Anti-P antibodies present in sera from patients with chronic Chagas heart disease (cChHD) recognize peptide R13, EEEDDDMGFGLFD, which encompasses the C-terminal region of the Trypanosoma cruzi ribosomal P1 and P2 proteins. This peptide shares homology with the Cterminal region (peptide H13 EESDDDMGFGLFD) of the human ribosomal P proteins, which is in turn the target of anti-P autoantibodies in systemic lupus erythematosus (SLE), and with the acidic epitope, AESDE, of the second extracellular loop of the ␤ 1 -adrenergic receptor. Anti-P antibodies from chagasic patients showed a marked preference for recombinant parasite ribosomal P proteins and peptides, whereas anti-P autoantibodies from SLE reacted with human and parasite ribosomal P proteins and peptides to the same extent. A semi-quantitative estimation of the binding of cChHD anti-P antibodies to R13 and H13 using biosensor technology indicated that the average affinity constant was about 5 times higher for R13 than for H13. Competitive enzyme immunoassays demonstrated that cChHD anti-P antibodies bind to the acidic portions of peptide H13, as well as to peptide H26R, encompassing the second extracellular loop of the ␤ 1 adrenoreceptor. Anti-P antibodies isolated from cChHD patients exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats, which resembles closely that of anti-␤ 1 receptor antibodies isolated from the same patient. In contrast, SLE anti-P autoantibodies have no functional effect. Our results suggest that the adrenergicstimulating activity of anti-P antibodies may be implicated in the induction of functional myocardial impairments observed in cChHD.

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Molecular expression cloning techniques have revealed that patients with chronic Chagas' heart disease (cChHD) present a strong humoral response against the cloned C-terminal portions of the four Trypanosoma cruzi ribosomal P proteins TcP1, TcP2 alpha (TcP2b), TcP2 beta (TcPJL5), and TcP0. This protein family presents several features that may be important in the immunopathology of Chagas disease. Their exposed location on the ribosome, and the amplification of their parasite-specific, Ser free C-terminal domain, generate a strong anti-parasite P response that may induce anti-P autoimmunity. Evidences indicate that the serological pattern of the anti-P response from chagasic patients may be the consequence of a chronic immunization with T. cruzi ribosomal antigens.

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Infection with Trypanosoma cruzi develops in three phases: acute, indeterminate or asymptomatic, and chronic phase (with cardiac or digestive manifestations). Moreover, transmission may occur from infected mothers to newborn, the so-called congenital form. In the present study, humoral responses against T. cruzi total extract and against the 13 amino acid peptide named R-13 derived from the parasite ribosomal P protein, previously described as a possible marker of chronic Chagas heart disease, were determined in chagasic patients and in blood bank donors from endemic areas. While in sera from acute phase, only IgM anti-T.cruzi response was observed, both IgM and IgG anti-T. cruzi antibodies were detected in sera from congenitally infected newborns. The percentage of positive response in sera from blood bank donors was relatively high in endemic regions. Antibodies against the R-13 peptide were present in a large proportion of cardiac chagasic patients but were totally lacking in patients with digestive form of Chagas' disease. Furthermore, anti-R-13 positive responses were detected in congenitally infected newborns.

Molecular expression cloning techniques revealed that patients with severe chronic Chagas heart disease showed a strong humoral response against the cloned C-terminal portion of the Trypanosoma cruzi ribosomal P2beta protein, previously named JL5. The main linear epitope of this polypeptide was mapped to the 13 C-terminal amino acid sequence EEEDDDMGFGLFD (named R13), which is almost identical to the mammalian ribosomal P consensus sequence EESDDDMGFGLFD (named H13). Enzyme-linked immunosorbent assay measurements demonstrated that sera from patients with chronic Chagas heart disease presented a very specific anti-P humoral response with high anti-R13, but low H13 antibody levels. We attempted to develop an animal model that would reproduce, at least partially, two features of the human infection: (1) the serological pattern of the anti-P response, and (2) specific cardiac symptoms. To this effect, mice were immunized with T. cruzi P2beta recombinant protein. Immunization reproduced the typical anti-P antibody profile defined for chronic infections, but did not induce cardiac inflammatory lesions. However, it altered significantly the electrocardiograms of immunized mice. It is suggested that this assay represents a functional test for assessing the biological activity of antibodies against T. cruzi ribosomal P protein on cardiac muscle.

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