Do commercial serologic tests for Trypanosoma cruzi infection detect Mexican strains in women and newborns?

We sought to determine the serological test that could be used for Trypanosoma cruzi seroprevalence studies in Mexico, where lineage I predominates. In a previous study among pregnant women and their newborns in the states of Yucatan and Guanajuato, we reported a 0.8-0.9% of prevalence for T. cruzi -specific antibodies by Stat-Pak and Wiener ELISA. We have expanded this study here by performing an additional non-commercial ELISA and confirming the seropositives with Western blot, using whole antigens of a local parasite strain. We found a seroprevalence of 0.6% (3/500) in Merida and 0.4% in Guanajuato (2/488). The 5 seropositive umbilical cord samples reacted to both non-commercial ELISA and Western blot tests, and only 1 of the maternal samples was not reactive to non-commercial ELISA. A follow-up of the newborns at 10 mo was performed in Yucatan to determine the presence of T. cruzi antibodies in children as evidence of congenital infection. None of the children was seropositive. One newborn from an infected mother died at 2 wk of age of cardiac arrest, but T. cruzi infection was not confirmed. The T. cruzi seroprevalence data obtained with both commercial tests (Stat-Pak and ELISA Wiener) are similar to those from non-commercial tests using a local Mexican strain of T. cruzi.     

Carbohydrate epitopes are responsible for antibody cross-reactivity in Trypanosoma cruzi-infected mice.

Anti-Trypanosoma cruzi antibodies can be eluted from western blots of T. cruzi antigens and thereby are fractionated on the basis of the electrophoretic mobility of the antigens to which they bind. Antibodies fractionated by these methods can bind antigens with electrophoretic mobility different from those antigens from which they are eluted. Such antibodies thus are considered cross-reactive. Studies in which the target antigens are reacted with sodium periodate to destroy carbohydrate epitopes prior to exposure to the eluted antibodies revealed that antibodies are produced that bind to both carbohydrate and noncarbohydrate epitopes on western blots, but that most of the cross-reactive antibodies are directed toward carbohydrate moieties.     

Antigenic make-up of Trypanosoma cruzi culture forms: identification of a specific component.

A comparison of Trypanosoma cruzi water soluble antigens with those of stercorarian and salivarian trypanosomes, and Leishmania using immunoprecipitation in gels and immunoelectrophoresis, with the aid of hyperimmune rabbit serum and heterologous adsorptions showed the following. 1) There is a high complexity of soluble antigens of T. cruzi and T. rangeli. 2) At the intraspecific level our results demonstrated the antigenic stability of T. cruzi when maintained in vitro, and that there was quantitative antigenic consistency of the culture forms of different strains of T. cruzi from diverse geographic and parasite sources. At the interspecific level, the antigenic relationships between T. cruzi and the other Trypanosomatidae were established, as follows: 6/10ths of the antigens are shared by stercorarian species (T. dionisii, T. rangeli); 4/10ths by a salivarian trypanosome (T. brucei); and 3/10ths by Leishmania (L. donovani, L. mexicana). 3) Among the 4/10ths of antigenic components specific to T. cruzi, one component was characterized by its antigenicity and immunogenicity in natural and experimental infections, and in immunization experiments; this component was specific to T. cruzi when compared to the other Trypanosomatidae antigens.     

Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite

Polyclonal antibodies obtained against antigenic proteins encoded by six recombinant DNA clones of Trypanosoma cruzi were used for the ultrastructural localization of the respective antigens in thin sections of parasites (epimastigote, amastigote and trypomastigote forms of T. cruzi) embedded at low temperature in Lowicryl K4M resin. Antigens of high molecular weight containing tandemly repeated amino acid sequence motifs and recognized by sera from patients with Chagas' disease, were located only in the flagellum, where it contacts the parasite cell body. Other antigens were located on the surface of the parasite while still others were found within the flagellar pocket, as is the case with an antigen released during the acute phase of Chagas' disease. Thus, we conclude that some of the T. cruzi proteins which are antigenic in human infections are located in defined regions of the parasite. Some of the antigens were not expressed to the same extent in the three different developmental stages of the parasite.     

