Cellular immune responses of chagasic patients to antigens derived from different Trypanosoma cruzi strains and clones.

The reactivity of peripheral blood mononuclear cells (PBMN) from 62 chagasic patients to antigens prepared with different Trypanosoma cruzi strains and clones belonging to different zymodemes was evaluated by the incorporation of 3H-thymidine into DNA. Standardization of experimental conditions was carried out by establishing the proper antigen concentration (15-20 micrograms protein), the adequate period of time (5-6 days) and the best cell concentration (300,000/well). Individual analysis of 62 patients showed 2 distinct patterns of cellular response. One group of patients (32%) had low cellular responses to all antigens tested while the remaining patients had high response to at least 1 of the antigens. No relationship of the immune responsiveness to the patients' clinical forms could be established. In addition, the PBMN response to different strain and clone antigens was not statistically significant. Thus, it appears that the cellular response induced by any particular clone or strain represents an expression of the stimulation of their common antigenic make-up.

Idiotypic/anti-idiotypic interactions in schistosomiasis and Chagas' disease.

Immunoaffinity-purified antibodies against soluble Schistosoma mansoni egg antigens (SEA) were isolated from the sera of patients with schistosomiasis mansoni. Similarly, antibodies against Trypanosoma cruzi epimastigote antigens were obtained from sera of patients with Chagas' disease. These antibody preparations were used in culture to demonstrate the presence of anti-idiotypic T lymphocytes in peripheral blood mononuclear cell preparations from patients with either schistosomiasis mansoni or Chagas' disease, or with both of these infections. Only cells from patients with schistosomiasis or both infections proliferated upon exposure to the anti-SEA antibodies. Conversely, only cells from patients with Chagas' disease or both infections responded to anti-epimastigote antibodies. Western blot analysis of SEA and epimastigote antigens, developed by patients' sera or by immunoaffinity-purified antibody preparations, substantiated that anti-SEA immunoaffinity-purified antibodies only reacted with components of SEA, and anti-epimastigote immunoaffinity-purified antibodies only reacted with components of epimastigote antigenic preparation. These studies demonstrate the presence of anti-idiotypic T lymphocytes in the peripheral blood of patients with schistosomiasis or Chagas' disease which are specific for idiotypes generated during these infections.

Use of Trypanosoma cruzi purified glycoprotein (GP57/51) or trypomastigote-shed antigens to assess cure for human Chagas' disease.

With the exception of assays for the detection of antibodies promoting complement-mediated lysis of Trypanosoma cruzi, serologic tests have generally failed to assess the effectiveness of chemotherapy for Chagas' disease. Conventional serology, although useful for the diagnosis of infection, is not capable of determining which patients have been cured. Here we demonstrate that a high proportion of antibodies detected by conventional serology (using fixed epimastigotes or trypomastigotes or crude extracts obtained therefrom) are directed against the carbohydrate residue galactosyl alpha 1- > 3 galactose (Gal alpha 1- > 3 Gal), a determinant also recognized by antibodies from noninfected healthy volunteers. In a study of 14 cured patients with long-term followup, we found that the persistently positive reactions detected using conventional serology were largely eliminated following immunoadsorption with melibiose. Because of their wide distribution among microorganisms of intestinal and pulmonary microflora, these carbohydrate determinants may keep stimulating lymphocytes previously primed by T. cruzi Gal alpha 1- > 3 Gal epitopes, thereby accounting for false-positive results in cured patients. Consistent with this proposition, enzyme-linked immunosorbent assays performed with two distinct T. cruzi antigen preparations that lack the Gal alpha 1- > 3 Gal epitope, namely purified GP57/51 and trypomastigote-shed antigens, were indeed capable of determining a cure after chemotherapy, albeit to a different degree. Collectively, the data indicate that conventional immunoassays prepared with highly specific T. cruzi antigens can be useful in the assessment of a cure after chemotherapy.

Effect of specific chemotherapy on the levels of lytic antibodies in Chagas's disease.

Clinical trials with compounds active in Chagas's disease have shown that after treatment parasitological diagnostic methods (xenodiagnosis) become repeatedly negative whereas conventional serology (immunofluorescence and complement fixation tests) persists steadily positive. Consequently, assessment of cure still remains controversial. This paper reports the influence of specific treatment on antibodies involved in the conventional serological diagnosis and on antibodies which bind to the living bloodstream forms and are related to host resistance. Antibodies lytic to Trypanosoma cruzi bloodstream stages were detected, through a complement-mediated lysis (CML) test, in: (a) 100% of 28 untreated patients; (b) 94% of a group of 21 treated patients in whom conventional serology remained positive, including those with persistently negative xenodiagnosis; (c) 0% of 17 normal controls. In some patients treated with a nitrofuran derivative (nifurtimox) or with a 2-nitroimidazole derivative (benznidazol), CML test became gradually negative whereas conventional serology continued to be positive. Finally, in five patients treated with benznidazol, serological tests, CML and xenodiagnosis became regularly negative, strongly suggesting parasitological cure. Those findings demonstrate a dissociation between the antibodies mediating serological diagnosis and those directed against living bloodstream parasites. Moreover, since in some patients both types of antibodies disappeared after treatment, the results suggest that cure of Chagas's disease should be based not only on negative xenodiagnosis but also on the elimination of specific antibodies detectable by conventional serology and CML test.

