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(The FASEB Journal. 1998;12:1551-1558.)
© 1998 FASEB

RESEARCH COMMUNICATION

Functionally active cardiac antibodies in chronic Chagas' disease are specifically blocked by Trypanosoma cruzi antigens

Masako Oya Masudaa, Mariano Levinc, Selma Farias De oliveiraa, Patricia C. Dos Santos Costaa, Pablo Lopez Bergamic, Norma A. Dos Santos Almeidaa, Roberto Coury Pedrosab, Ines Ferraric, Johan Hoebeked, and Antonio Carlos Campos de Carvalhoa,1

a Instituto de Biofisica Carlos Chagas Filho, CCS, UFRJ, Brazil
b Hospital Universitário Clementino Fraga Filho, CCS, UFRJ, Rio de Janeiro, RJ, Brazil
c Instituto de Investigaciones en Ingenieria Genética y Biología Molecular (INGEBI), 1428 Buenos Aires, Argentina
d Laboratoire d'Enzymologie et de Chimie de Protéins, Centre National de la Recherche Scientifique, F-37032 Tours, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies of chronic chagasic patients have been shown to interfere with electric and mechanical activities of cardiac embryonic myocytes in culture and with whole mammalian hearts. A mechanism proposed for this effect involves interaction of the antibodies with G-protein-linked membrane receptors, thus leading to activation of beta adrenergic and muscarinic receptors; more specifically, IgG of chagasic patients would interact with the negatively charged regions of the second extracellular loop of these receptors. We performed competition experiments to test this hypothesis. We evaluated the effect of sera/IgG from patients previously known to depress electrogenesis and/or atrioventricular conduction in isolated rabbit hearts after incubation with live and lysed parasites, the peptide corresponding to the second extracellular loop (O2) of the M2 receptor, and different peptides derived from two ribosomal proteins of T. cruzi: P0 and P2ß. Our results indicate that 1) the antigenic factor inducing the functionally active IgGs in the chagasic patients is probably an intracellular T. cruzi antigen; 2) IgG/serum is interacting with the O2 region of the M2 receptor in the rabbit heart; and 3) the negative charges present in the ribosomal proteins of T. cruzi are important in mediating the interaction between the patients' serum/IgG and the receptor.—Masuda, M. O., Levin, M., Farias de Oliveira, S., dos Santos Costa, P. C., Bergami, P. L., dos Santos Almeida, N. A., Pedrosa, R. C., Ferrari, I., Hoebeke, J., Campos de Carvalho, A. C. Functionally active cardiac antibodies in chronic Chagas' disease are specifically blocked by Trypanosoma cruzi antigens. FASEB J. 12, 1551–1558 (1998)


Key Words: immunoglobulin • heart block • ribosomal P proteins • muscarinic receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CHAGAS' DISEASE IS the result of a parasitic infection by the hemoflagellated protozoan Trypanosoma cruzi (1). The disease has acute and chronic phases characterized by distinct signs and symptoms, separated by an indeterminate period, which can last for many decades, when patients are clinically asymptomatic. The gastrointestinal tract and heart are the main targets of the disease; during the chronic phase, dilation of these organs, which constitute the mega syndromes, is a distinct feature of Chagas' disease.

The acute phase is characterized by an active infection, with the presence of circulating parasites in blood and many tissues. The heart, when affected, shows signs of inflammation and myocardial damage (2, 3). Usually the clinical manifestations of this phase are very mild, and the disease may often go undetected (4). In contrast, the chronic phase of Chagas' disease displays exuberant symptomatology, but live parasites are rarely found in blood or tissues. Cardiac manifestations include electrical and mechanical disturbances that range from conduction defects and arrhythmias to congestive heart failure, cardiomegaly, and sudden death (5, 6).

Many hypothesis have emerged to explain the cardiac attack during the chronic phase (7). Autoimmunity has been proposed as a key factor from the very beginning due to the virtual absence of parasites in blood or affected tissues. Through the years, an impressive number of publications have presented experimental evidence favoring humoral or cell-mediated autoimmune mechanisms in the pathogenesis of the chronic cardiac disease (reviewed by Brenner, ref 7).