The natural heterogeneity of trypanosoma cruzi: Biological and medical implications

Trypanosoma cruzi is a heterogeneous group of parasites. The imposition of natural or artificial pressures can result in the selection of subsets of the population with concomitant changes in characteristics used to evaluate the group. In order to ascertain the extent of heterogeneity, stocks of single-cell clones were prepared from various sources. Selected cell biological, biochemical, immunochemical, parasitological, and histopathological parameters of these clones have been studied. A ten-fold difference in the rate of growth of the epimastigote stage of T cruzi clones has been observed. The extracellular growth rates of the clones correlate with the rate of growth of the obligate intracellular amastigote stage and consequently, the length of intracellular cycle of the parasite. A 40% difference in the amount of total DNA/parasite has been found between clones. Although the amount of DNA/kinetoplast and nucleus varies between clones, the major contribution to the differences in total DNA/parasite appears to be the nucleus. From 16 to 35 antigens have been demonstrated in the T cruzi clones assayed to date. Five to seven of these antigens are common to all of the stocks assayed. However, both isolate- and clone-specific antigens have also been demonstrated. The susceptibility of inbred strains of mice to T cruzi clones varies with the clone of the parasite. These data imply that the genetics of the parasite as well as the host modulate both the course and outcome of a T cruzi infection. The influence of monosaccharides on the receptor-mediated infection of vertebrate cells by trypomastigotes of T cruzi also varies between clones. The implications of these findings upon our concept and understanding of present and future problems in Chagas disease are discussed.     

EVI antibodies in patients with Chagas' disease: relationship with anti-Trypanosoma cruzi immunoglobulins and effects of specific treatment

Antibodies against heart vascular structures and striated muscle cells interstitium (EVI antibodies) persist in Chagas' disease patients who had been cured by specific treatment as demonstrated by negative xenodiagnosis, conventional serology (CS) and complement mediated lysis (CoML). On the other hand, EVI antibodies are either present or absent in treated patients presenting positive CS but negative CoML. Since CoML detects antibodies associated to resistance, EVI antibodies are not likely to participate in the control of T. cruzi infections although they might be induced by cross-reacting antigens of heart cells and the parasite. They are neither necessarily related to antibodies responsible for CS. Absorption with T. cruzi and heart tissue confirms the suggestion that EVI antibodies are induced by a number of antigenic determinants, most from heart structures with a minor participation of T. cruzi antigens.     

Trypanosoma cruzi: expression of antigens on the membrane surface of parasitized cells

A direct immunofluorescent antibody test with an anti-Trypanosoma cruzi F(ab')2 conjugate was used to demonstrate antigens of T. cruzi on the membrane surface of intact live or fixed macrophages and L929 mouse fibroblasts infected with the organism. Antigens were demonstrated in 5 to 50% of infected cells, and their presence was not directly related to the number of intracellular organisms. Cells with as few as four intracellular amastigotes had demonstrable surface antigens, whereas some cells with as many as twelve or more organisms did not. Capping of antigen-antibody complexes was noted to begin a few minutes after the addition of the anti-T cruzi F(ab')2 conjugate; by 30 min, most of the parasitized cells had eliminated the complexes, and no surface antigen of parasitic nature could be demonstrated. Although capping may have caused a negative result in a previously positive cell, other mechanisms may be involved, because antigens were not demonstrated in some heavily parasitized cells examined immediately after completion of the test. Treatment of the infected cells with trypsin or chymotrypsin resulted in the absence of demonstrable parasite antigens on the cell membrane surface. However, the antigens were again demonstrated 12 hr after the enzymes were removed. The reappearance of parasite antigens on the surface of infected cells was prevented by treatment of the monolayers with puromycin or tunicamycin. A T cell-enriched population of spleen lymphocytes from mice chronically infected with T. cruzi recognized the membrane-bound antigens and proceeded to destroy the host cell and the intracellular organisms. In this process, noninfected cells were also destroyed, possibly because they were coated with antigens released from intact infected cells or from infected cells that had been lysed by the action of the sensitized lymphocytes or their products.     

Experimental American leishmaniasis and Chagas' disease in the Brazilian squirrel monkey: effect of dual infection on antibodies to parasite antigens.

