treated infected mice displayed a 100% survival rate in contrast to the 60% survival rate commonly observed in untreated infected mice. These data are consistent with curcumin modulating infection-induced changes in signaling pathways involved in inflammation, oxidative stress, and apoptosis. These data suggest that curcumin and its derivatives could be a suitable drug for the amelioration of chagasic heart disease.     

Splenectomy increases mortality in murine Trypanosoma cruzi infection

The spleen is a secondary lymphoid organ that harbours a variety of cells such as T and B lymphocytes and antigen‐presenting cells important to immune response development. In this study, we evaluated the impact of spleen removal in the immune response to experimental Trypanosoma cruzi infection. C57BL/6 mice were infected with Y strain of the parasite and infection was followed daily. Mice that underwent splenectomy had fewer parasites in peripheral blood at the peak of infection; however, mortality was increased. Histological analysis of heart and liver tissues revealed an increased number of parasites and inflammatory infiltrates at these sites. Spleen removal was associated with reduction in IFN‐γ and TNF‐α production during infection as well as with a decrease in specific antibody secretion. Haematological disorders were also detected. Splenectomized mice exhibited severe anaemia and decreased bone marrow cell numbers. Our results indicate that spleen integrity is critical in T. cruzi infection for the immune response against the parasite, as well as for the control of bone marrow haematological function.     

DNA-Based immunization with Trypanosoma cruzi complement regulatory protein elicits complement lytic antibodies and confers protection against Trypanosoma cruzi infection

A complement regulatory protein (CRP) of Trypanosoma cruzi was evaluated as a vaccine candidate in a murine model of experimental T. cruzi infection. Recombinant CRP derived from an Escherichia coli expression system and a plasmid encoding the full-length crp structural gene under the control of a eukaryotic promoter were used to immunize BALB/c mice. Immunization with both protein and DNA vaccines resulted in a Th1-type T-cell response, comparable antibody titers, and similar immunoglobulin G isotype profiles. Only mice immunized with the crp DNA plasmid produced antibodies capable of lysing the parasites in the presence of complement and were protected against a lethal challenge with T. cruzi trypomastigotes. These results demonstrate the superiority of DNA immunization over protein immunization with the recombinant CRP. The work also supports the further investigation of CRP as a component of a multigene, anti-T. cruzi DNA vaccine.     

Altered thymocyte migration during experimental acute Trypanosoma cruzi infection: combined role of fibronectin and the chemokines CXCL12 and CCL4

We previously showed migration disturbances in the thymus during experimental infection with Trypanosoma cruzi, the causative agent of Chagas disease. These changes were related to the enhanced expression of extracellular matrix ligands and receptors, leading to the escape of immature cells to the periphery. Here, we analyzed the expression and role of selected chemokines (CXCL12 and CCL4) and their receptors (CXCR4 and CCR5) in regulating thymocyte migration in conjunction with extracellular matrix during acute T. cruzi infection. We found increased chemokine deposition in the thymus of infected mice when compared to controls, accompanied by enhanced co-localization with fibronectin as well as up-regulated surface expression of CXCR4 and CCR5 in thymocytes. We also noticed altered thymocyte migration towards the chemokines analyzed. Such an enhancement was even more prominent when fibronectin was added as a haptotatic stimulus in combination with a given chemokine. Our findings suggest that thymocyte migration results from a combined action of chemokines and extracellular matrix (ECM), which can be altered during pathological conditions such as T. cruzi infection, and may be at the origin of the changes in the T cell repertoire seen in this pathological process.     

Trypanosoma cruzi infection enhances polyreactive antibody response in an acute case of human Chagas' disease

The kinetics of antibody response in an acute case of human Chagas' disease was investigated. Hypergammaglubulinaemia appeared at day 17 of infection, and persisted after 66 days of infection, at which time parasitaemia became undetectable. Titration of immunoglobulins showed that the three principal isotypes were involved in the response, emphasizing polyclonal B cell activation. Total IgA was detected before total IgM, and the killer before total IgG. High titres of autoantibodies were found among IgM and IgG subclasses. IgA was also the first isotype to be detected among specific anti-Trypanosoma cruzi antibodies. However, the maximal parasite antibody response was attained after 30 days of infection for all isotypes. With regard to possible cross-reactivity between molecules of host and parasite, adsorption experiments on T. cruzi-specific immunosorbent were designed. Specific antibodies, present in the eluates, also recognized natural antigens, especially laminin. In order to characterize the α-galactose epitope of laminin, adsorption experiments on sheep erythrocytes were performed, and revealed the possible presence of another epitope on the glycoprotein. Our results indicate that in the case of Chagas' disease investigated here, polyclonal activation occurred; moreover, they suggest that molecular mimicry may play a role by increasing autoantibodies, probably via a parasite-driven mechanism.