Autoantibodies to adrenergic and muscarinic receptors have been found in immunoglobulin (IgG)2 fractions from blood samples of chronic chagasic patients (8–17). In many studies, binding of the autoantibodies to receptors has been demonstrated, and functional alterations in second messengers levels and beat rate of cultured cardiac cells have been measured. Recently we demonstrated that IgGs from a group of chronic chagasic patients with complex cardiac arrhythmias are functionally competent in inducing reduction in beat rate and atrioventricular conduction block in isolated whole rabbit hearts, and that these IgGs exert a muscarinic-like effect that can be blocked by atropine (17).

The acute and chronic phases of the disease respond differently to cloned parasitic proteins (18). While 90% of acute Chagas' patients present antibodies to a membrane-anchored, trypomastigote-specific protein (SAPA antigen), only 10% of chronic patients have anti-SAPA antibodies (19). On the other hand, 90–100% of the chronic patients tested presented IgG antibodies to intracellular parasite proteins (18, 20–22). Levin and co-workers (23) have found that many of the antibodies present in cardiomyopathic chronic chagasic patients' sera are directed against T. cruzi P ribosomal antigens. In addition, they have demonstrated a cross-reaction between an epitope within the T. cruzi P0 ribosomal antigen and extracellular epitopes mapped to the second extracellular loops of the adrenergic and muscarinic cardiac receptors (11, 14). More recently, Kaplan el al. (24) proved that antibodies against the T. cruzi P2ß ribosomal P protein (TcP2ß) directed against its COOH-terminal peptide, R13, also display functional autoreactive properties.

The targeted epitope in the cardiac membranes seems to be a stretch of negatively charged amino acids located in the second extracellular loop of ß1-adrenergic and M2-muscarinic receptors (10, 14, 25). We therefore decided to investigate whether the functionally active antibodies characterized in our previous study (17) were directed to intracellular antigens of T. cruzi and other protozoan parasites. We also tested the ability of these antibodies to recognize the second extracellular loop of the M2 muscarinic receptor and whether a cross-reactive mechanism between this region of the receptor and the P family of ribosomal proteins of T. cruzi was involved in the immune recognition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
ECG recordings in isolated rabbit hearts
The method for electrocardiogram (ECG) recording from isolated hearts has been described in detail (26). In brief, young rabbits weighing 1.5–2.0 kg were killed by cervical dislocation, and their hearts were rapidly removed and cannulated through the aorta for continuous perfusion of the coronary circulation with Tyrode solution (in mmol/l): NaCl, 127; KCl, 2.7; NaHCO3, 12; MgCl2, 0.5; glucose, 10; and CaCl2, 2.7, pH 7.2) at 36 ± 0.2°C. The hearts were immersed in warmed Tyrode in a water-jacketed glass flask; three glass electrodes filled with 1 mol/l NaCl were positioned inside the flask to obtain optimal electrocardiographic recordings. Two electrodes were connected to the differential input of a high gain amplifier (3A9, TEKTRONIX Inc, Beaverton, Oreg.) and the third was connected to ground. The experimental protocol consisted of control recordings for 15–20 min in Tyrode's solution, a 30 min perfusion with Tyrode containing serum (or IgG) from the chagasic patients or serum (or IgG) previously incubated with peptides or parasite antigens (see below), and return to Tyrode. Whenever possible, effects of pure fractions and of those incubated with the peptides or parasite antigens were tested in the same heart preparation, with a 30 min washout between application of the two fractions. The ECG was continuously monitored on an oscilloscope (561 TEKTRONIX, Inc.) and recorded on a chart recorder (2200, GOULD Inc, Glen Burnie, Md.); expanded records were taken every 5 min. Experiments were carried out only if no significant change in the ECG parameters was observed for the 15–20 min duration of the control recordings. The ECG analysis included P wave frequency, presence of atrioventricular conduction block, and other arrhythmias.

Serum purification
We used sera or IgG fractions obtained from two previously characterized chronic chagasic patients with complex arrhythmias (17). Fractionation was performed by serum precipitation with ammonium sulfate (40%), followed by overnight dialysis against phosphate buffer (pH 8.0) and chromatography in a DEAE-matrix under continuous addition of phosphate buffer (pH 8.0). Protein concentration in sera or IgG fractions was determined by the Bradford method (27). Final concentrations used in the experiments (after dilution in Tyrode) ranged from 0.62 to 1.44 mg/ml and 0.02 to 0.06 mg/ml for serum and IgG, respectively.