Adult, laboratory-bred squirrel monkeys (Saimiri sciureus) were infected with either Leishmania braziliensis braziliensis or L. b. panamensis and, 42 weeks later, they were challenge-infected with Trypanosoma cruzi. Another group of monkeys was infected with T. cruzi and challenged with L. b. braziliensis after 42 weeks. Immunoblotting was used to examine parasite antigens bound by antibodies in plasma obtained from the monkeys during the course of primary and challenge infections. During primary infections Leishmania-infected monkeys produced antibodies which bound to a number of Leishmania antigens, most notably a Leishmania antigen of 72 kDa, which were not recognized by antibodies produced by the monkeys given a primary infection of T. cruzi. These Leishmania-induced antibodies were no longer detectable 42 weeks after primary infections. However, when the Leishmania-infected monkeys were challenged with T. cruzi they once again produced antibodies capable of binding numerous Leishmania antigens, including the antigen of 72 kDa, which had not been recognized by antibodies produced by the monkeys with primary T. cruzi infections. A similar phenomenon was observed in T. cruzi-infected animals following Leishmania challenge.     

Infection of T lymphocytes by Trypanosoma cruzi. Possible role of CD3 and HLA-DR

The mechanisms by which the causative agent of Chagas' disease impair its host's immune response are of paramount importance but poorly understood. Results presented in this paper show for the first time that Trypanosoma cruzi trypomastigotes infect T lymphocytes in vitro and more interestingly in vivo, and that trypomastigotes released from infected cells are infectious. In addition treatment of purified human T lymphocytes with McAb against CD3 and HLA-DR antigens significantly inhibited parasite infection. T. cruzi antigens were detected on the membrane of infected T cells and could therefore represents targets for cytotoxic mechanisms. These results might have important consequences for the understanding of the dramatic disruption of immune response observed during Chagas' disease and more generally provide additional information on T lymphocyte infection by pathogens.     

Immunoblot analysis of trypomastigote excreted-secreted antigens as a tool for the characterization of Trypanosoma cruzi strains and isolates

An analysis of antibody recognition of Trypanosoma cruzi exoantigens by immunoblotting revealed a unique banding pattern that seems to be characteristic of each strain or isolate. Trypomastigote excreted-secreted antigens (TESA) present in supernatants of LLC-MK2 cells infected with 5 strains and 10 isolates of T. cruzi produced 13 different immunoblotting patterns. The same bands were observed when probed with acute-phase Chagas' disease serum or with serum from a rabbit immunized with the repetitive domain of T. cruzi transialidase recombinant protein (anti-shed acute-phase antigens). Three similar patterns were observed with TESA from 3 human isolates that probably belong to the same T. cruzi strain. When clone CL Brener, clone CL-14, and CL parental strain were analyzed, the same bands were observed, although they presented different biological behavior. These results suggest that immunoblotting analysis of TESA may be a useful tool for characterization of T. cruzi strains and isolates.     

Analysis of antibody cross-reactivity in experimental American trypanosomiasis.

Susceptible C3H/He mice were immunized with the avirulent Corpus Christi strain of Trypanosoma cruzi and subsequently infected with virulent Brazil stain organisms. Seventy days after infection sera were isolated and analyzed on western blots of electrophoretically separated T. cruzi antigens prepared from culture-form parasites (primarily epimastigotes). More than 25 bands were identified. The antibodies were fractionated by elution from various regions of western blots corresponding to average molecular weights of approximately greater than 130, 77, 70, 60, 48, or 38 kDa. Each of these antibody preparations was then incubated with strips of nitrocellulose containing all of the electrophoretically separated T. cruzi, and cross-reactivity was determined. Antibodies isolated from the 130-, 77-, and 70-kDa regions all cross-reacted with each other. Antibodies eluted from the 60-kDa region bound antigens in the 60-, 70-, and the 77-kDa regions. More importantly, antibodies eluted from every region bound antigens in the 70-kDa region. Conversely, antibodies eluted from this region bound to antigens in all of the other regions. These data indicate the presence of (a) common antigenic epitope(s) in T. cruzi infections in these mice that is predominantly found in the 70-kDa antigen-antibody complex on western blots.     

