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.     

CD40 ligation prevents Trypanosoma cruzi infection through interleukin-12 upregulation

Because of the critical role of the CD40-CD40 ligand (CD40L) pathway in the induction and effector phases of immune responses, we investigated the effects of CD40 ligation on the control of Trypanosoma cruzi infection. First, we observed that supernatants of murine spleen cells stimulated by CD40L-transfected 3T3 fibroblasts (3T3-CD40L transfectants) prevent the infection of mouse peritoneal macrophages (MPM) by T. cruzi. This phenomenon depends on de novo production of nitric oxide (NO) as it is prevented by the addition of N-nitro-L-arginine methyl ester, a NO synthase inhibitor. NO production requires interleukin (IL)-12-mediated gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) synthesis as demonstrated by inhibition experiments using neutralizing anti-IL-12, anti-IFN-gamma, and anti-TNF-alpha monoclonal antibodies (MAb). We found that an activating anti-CD40 MAb also directly stimulates IFN-gamma-activated MPM to produce NO and thereby to control T. cruzi infection. To determine the in vivo relevance of these in vitro findings, mice were injected with 3T3-CD40L transfectants or 3T3 control fibroblasts at the time of T. cruzi inoculation. We observed that in vivo CD40 ligation dramatically reduced both parasitemia and the mortality rate of T. cruzi-infected mice. A reduced parasitemia was still observed when the injection of 3T3-CD40L transfectants was delayed 8 days postinfection. It was abolished by injection of anti-IL-12 MAb. Taken together, these data establish that CD40 ligation facilitates the control of T. cruzi infection through a cascade involving IL-12, IFN-gamma, and NO.     

Suppression of human lymphocyte responses by Trypanosoma cruzi.

Virtually nothing is known about the basis for the immunosuppression associated with human T. cruzi infection. We have used an in vitro system to explore this effect. Incubation of human peripheral blood mononuclear cells (PBMC) with blood forms of T. cruzi abrogated their responses to suboptimal, optimal and supraoptimal doses of Con A, PHA or PWM, whether or not monocytes were depleted. Killed parasites were not suppressive. Maximal suppression (74%) occurred when the parasites were present during the entire culture period (96 hr), although significant suppression (33%) was seen when the organisms were added 24, 48 or 72 hr after initiation, suggesting that the early stages of lymphocyte activation had been impaired and that a second generation of cells was also affected. The 4-day supernatant medium of a T. cruzi suspension supported PBMC responses to Con A as well as medium incubated alone, indicating that suppression did not result from parasite removal of essential nutrients. Furthermore, 96 hr after mitogenic stimulation, the proportions of viable PBMC in cultures containing or lacking the parasites were comparable. Although T. cruzi binds Con A and PHA, this absorption was not the cause of reduced responsiveness since optimal concentrations of Con A and PHA remained in solution under our conditions. Levels of IL-2 in PHA-stimulated PBMC cultures were markedly reduced in the presence of T. cruzi. However, exogenous IL-2 failed to restore lymphocyte responsiveness. T. cruzi neither absorbed nor inactivated IL-2. Thus, the noted suppression appeared to involve at least deficient production and utilization of IL-2.     

Contact sensitivity responses in mice infected with Trypanosoma cruzi.

Mechanisms of depression of contact sensitivity responses in C57BL/10 mice infected with Trypanosoma cruzi were studied. Cellular involvement during sensitization with oxazolone was investigated in mice acutely infected with T. cruzi. Contact sensitivity was not expressed in mice during the latter stages of the acute infection. Spleen cells from sensitized, infected mice which were unable to respond to oxazolone could confer contact sensitivity upon normal syngenic mice as effectively as spleen cells from uninfected, sensitized donors. The ability of mice infected with T. cruzi to respond to an eliciting dose of oxazolone was significantly improved when macrophages from normal syngenic donors were administered to them at the time of skin test. When either normal or infected mice were used as recipients of lymphocytes from sensitized donors, the normal mice responded significantly better than did infected mice after administration of an eliciting dose of oxazolone. An increase in pyroninophilic cells was observed in draining lymph nodes after application of a sensitizing dose of oxaxolone to the ears of either normal or acutely infected mice. These results indicate that suppression of contact sensitivity during acute T. cruzi infection is directed toward the efferent arm rather than the afferent arm of the response.     

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)     

Interleukin-12-mediated resistance to Trypanosoma cruzi is dependent on tumor necrosis factor alpha and gamma interferon.