Incubation with parasites
Sera were incubated on a 1:1 volume basis with a suspension of live T. cruzi (Y strain) trypomastigotes obtained from blood of infected mice or Leishmania brasiliensis obtained from cultures in artificial medium. Parasites were ressuspended in phosphate-buffered solution at 2 x 106 parasites/ml after extensive washing with this buffer. Incubation was performed overnight at 4°C or for 1 h at 37°C. Incubation was also carried out under identical conditions with parasites that were previously lysed by osmotic shock and sonication.

Peptides
Sequences of the peptides used for the competition experiments are shown in Fig. 1. The first sequence corresponds to the second extracellular loop of the muscarinic M2 receptor. P0–14 and P0–13 are derived from the P0 ribosomal protein and the R peptides are derived from the P2ß ribosomal proteins of T. cruzi. HSC70 is a peptide derived from the human heat shock protein of 71 kDa. All peptides were synthesized in an automatic peptide synthesizer from Applied Biosystems (Foster City, Calif.) by F-moc technique, and their purity was checked by high-performance liquid chromatography after cleavage and desalting on a P2 column (Bio-Rad, Hercules, Calif.).



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  		Figure 1. Amino acid sequences of peptides corresponding to the second extracellular loop of the M2-muscarinic receptor, to different T. cruzi ribosomal proteins, and to HSC-70, as identified.

Recombinant protein
Recombinant P2ß protein was obtained from bacteria transformed with the pMal-c2 plasmid containing an almost complete copy of P2ß, as previously described (15).

Incubation with peptides
When peptides (or recombinant protein) were incubated with sera from the patients, peptide concentration was 1 mg/ml (in serum). In the case of IgG fractions, peptides were added in equimolar concentrations. Assuming a mean molecular mass of 150,000 daltons for IgG, the molar concentration of our IgG samples ranged from 167 to 400 nM. Since the amount of specific anti-receptor autoantibodies is generally less than 1% of the total IgG fraction (14, 28), the addition of peptide on a molar basis should be sufficient to saturate all anti- receptor antibody binding sites. Serum or IgG fractions were incubated with peptides overnight at 4°C. In control experiments, either serum or IgG fractions were incubated with maltose binding protein (MBP), a peptide derived from the human heat shock protein (HSC70), and polyglutamic acid.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Functionally active antibodies in chronic chagasic sera specifically recognize intracellular T. cruzi but not Leishmania antigens
As an initial test of the cross-reactivity between cardiac and T. cruzi antigens, we incubated sera of two chronic chagasic patients (HB1 and HB2), previously known to decrease heart rate and/or block AV conduction in Langendorff perfused rabbit hearts with trypomastigotes obtained from infected mice.

Figure 2 shows the typical result observed when normal rabbit hearts are perfused with serum (or IgG fraction) from patients HB1 and HB2 diluted in Tyrode solution. Both sera decrease sinus rate (from 130 to 112 bpm in panel A and from 146 to 135 in panel B) and induce second degree AV conduction blockade (17).



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  		Figure 2. Effects of serum of patients HB1 (A) and HB2 (B) on the electrogenesis of isolated rabbit hearts. Records in each pair were obtained from the same heart. Arrows indicate the P waves. The upper traces in each pair (control) represent ECGs recorded during Tyrode perfusion. Lower traces (S) were obtained in the presence of serum diluted 1:100 in Tyrode. Sera from both patients induce a decrease in sinus rate (increased PP interval) and second degree AV conduction block. Lower records in each pair were taken at 10 and 13 min of serum perfusion. Calibration bar: 1 s.