Lytic antibody titre as a means of assessing cure after treatment of Chagas disease: a 10 years follow-up study.

A complement-mediated lysis test (CoML) using living trypomastigotes was compared with conventional serological methods and with haemoculture. Over a 10 years follow-up period evidence was obtained which supported the view that chagasic patients, treated with nitroheterocyclic drugs, in whom CoML had reverted to negative, might be considered cured despite conventional serology remaining positive.

Hemocultures from chronic Chagasic patients using EDTA or heparin as anticoagulants.

Hemoculture tests, a method for the detection of Trypanosoma cruzi, were used to investigate the effects of the anticoagulants heparin or EDTA on the parasite growth in culture medium (liver infusion tryptose, LIT). Hemocultures from 13 patients with positive serology for chronic Chagas' disease performed in parallel with both anticoagulants resulted in a total of seven (54%) positive hemocultures, three positive with blood samples collected with EDTA (23%), two with heparin (15%) and two with both anticoagulants (15%). There was no significant difference between the number of positive tubes in blood samples collected with either heparin (11%) or with EDTA (13%), an indication that heparin does not block the growth of T. cruzi. However, the simultaneous use of both anticoagulants may improve the positivity index of the hemocultures.

IFN-gamma in human Chagas' disease: protection or pathology?

An apparently paradoxical role for IFN-gamma in human Chagas' disease was observed when studying the pattern of cytokine production by peripheral blood mononuclear cells (PBMC) obtained from two groups of chagasic patients after specific stimulation with Trypanosoma cruzi-derived antigens. The groups studied were 1) patients treated with benznidazole during the acute phase of Trypanosoma cruzi infection and 2) chronically infected untreated patients. In the treated group, higher levels of IFN-gamma were produced by PBMC from individuals cured after treatment when compared to non-cured patients. In contrast, in the chronically infected group (not treated) higher levels of IFN-gamma were produced by PBMC from cardiac patients in comparison with asymptomatic (indeterminate) patients. This apparently paradoxical role for IFN-gamma in human Chagas' disease is discussed in terms of the possibility of a temporal difference in IFN-gamma production during the initial stages of the infection (acute phase) in the presence or absence of chemotherapy. The maintenance of an immune response with high levels of IFN-gamma production during the chronic phase of the infection may favor cure or influence the development of the cardiac form of the disease.

[A longitudinal electrocardiogram study of Chagas' disease from the acute phase]. / Estudo longitudinal do electrocardiograma na doença de Chagas desde a fase aguda.

Several studies have been done to analyse the relationship between the characteristics of the electrocardiogram (ECG) and mortality in the several stages of the disease, using different methods like multiple case and longitudinal studies. We analysed the ECG from the acute stage up to twenty years of follow-up (+/- 9 years) in 42 patients with Chagas' disease to determine their evolution and it's value like an index for therapeutic evaluation. The 42 patients (18 female, 24 males) were originally from the north of the State of Minas Gerais (Brazil) and the initial stage was mainly in the first two decades of age. All bad cardiac involvement and received full specific treatment. We utilized the following criteria for the ECG analyses: Modified Minnesota Code for Chagas' disease; WHO/ISFC TASK FORCE for inter ventricular conduction disturbances and Pieretti criteria for inactive electrical areas. We conclude that: a) The electrocardiogram changes tend to get worse with evolution into the chronic stage; b) The electrocardiogram is not a good index for therapeutic valuation.

[Hemoculture: sensitive technique in the detection of Trypanosoma cruzi in chagasic patients in the chronic phase of Chagas disease]. / Hemocultura: técnica sensível na detecção do Trypanosoma cruzi em pacientes chagásicos na fase crônica da doença de chagas.

The sensitivity of hemocultures, performed once or three times, was investigated in 52 patients in the chronic phase of Chagas disease. Modifications were introduced in the technique such as, processing the blood more rapidly, gently homogenizing the cultures and examining them after 120 days of culture. Our results show a high percent age of positivity i.e. 79% and 94% of patients submitted, respectively, to one or three tests. No significant differences related to the patients age were detected, which varied from 14 to 82 years old. Our results demonstrate that hemoculture is a sensitive method for the parasitological diagnosis of Chagas disease and is ideal for monitoring cure in treated patients.

[Soluble antigens released by Trypanosoma cruzi trypomastigotes used in ELISA to detect cure in chagasic patients following specific treatment]. / Antígenos solúveis liberados por tripomastigotas de Trypanosoma cruzi utilizados no teste de ELISA para detectar cura em pacientes chagásicos após tratamento específico.