The aim of this study was to determine if interleukin-12 (IL-12) has a role in the immune response to Trypanosoma cruzi. Infection of BALB/c mice with the virulent Tulahuen strain of T. cruzi is characterized by a high-level parasitemia, pathology in the heart associated with the presence of amastigotes, and death during the acute phase of the disease. Administration of IL-12 to BALB/c mice infected with T. cruzi resulted in a reduced parasitemia and a significant delay in the time to death compared with those for infected controls. This protective effect was correlated with increased levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in serum. To determine if these cytokines were involved in the protective effects of IL-12, we treated infected mice with IL-12 alone or in combination with monoclonal antibodies specific for IFN-gamma or TNF-alpha. These antibodies antagonized the protective effect of exogenous IL-12. Treatment of infected mice with a polygonal antibody specific for IL-12 resulted in a significant increase in parasitemia but did not affect the time to death. These latter studies demonstrate a role for endogenous IL-12 in resistance to T. cruzi. Together, our data identify an IL-12-mediated mechanism of resistance to T. cruzi, which is dependent on IFN-gamma and TNF-alpha.     

Suppression of antibody responses in humans infected with Trypanosoma cruzi.

Peripheral blood leukocytes from patients serologically positive for Chagas' disease were examined for their ability to respond to heterologous antigens in vitro. It was found that mononuclear cells from chagasic patients had greatly reduced ability to respond to sheep erythrocytes (SRBC) as compared with peripheral blood mononuclear cells (PBMC) from control subjects. The reduction in anti-SRBC antibody activity was independent of antigen dose and was not a result of differences in antibody response kinetics. Depletion of plastic-adherent result of differences in antibody response kinetics. Depletion of plastic-adherent cells from the PBMC of patients did not affect the suppressed state of the nonadherent lymphocytes. No relationship was evident between the duration of Trypanosoma cruzi infection and the degree of humoral responsiveness.     

Suppression of cellular responses in mice during Trypanosoma cruzi infections.

Immunosuppression has been reported to occur in several protozoan parasitic infections. The significance of this suppression on host resistance or on parasite avoidance of immune destruction has not, however, been determined. In the present study two strains of mice that differ with respect to resistance to Trypanosoma cruzi were examined during the course of infection for differences in expression of suppression of blastogenic responses to phytohemagglutinin and an antigen preparation of these parasites. It was found that in vitro blastogenic responses were suppressed in both strains of mice: on day 12 for C57BL/6 mice (resistant strain) and on day 17 for C3H(He) mice (susceptible strain). Neither C3H(He) nor C57BL/6 lymph node cells (LNC) responded to a crude sonically treated antigen of these parasites, although C57BL/6 LNC were inhibited by this antigen later in infection. There was no abrogation of suppression of LNC responses late in infection, when decreases in spleen weight or total spleen cell numbers occurred, in the resistant C57BL/6 mice. LNC from normal uninfected mice were found to be completely suppressed in responsiveness to phytohemagglutinin when cultured at a 2:1 ratio with LNC from C57BL/6 mice with 18-day infections. Attempts to characterize the cell type responsible for this suppression showed it to be a non-thy 1.2-bearing, nylon wool-adherent cell.     

Myocardial expression of endothelin-1 in murine Trypanosoma cruzi infection.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of myocarditis and chronic cardiomyopathy and is accompanied by microvascular spasm and myocardial ischemia. We reported previously that infection of cultured endothelial cells with T. cruzi increased the synthesis of biologically active endothlein-1 (ET-1). In the present study, we examined the role of ET-1 in the cardiovascular system of CD1 mice infected with the Brazil strain of T. cruzi and C57BL/6 mice infected with the Tulahuen strain during acute infection. In the myocardium of infected mice myonecrosis and multiple pseudocysts were observed. There was also an intense vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium. Immunohistochemistry studies employing anti-ET-1 antibody revealed increased expression of ET-1 that was most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for preproET-1, endothelin converting enzyme and ET-1 were observed in the same myocardial samples. Plasma ET-1 levels were significantly elevated in infected CD1 mice 10-15 days post infection. These observations suggest that increased levels of ET-1 are a consequence of the initial invasion of the cardiovascular system and provide a mechanism for infection-associated myocardial dysfunction.     