When the serum was incubated with live trypomastigotes, inhibition of the serum effect was not observed, as illustrated in Fig. 3A. In the first record (control), an ECG was obtained in Tyrode solution and shows a regular sinus rhythm. The second record (S/tryp-alive) was obtained during perfusion with Tyrode solution containing serum of patient HB1 previously incubated with the live trypomastigotes, as described in Materials and Methods. The result was similar to that observed during perfusion with this same serum without preincubation with the parasite at the same dilution (1:100 in Tyrode): a reduction in heart rate and a clear second degree AV block. This result suggests that surface antigens of the parasite do not cross-react with cardiac antigens targeted by the functionally active antibodies present in chronic chagasic patients' sera. In contrast, when serum from the same patient was incubated with lysed trypomastigotes, inhibition of the serum effect by the parasite was evident, as shown in Fig 3B. The upper record (control) was obtained under Tyrode perfusion and the lower (S/tryp-lysed) during perfusion with serum from patient HB1, preincubated with lysed parasites. No conduction block or decrease in beat rate was present under this condition, indicating the ability of intracellular T. cruzi antigens to functionally block active antibodies present in the patient's serum. Similar results were obtained when serum from the other patient in this study was used.



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  		Figure 3. Incubation of serum with trypomastigotes. ECG records obtained from isolated rabbit hearts under Langendorff perfusion. Records in each pair were obtained from the same heart. Control: represents the record obtained during perfusion with Tyrode solution. S/tryp-alive: record obtained during perfusion with Tyrode solution containing serum from patient HB1 preincubated overnight with 2 x 106 live trypomastigotes at 4°C [final dilution of serum: 1:100 (v:v) dilution]. Note a second degree AV conduction block indicated by the presence of one QRS complex for two P waves. S/tryp-lysed: record obtained during perfusion with Tyrode containing the same serum sample preincubated overnight with 2 x 106 lysed trypomastigotes, at 4°C. The serum completely lost the ability to induce AV conduction block. Calibration bar: 1 s.

To test for the specificity of this cross-reactive response, we repeated the experiments with sera from both patients that had been incubated with live and lysed trypomastigotes of L. brasiliensis, using the same protocol as for T. cruzi trypomastigotes. Incubation with either live or lysed Leishmania did not affect the ability of the sera to decrease sinus rate and block AV conduction in the isolated rabbit hearts (not shown).

Functionally active antibodies in chronic chagasic sera recognize the cardiac muscarinic receptor
Although previous studies had indicated the presence of antibodies with an important adrenergic effect in the serum of chagasic patients (8, 11), our own work (17) detected mainly a muscarinic-like effect in IgG or serum samples obtained from chronic chagasic patients with complex cardiac arrhythmias. Therefore, in searching for the cardiac antigen(s) being recognized by the antibodies in the sera of the two patients used in this study, we incubated them with a peptide corresponding to the second extracellular loop of the human M2 muscarinic receptor. For both patients, incubation of serum or IgG fraction with the peptide completely inhibited the muscarinic-like effect, as illustrated in Fig. 4. Compare the control record during Tyrode perfusion with the record obtained during perfusion with the IgG fraction from patient HB1 in Tyrode (0.06 mg/ml). A second degree AV block is induced by IgG (Fig 4A). When this IgG fraction (same concentration as above, but applied to a different heart) has been incubated with the peptide corresponding to the second extracellular loop of M2, on a 1:1 molar basis, the fraction loses its ability to block AV conduction, as illustrated in Fig 4B (compare the control in Tyrode to the IgG+/M2 record), thus confirming the hypothesis that antibodies in our patients' sera are interacting with the second extracellular loop of the muscarinic receptor.



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  		Figure 4. Incubation of IgG fraction with the peptide corresponding to the second extracellular loop of M2- muscarinic receptor. IgG+: typical ECG record obtained during perfusion with IgG (0.06 mg/ml) from patient HB1; second degree AV block is evident. The same IgG fraction, in the same concentration (tested in another heart), completely lost its ability to induce AV block when preincubated with the peptide corresponding to the second extracellular loop of M2-muscarinic receptor (IgG+ and M2). Calibration bar: 1 s.

Functionally active antibodies in chronic chagasic sera cross-react with T. cruzi P-ribosomal proteins
Considering that chagasic patients with cardiomyopathy present high levels of antibodies to T. cruzi P- ribosomal proteins, and to further test the hypothesis that cross-reactivity between cardiac and T. cruzi derived antigens is responsible for the generation of the antibodies that induce the muscarinic-like response in the sera of our patients, we incubated either serum or IgG fractions from patients HB1 and HB2 with recombinant protein and peptides derived from T. cruzi ribosomal proteins, as described in Materials and Methods.