Two soluble antigens isolated from T. cruzi were evaluated by ELISA for the diagnosis of chronic infection and assessment of cure after specific chemotherapy, namely, supernatants from parasite cell-cultures (SpAg) and trypomastigote excretory/secretory antigens (ESAg). Among the treated patients, a group defined as "dissociated" had been monitored for 3 to 10 years and displayed negative hemocultures and tests of trypomastigote complement-mediated lysis persistently negative although positive by conventional serology (indirect fluorescence with epimastigote, mainly). A negative lysis test indicates parasite elimination by the patient. Our ELISA results showed that among the non-treated chagasic controls the SpAg e ESAg detected 93% and 100%, of the cases, respectively. However, only 28% of sera from the dissociated group, considered as cured, were positive by ELISA using SpAg whereas all of such sera were negative using ESAg. Therefore ELISA with ESAg seems to be ideal to replace the complement-mediated lysis test with the aim to define cure after treatment of the chronic infection by T. cruzi.

Human and mouse sera recognize the same polypeptide associated with immunological resistance to Trypanosoma cruzi infection.

Effective immunological resistance to challenge with trypomastigotes of Trypanosoma cruzi has been linked to antibodies which are specific for determinants on live, but not fixed, trypomastigotes. In man and mouse, these antibodies can be detected specifically by viable immunofluorescence (VIF) and complement mediated lysis (CML) assays. VIF/CML positive sera from chagasic patients or experimentally infected mice recognize the same trypomastigote specific surface polypeptides of apparent Mr 70-160 kDa. VIF/CML negative chagasic sera fail to react with polypeptides of Mr 120, 145 and 160 kDa, whereas negative mouse sera lack antibodies to the 160 kDa component alone. Taken together, these clinical and experimental data suggest that the 160 kDa polypeptide should be tested for its potential in immunoprophylaxis.

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.

Identification and partial characterization of Trypanosoma cruzi antigens recognized by T cells and immune sera from patients with Chagas' disease.

Trypanosoma cruzi antigenic specificities involved in human T-cell and antibody responses were compared in chronic chagasic patients affected with cardiomyopathy (C) or with the indeterminate form (I), the asymptomatic form of chronic Chagas' disease. T-cell Western blotting (immunoblotting) was performed to identify the most active antigens in epimastigote extracts (EPI-Ag). The patterns of peripheral blood mononuclear cell (PBMC) and T-cell proliferative responses induced by fractions blotted to nitrocellulose were heterogeneous, but the computation of their frequency distribution disclosed some important antigen specificities. Molecules ranging from 100 to 150 kilodaltons (kDa) were frequently stimulatory to PBMC from I patients (5 of 8 cases) and were less so when confronted with C patient (1 of 7 cases) lymphocytes. In contrast, both groups of patients actively responded to fractions ranging from 48 to 57 and 28 to 32 kilodaltons (kDa). The Western immunoblotting patterns of antibody reactivity displayed by 17 C and 15 I patients were also similar, yielding outstanding staining in the molecular mass ranges of 70 to 80 and 43 to 57 kDa. The latter antigen complex was recognized by 100% of the 32 chronic Chagas' disease serum specimens tested and closely corresponded to the migratory position recognized by T cells of most patients tested. The identification of the active molecules contained in the 43- to 57-kDa region was sought, with a focus on GP57/51, an antigen with well-established serodiagnostic properties. Immunoblotting analysis of EPI-Ag with a monoclonal antibody to GP57/51 confirmed its presence within the predicted molecular weight region. Highly purified GP51 was then used to demonstrate directly its capacity to promote specific PBMC proliferative responses in vitro. Data computed from a survey with 12 patients have shown a linear correlation (r = 0.93) between PBMC responses to EPI-Ag and to purified GP51, suggesting that the immune response to this particular glycoprotein may be an important component of human immune responses against T. cruzi.

Idiotype stimulation of T lymphocytes from Trypanosoma cruzi-infected patients.

Anti-Trypanosoma cruzi epimastigote antibodies (anti-epi) from pooled and individual sera from patients with chronic Chagas' disease were purified on immunoaffinity columns of epimastigotes antigens (epi) coupled to activated Sepharose 4B. SDS-PAGE analysis of purified anti-epi preparations showed only the presence of human IgG H and L chains. These antibodies preparations showed similar Western blotting profiles as the sera pools from which they originated. The main polypeptides recognized by anti-epi had apparent molecular masses 31, 46, 51, 75 and 85 kDa. No difference in these patterns were detected between anti-epi from pooled sera of cardiac (anti-epiC) and indeterminate (anti-epiI) clinical forms. Anti-epi preparations (20 to 60 micrograms/ml) of pooled and individual sera stimulated proliferation of homologous and autologous PBMN or T-lymphocyte-enriched population. The stimulatory ability was dependent upon the PBMN-anti-epi combinations. There is no direct correlation between the level of PBMN response to epi and anti-epi stimuli. Comparison of the stimulatory activities of anti-epiC vs anti-epiI on PBMN of either cardiac or indeterminate group of patients indicate that anti-epiC is significantly more active than anti-epiI (p less than 0.025). These data demonstrate the presence of auto-anti-idiotypic-T cells in chagasic patients and lead to the possibility that idiotype/anti-idiotype interactions may play a role in determining the pathogenesis of chagasic cardiopathy.