IgE dependent autoimmune response. Effect of Trypanosoma cruzi infection

The autoimmune response to mouse accessory glands (MAG) was investigated in male BALB/c mice immunized with different doses of chemically modified mouse accessory glands (MMAG) and complete Freund's adjuvant (CFA). This autoimmune response was studied at several time intervals using the skin test with MAG. It was found that 5 mg of MMAG induced on the day 15 an autoimmune response detected by specific skin test at 20 min., 3 h and 24 h. The results of the immediate type hypersensitivity (ITH) were higher than those with the other skin tests. In order to study the type of immunoglobulin involved, the ITH was also analyzed by passive cutaneous anaphylaxis (PCA) at different time intervals with treated and untreated sera at 56 degrees C. The findings suggest the presence of reaginic antibodies, IgE being the major antibody as detected by enzime linked immunosorbent assay (ELISA). The MAG was subsequently fractionated using Sephandex G-100 and the fractions thus obtained (FI,FII and FIII) were used to challenge mice immunized with MMAG. It was found that FI was the only fraction which revealed an ITH similar to that revealed by MAG. The effect of infection with Trypanosoma cruzi on the autoimmune response to MAG was analyzed with different mouse groups intraperitoneally treated with 2 x 10(3) blood trypomastigotes/animal at several time intervals: namely, on days -5, 0, +5 and +10 with respect to the immunization with MMAG. The autoimmune response to MAG showed suppression when the animals received the parasites on the same day as the autoantigen.     

Interleukin-12 mediates resistance to Trypanosoma cruzi in mice and is produced by murine macrophages in response to live trypomastigotes.

Host resistance to infection by Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the first week of infection in mice, NK cell-derived gamma interferon (IFN-gamma) is involved in controlling intracellular parasite replication, mainly through the induction of NO biosynthesis by activated macrophages. Interleukin-12 (IL-12) has been shown to be a powerful cytokine in inducing IFN-gamma synthesis by NK cells, as well as in mediating resistance to different intracellular protozoa. We have therefore studied the ability of T. cruzi to elicit IL-12 synthesis by macrophages and the role of this cytokine in controlling parasite replication during acute infection in mice. Our results show that macrophages cultured in the presence of live trypomastigote forms (but not epimastigotes) release IL-12 that can induce IFN-gamma production by normal spleen cells. IL-12 was detected in as little as 12 h after the addition of the trypomastigotes, and the level of IL-12 peaked at 48 h after the initial macrophage-parasite incubation. The addition of anti-IL-12 monoclonal antibody to macrophage-trypomastigote supernatants dose-dependently inhibited IFN-gamma production by naive splenocytes. Finally, the in vivo role of IL-12 in resistance to infection by T. cruzi was analyzed. Mice treated with anti-IL-12 monoclonal antibody had significantly increased parasitemia and mortality in comparison with those of control infected mice treated with control antibody. Together, these results suggest that macrophage-derived IL-12 plays a major role in controlling the parasitemia in T. cruzi-infected mice and that the animal's resistance during the acute phase of infection may, at least in part, be a consequence of postinfection levels of IL-12.     

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.     

Differential regulation of lymphoproliferative responses to Trypanosoma cruzi antigen in patients with the cardiac or indeterminate form of Chagas disease

In the search to identify differences in the immunological response between patients with the indeterminate or cardiac form of Chagas disease, trypomastigote-specific peripheral blood mononuclear cell (PBMC) proliferative responses were studied. Suppression of lymphoproliferation occurred in both groups of patients, being more intense in those with the cardiac form. By adding to the cultures neutralizing mAbs anti-IFN-gamma, anti-IL-4, anti-IL-13, or anti-IL-10, indomethacin to block prostaglandin synthesis, NMMA as inhibitor of nitric oxide (NO) synthesis, or glutathione-peroxidase as H(2)O(2) scavenger, it was found that indomethacin augmented lymphoproliferation in both groups of patients. However, anti-IL-10 treatment increased proliferation only in PBMC cultures from patients with the cardiac form, indicating that in these patients, IL-10 was suppressing the immune response. These patients also had higher IL-10 levels in unstimulated cultures and higher PGE(2) levels in stimulated cultures. The results evidence that IL-10 regulates parasite-specific T cell responses in patients with the cardiac form, whereas regulation by prostaglandins (PG) occurs in patients with either cardiac or indeterminate form of the disease.     

Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection

In experimental Chagas' disease, lymphocytes from mice infected with Trypanosoma cruzi show increased apoptosis in vivo and in vitro. Treatment with a pan-caspase blocker peptide inhibited expression of the active form of effector caspase-3 in vitro and rescued both B and T cells from cell death. Injection of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone, but not a control peptide, reduced parasitemia and lymphocyte apoptosis in T. cruzi-infected mice. Moreover, treatment with caspase inhibitor throughout acute infection increased the absolute numbers of B and T cells in the spleen and lymph nodes, without affecting cell infiltrates in the heart. Following treatment, we found increased accumulation of memory/activated CD4 and CD8 T cells, and secretion of IFN-gamma by splenocytes stimulated with T. cruzi antigens. Caspase inhibition in the course of infection reduced the intracellular load of parasites in peritoneal macrophages, and increased the production of TNF-alpha and nitric oxide upon activation in vitro. Our results indicate that inhibition of caspases with a pan-caspase blocker peptide improves protective type-1 immune responses to T. cruzi infection. We suggest that mechanisms of apoptosis are potential therapeutic targets in Chagas' disease.     

Genetic immunization elicits antigen-specific protective immune responses and decreases disease severity in Trypanosoma cruzi infection

Immunity to Trypanosoma cruzi requires elicitation of humoral and cell-mediated immune responses to extracellular trypomastigotes and intracellular amastigotes. In this study, the effectiveness of the T. cruzi trans-sialidase family (ts) genes ASP-1, ASP-2, and TSA-1 as genetic vaccines was assessed. Immunization of mice with plasmids encoding ASP-1, ASP-2, or TSA-1 elicited poor antigen-specific cytotoxic-T-lymphocyte (CTL) activity and T. cruzi-specific antibody responses. Codelivery of interleukin-12 and granulocyte-macrophage colony-stimulating factor plasmids with antigen-encoding plasmids resulted in a substantial increase in CTL activity and antibody production and in increased resistance to T. cruzi infection. In pooled results from two to four experiments, 30 to 60% of mice immunized with antigen-encoding plasmids and 60 to 80% of mice immunized with antigen-encoding plasmids plus cytokine adjuvants survived a lethal challenge with T. cruzi. In comparison, 90% of control mice injected with empty plasmid DNA died during the acute phase of infection. However, the pool of three ts genes provided no greater protection than the most effective single gene (ASP-2) either with or without coadministration of cytokine plasmids. Importantly, the extent of tissue parasitism, inflammation, and associated tissue damage in skeletal muscles during the chronic phase of T. cruzi infection in mice immunized with antigen-encoding plasmids plus cytokine adjuvants was remarkably reduced compared to mice immunized with only cytokine adjuvants or empty plasmid DNA. These results identify new vaccine candidates and establish some of the methodologies that might be needed to develop effective vaccine-mediated control of T. cruzi infection. In addition, this work provides the first evidence that prophylactic genetic immunization can prevent the development of Chagas' disease.     

Heterophil nature of EVI antibody in Trypanosoma cruzi infection

Previous findings implying the autoreactive capability of circulating complement-fixing (EVI) antibodies in patients with Chagas' disease (American trypanosomiasis), under both in vitro and in vivo conditions have been irreproducible. The presence of the tissue antigens involved in the reactivity with EVI antibodies is exclusively confined to nonhuman substrates, suggesting the heterophil nature of these antibodies which, on the other hand, have also been described in human sera collected in areas free from Chagas' disease. This would preclude any direct pathogenic effect of EVI antibodies when present in the circulation, though they may be useful immunological markers of unsuspected Trypanosoma cruzi infection in endemic areas.     

Use of parasite antigens and interleukin-2 to enhance suppressed immune responses during Trypanosoma cruzi infection in mice.

Mice infected with Trypanosoma cruzi exhibit an early and profound suppression of parasite-specific and nonspecific immune responses. Earlier studies have shown that this suppression is due, at least in part, to suppressor macrophages, deficiency in production of interleukin-2 (IL-2), and reduced T helper (Th)-cell activity. In the present study, the effect of exogenously supplied IL-2 on enhancement of parasite-specific Th-cell activity, anti-parasite immunoglobulin G (IgG) and IgM antibody levels, parasitemia, and longevity was examined in infected mice. The results showed that administration of IL-2 with and without antigenic stimulation with trinitrophenylated T. cruzi significantly enhanced parasite-specific IgM and IgG levels. Injection of IL-2 and trinitrophenylated T. cruzi together significantly enhanced parasite-specific Th-cell activity and was more effective in enhancement of parasite-specific antibody levels. In addition, it was found that IL-2 alone had a rapid and lasting effect in reducing parasitemia. These results suggest that deficiency in IL-2 may play a major role in host susceptibility to T. cruzi.