Results are illustrated in Fig. 5 and Fig. 6. Figure 5shows the ECG recordings obtained with IgG incubated with P2ß recombinant protein and derived peptides. Incubation with the carrier protein alone (MBP) does not prevent the induction of AV block by the IgG (Fig 5A). On the other hand, incubation of serum with the recombinant protein (MBP+P2ß) blocks its ability to induce AV conduction blockade (Fig 5B). Figure 5C, D shows the results of similar experiments carried out with IgG fractions from patients HB1 and HB2 incubated on a molar basis with peptides R13 and R7, respectively. Whereas R13 was able to inhibit the AV block and bradycardia induced by the IgG fraction ( Fig. 5C), R7 was not ( Fig. 5D).



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  		Figure 5. Incubation of IgG fraction with P2ß and related peptides. A) The carrier protein MBP does not interfere with the IgG effect in inducing bradycardia and second degree AV conduction block. Control: during Tyrode perfusion. IgG+/MBP: during perfusion with IgG from patient HB1 incubated overnight with MBP. B) The fusion protein MBP-S23 blocks the effect of chagasic IgG. Control: ECG obtained in Tyrode solution. IgG+/P2ß: during perfusion with IgG from patient HB1 incubated with the fusion protein MBP-S23 at equimolar concentration. C) Incubation with R13 peptide blocks the effect of chagasic IgG. Control: in Tyrode solution. IgG+/R13: perfusion with IgG from patient HB1 incubated overnight at 4°C with peptide R13 at equimolar concentration. D) Incubation with R7 peptide fails to block the effect of chagasic IgG. Control: in Tyrode solution. IgG+/R7: perfusion with IgG from patient HB2 incubated overnight at 4°C with the peptide R7 at equimolar concentration. Calibration bar: 1 s.



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  		Figure 6. Incubation of chagasic serum with peptides from the P0 family of T. cruzi ribosomal proteins. Both P0–14 (S/P0–14) and P0–13 (S/P0–13) abolished the serum ability to induce bradycardia and AV conduction block. A, B) Upper records: control, in Tyrode solution; lower records: perfusion with serum from patient HB1 (diluted 1:100 v:v in Tyrode) incubated overnight at 4°C with 1 mg/ml of peptides P0–14i and P0–13. Calibration bar: 1 s.

Incubation of serum from patient HB1 with P0–14 or P013, peptides derived from the P0 ribosomal protein of T. cruzi, also leads to protection against AV block, as illustrated in Fig. 6A, B.

Similar results were obtained with recombinant protein and peptides incubated with the serum from the other patient used in our study, but are not illustrated in the figures.

Taken together, these experiments show that as long as the peptides derived from T. cruzi ribosomal proteins retain a stretch of negative charges present in their carboxy terminus, they are competent to inhibit the muscarinic-like action of the antibodies derived from the patients' sera. Incubation of sera or IgG from both patients with the HSC70 peptide or polyglutamic sequences could not prevent the AV block (not shown), indicating that the charge effect is not unspecific.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Autoimmune mechanisms have long been implicated in the pathological changes associated to the chronic phase of Chagas' disease. Indeed, numerous studies have described the presence of antibodies to various autoantigens present in different organs and tissues in the serum of chronic Chagasic patients. However, in few instances a clear association has been established between the presence of these autoantibodies and their ability to induce the pathological alterations characteristic of the chronic phase of the disease. Moreover, the presence of autoantibodies has never been clearly shown to be a cause or consequence of the disease. We now present evidence that not only shows a direct effect of chagasic anti-T. cruzi antibodies in the electrogenesis and conduction of the heart impulse, but also points to a cross-reactive mechanism between P-ribosomal proteins of the parasite and M2 muscarinic receptors of the cardiac surface membrane as probable cause for the pathological immune response.

The presence in chronic chagasic patients of antibodies to G-protein-coupled receptors has been clearly and extensively demonstrated by the work of Sterin-Borda and collaborators (8, 9). More recently, it has been shown that in different cardiomyopathies, including chagasic, the targeted epitopes in these receptors are regions of the second extracellular loop (29). In chagasic cardiomyopathy, the immunodominant epitope seems to involve the region of the second extracellular loop, which is rich in negatively charged amino acid residues (14). This type of recognition makes the immune response specific to ß1-adrenergic and M2-muscarinic, but not to the ß2-adrenergic, receptors. In addition, P-ribosomal proteins of T. cruzi have been described as a major antigenic component of the immune response in chronic chagasic patients with heart disease. Levin et al. (30) and Ferrari et al. (11) have demonstrated that antibodies to the P0 ribosomal protein of T. cruzi recognize the extracellular loop of the human ß1-adrenergic receptor.