Heat-killed Trypanosoma cruzi induces acute cardiac damage and polyantigenic autoimmunity

Chagas heart disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially fatal cardiomyopathy often associated with cardiac autoimmunity. T. cruzi infection induces the development of autoimmunity to a number of antigens via molecular mimicry and other mechanisms, but the genesis and pathogenic potential of this autoimmune response has not been fully elucidated. To determine whether exposure to T. cruzi antigens alone in the absence of active infection is sufficient to induce autoimmunity, we immunized A/J mice with heat-killed T. cruzi (HKTC) emulsified in complete Freund's adjuvant, and compared the resulting immune response to that induced by infection with live T. cruzi. We found that HKTC immunization is capable of inducing acute cardiac damage, as evidenced by elevated serum cardiac troponin I, and that this damage is associated with the generation of polyantigenic humoral and cell-mediated autoimmunity with similar antigen specificity to that induced by infection with T. cruzi. However, while significant and preferential production of Th1 and Th17-associated cytokines, accompanied by myocarditis, develops in T. cruzi-infected mice, HKTC-immunized mice produce lower levels of these cytokines, do not develop Th1-skewed immunity, and lack tissue inflammation. These results demonstrate that exposure to parasite antigen alone is sufficient to induce autoimmunity and cardiac damage, yet additional immune factors, including a dominant Th1/Th17 immune response, are likely required to induce cardiac inflammation.     

Immunological characterization of antigens released by Trypanosoma cruzi-infected cells.

Chagas' disease, caused by Trypanosorna cruzi, is characterized by the appearance of pathological lesions in the heart and other tissues during the chronic phase. The mechanisms responsible for such damage are still unclear. In the vertebrate host, T. cruzi replicates intracellularly before transforming from amastigotes into trypomastigotes. The infected host cell then lyses, releasing the cytoplasmic contents and the parasites that shed membrane glycoproteins soon after release. The sum of all these components we have termed released antigen (Rag). We characterized antigens, released in vitro by fibroblasts infected with T. cruzi, obtained by concentrating conditioned serum-free culture media. The results demonstrate that Rag contains a complex protein mixture including stage-specific T. cruzi antigens (Ssp-1, -2, -4), glucose-regulated protein (Grp) 78h, and peptides recognized by the monoclonal antibody 2B10. These peptides exhibit neuraminidase activity and are expressed by intracellular and 10-20% of released trypomastigotes. Additionally, Rag is recognized by sera from T. cruzi-infected mice and human chagasic patients. Rag also stimulates in vitro production of interferon-gamma by splenocytes from resistant C57B1/6 and susceptible BALB/c infected mice and interleukin-4 by splenocytes from BALB/c infected mice. Altogether these results indicate that Rag is immunologically relevant and could contribute to pathogenesis of T. cruzi infection.     

Rapid determination of Trypanosoma cruzi urinary antigens in human chronic Chagas disease by agglutination test

Detection of Trypanosoma cruzi in man becomes particularly difficult during the chronic stage of Chagas disease because of the low parasitemia. We were able to develop a simple and straightforward method for determining the concentration of T. cruzi antigens in urine using nitrocellulose micellar suspension (Nitrocell-Mr, Polychaco Argentina) and for their subsequent detection through a "latex" type agglutination test. The latex used was an esferocell nitrocellulose suspension (Esferocell-Mr, Polychaco). Specific antigens for T. cruzi were detected in 54 of 58 urine samples from chronic chagasic patients. The antigens characterized by affinity chromatography and SDS-PAGE were glycoproteins with apparent molecular weights (and pIs) of 100 kDa (pI 5 to 5.5), 80 kDa (pI 6.0), and 50 kDa (pI 6.5 to 7.0). This method is practical and fulfills the requirement of large-scale epidemiological studies. It is also helpful in cases of conflictive serology.     

Immunopathology of cardiomyopathy in the experimental Chagas disease

The mechanisms by which Trypanosoma cruzi causes cardiomyopathy and induces neuronal destruction are discussed in this paper. The results suggest that autoimmunity in the chronic phase is the main cause of the progressive cardiac destruction, and that autoreactivity is restricted to the CD4+ T cell compartment. During the acute phase, the neuronal and cardiac fiber destruction occurs when ruptured parasite nests release T. cruzi antigens that bind to the cell surface in the vicinity which become targets for the cellular and humoral immune response against T. cruzi. The various factors involved in the genesis of autoimmunity in chronic T. cruzi infection include molecular mimicry, presentation of self-antigens and imbalance of immune regulation.     