Our previous work (17) pointed to a predominant muscarinic-like response in the interaction between human chagasic antibodies and the isolated rabbit heart perfused by the Langendorff technique. Even when the hearts were previously atropinized to inhibit a muscarinic-like effect, rarely did we observe an adrenergic effect when perfusing the isolated hearts with patients' sera or IgGs (unpublished results). In contrast to our findings, a ß1-adrenergic effect was predominant when patients' sera or IgGs were tested in cultures of neonatal rat cardiac myocytes (11, 24). We attribute this discrepancy to the great variability in the expression of muscarinic receptors in cultured cardiac myocytes; the response of these cultures to carbachol is extremely dependent on time in culture. Alternatively, different targets (muscarinic, adrenergic, and other receptor types) may be recognized by these sera, and our system may be poorly sensitive to an adrenergic- like effect. At any rate, until recently the emphasis in the humoral immune response in Chagas' disease was directed to the ß1-adrenergic receptors, even though the clinical findings in chagasic patients more closely resemble a vagal syndrome. In our view, the whole heart model is more suited for the evaluation of the effects of the antibodies present in the sera of chronic chagasic patients because it reveals the muscarinic-like activation as the major alteration induced by the chagasic sera. In support of this view, two recent publications from Goin et al. (16) and Ferrari et al. (31) emphasize the participation of anti-M2 antibodies in the immune response of chagasic patients. In fact, Elies et al. (14) find the anti-M2 response to be predominant according to enzyme-linked immunoassay.

The cross-reactivity between T. cruzi ribosomal proteins and membrane cardiac receptors was originally proposed by Ferrari et al. (11) for the ß1-adrenergic receptor and the P0 proteins. The results presented here extend this cross-reactivity to the M2- muscarinic receptor, in agreement with the work of Elies et al. (14), and generalize it to P0 and P2ß ribosomal proteins of T. cruzi, compatible with results presented by Kaplan et al. (24). The results also suggest that this cross-reactive mechanism is very specific, since only T. cruzi and not Leishmania trypomastigotes were able to inhibit the chagasic antibodies' actions on the heart. Furthermore, our results strongly support the contention of Hoebeke and co- workers (29) that the negative charges in the second extracellular loop of these receptors are indeed the major antigenic determinants for the functionally active antibodies present in chronic chagasic patients' sera. Finally, our findings strengthen the hypothesis of a cross-reactive mechanism as the basis for the immune recognition of cardiac surface antigens, suggesting that antibodies recognizing G-protein-coupled receptors in the heart may be the cause of at least some of the pathological findings associated with the chronic phase of Chagas' disease. It is interesting that Lopez Bergami et al. (15) reported that immunization of mice with recombinant T. cruzi P2ß ribosomal protein induces alterations in the ECG, whereas Matsui et al. (32) report morphological cardiomyopathic changes in rabbits immunized with peptides derived from the second extracellular loop of the ß1-adrenergic and M2-muscarinic receptors.


   ACKNOWLEDGMENTS
 
Financial support was provided by FUNDAÇÃO VITAE, CEPG-UFRJ, CNPq, FINEP, FAPERJ and PRONEX (grant 41.96.0869.00). Drs. Masuda and Campos de Carvalho are career investigators for CNPq. We wish to acknowledge the valuable contributions of Cristina Borges e Sá and Paulo R. Costa for collecting blood from the patients and of Daisy Avanzi and Eliane Modesto for technical assistance in isolation of IgG.


   FOOTNOTES
 
1 Correspondence: Instituto de Biofisica Carlos Chagas Filho, CCS, bloco G, Ilha do Fundão 21949–900, Rio de Janeiro, RJ, Brasil. E mail: acarlos{at}ibccf.biof.ufrj.br

2 Abbreviations: ECG, electrocardiogram; Ig, immunoglobulin; MBP, maltose binding protein; SAPA, membrane-anchored, trypomastigote-specific protein.

Received for publication March 23, 1998. Accepted for publication May 29, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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