Antigenic analysis of Trypanosoma cruzi strains by crossed immunoelectrophoresis: demonstration and isolation of antigens particular to some strains

Antigenic differences among soluble extracts of Y, CL, SF, and Colombian strain epimastigotes of Trypanosoma cruzi have been demonstrated by crossed immunoelectrophoresis with an intermediate gel containing the heterologous antiserum. Using crossed immunoelectrophoresis with the homologous antiserum, over 30 precipitin lines could be demonstrated for each strain and, even though marked differences were observed in experimental infections, the strains shared a significant number of antigens. In addition, some strain-particular antigens were isolated using affinity chromatography. These antigens could be valuable in the study of biological, immunological, and pathological characteristics of experimental and natural T. cruzi infections.     

Comparative evaluation of therapeutic DNA vaccines against Trypanosoma cruzi in mice

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem in most of Latin America. A key priority is the development of new treatments, due to the poor efficacy of current ones. We report here the comparative evaluation of therapeutic DNA vaccines encoding various T. cruzi antigens. ICR mice infected with 500 parasites intraperitoneally were treated at 5 and 12 days postinfection with 20 microg of plasmid DNA encoding T. cruzi antigens TSA-1, TS, ASP-2-like, Tc52 or Tc24. Treatment with plasmid encoding TS and/or ASP-2-like antigens had no significant effect on parasitemia or survival. Treatment with Tc52 DNA significantly reduced parasitemia, as well as cardiac parasite burden, and improved survival, although myocarditis was not significantly affected. Finally, treatment with plasmids encoding Tc24 and TSA-1 induced the most complete control of disease as evidenced by significant reductions in parasitemia, mortality, myocarditis and heart parasite burden. These data demonstrate that therapeutic vaccine efficacy is dependent on the antigen and suggest that DNA vaccines encoding Tc24, TSA-1, and Tc52 represent the best candidates for further studies of a therapeutic vaccine against Chagas disease.     

Comparison of genes encoding Trypanosoma cruzi antigens

Trypanosoma cruzi, the causative agent of Chagas disease, simultaneously expresses several different surface antigens. This contrasts sharply with blood-stream forms o f the African trypanosomes, which display only one variant surface glycoprotein at a time. Over the past few years, the genes coding for a number of T. cruzi proteins recognized by sera from patients have been cloned and at least partially sequenced. However, some of those discovered in more than one laboratory have been given different names. Here, Carlos Frasch, Juan Cazzulo, Lena Aslund and UIf Pettersson try to systematize the literature available on these antigens, including what is known about their localization and function.     

Suppressive substance produced by T cells from mice chronically infected with Trypanosoma cruzi. II. Partial biochemical characterization

Culture supernatants of splenic T cells from susceptible CBA mice chronically infected with Trypanosoma cruzi contain a suppressive substance which can inhibit the induction of delayed-type hypersensitivity (DTH) to a wide range of antigens. The suppressive substance is distinct from T. cruzi antigen inasmuch as the supernatant depleted of any residual T. cruzi antigen by an affinity column still retains the suppressive activity, whereas addition of T. cruzi antigens to control supernatant did not confer suppressive function. The suppressive supernatant does not contain detectable levels of IL-1, IL-2, IL-3, or IFN-gamma but a modest level of IL-1 and IL-2 inhibitory activities. However, both these inhibitory activities elute at a different position from the DTH suppressive activity on gel filtration. The DTH suppressive activity is heat labile (1 h, 56 degrees C), cryostable, but destroyed by trypsin treatment. It binds to ricin but not to lentil lectin. Sepharose 4B gel filtration and HPLC analysis in mild chaotropic agents (urea, ethylene glycol) demonstrate that the suppressive substance has an apparent Mr of 30 to 60 kDa, but full DTH-suppressive activity is retained only in an aggregated form.     

Suppression of cell-mediated immunity in experimental Chagas' disease.

The effect of acute infection with the Tulahuén strain of Trypanosoma cruzi on the cellular immune response in Swiss mice was studied. Mice were immunized with either Freund's complete adjuvant or oxazolone, a skin sensitizing agent, and subsequently skin-tested with either BCG protoplasm or oxazolone to detect delayed hypersensitivity. Depression of the response to these antigens was observed in infected mice during the stage of marked parasitemia. Mice which were responsive to oxazolone before infection lost their ability to respond as the infection progressed. When immunized with live attenuated T. cruzi before infection with virulent organisms, mice developed a greater than normal sensitivity to oxazolone and survived infection. These experiments do not conclude whether immunosuppression due to infection with T. cruzi is directed toward induction or expression of the cell-mediated immune response to the antigens employed.