RESUMO
Monoclonal antibodies were raised against a recombinant ribosomal P2beta protein of Trypanosoma cruzi. One of these reacted with the C terminus of this protein (peptide R13, EEEDDDMGFGLFD) and epitope mapping confirmed that this epitope was the same as the one defined by the serum of immunized mice, and similar to the previously described chronic Chagas' heart disease (cChHD) anti-P epitope. Western blotting showed that the monoclonal antibody recognized the parasite ribosomal P proteins, as well as the human ribosomal P proteins. Electron microscopy showed that it stained different structures in parasite and human cells. Interestingly, surface plasmon resonance measurements indicated that the affinity for the parasite ribosomal P protein epitope (R13) was five times higher than for its human counterpart (peptide H13, EESDDDMGFGLFD). Since the human epitope contained an acidic region (EESDD) similar to the AESDE peptide recognized by cChHD patients in the second extra-cellular loop of the human beta1-adrenergic receptor, the biological activity of the antibody was assessed on neonatal rat cardiomyocytes in culture. The monoclonal antibody had an agonist-like effect. These results, together with the fact that the monoclonal reacted in Western blots with the different isoforms of the heart beta1-adrenergic receptor, confirm the possible pathogenic role of antibodies against the parasite ribosomal P protein based on their cross-reaction with the human beta1-adrenergic receptor.
Assuntos
Anticorpos Antiprotozoários/imunologia , Autoanticorpos/imunologia , Proteínas de Protozoários/imunologia , Receptores Adrenérgicos beta 1/imunologia , Proteínas Ribossômicas/imunologia , Trypanosoma cruzi/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Células Cultivadas , Cardiomiopatia Chagásica/imunologia , Reações Cruzadas , Células HeLa , Humanos , Epitopos Imunodominantes/imunologia , Miocárdio/imunologia , Coelhos , Ratos , Ratos Wistar , Trypanosoma cruzi/ultraestruturaRESUMO
A complement regulatory protein (CRP) of Trypanosoma cruzi was evaluated as a vaccine candidate in a murine model of experimental T. cruzi infection. Recombinant CRP derived from an Escherichia coli expression system and a plasmid encoding the full-length crp structural gene under the control of a eukaryotic promoter were used to immunize BALB/c mice. Immunization with both protein and DNA vaccines resulted in a Th1-type T-cell response, comparable antibody titers, and similar immunoglobulin G isotype profiles. Only mice immunized with the crp DNA plasmid produced antibodies capable of lysing the parasites in the presence of complement and were protected against a lethal challenge with T. cruzi trypomastigotes. These results demonstrate the superiority of DNA immunization over protein immunization with the recombinant CRP. The work also supports the further investigation of CRP as a component of a multigene, anti-T. cruzi DNA vaccine.
Assuntos
Doença de Chagas/prevenção & controle , Proteínas Inativadoras do Complemento/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Citocinas/biossíntese , Imunização , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Proteínas de Protozoários/genética , Vacinas Sintéticas/imunologiaRESUMO
Antibodies against the Trypanosoma cruzi ribosomal P2beta protein (TcP2beta) have been associated with the chronic cardiac pathology of Chagas' disease in humans. Using synthetic peptides spanning the entire TcP2beta molecule, we investigated their epitope recognition by antibodies from mice chronically infected with T. cruzi and from mice immunized with two recombinant TcP2betas. We found clear differences in epitope recognition between antibodies from T. cruzi-infected mice and mice immunized with two different recombinant TcP2betas associated with different schedules of immunization. Major epitopes recognized by antibodies from mice immunized with recombinant glutathione S-transferase (GST) or histidine (Hist) fusion TcP2beta (GST-TcP2beta or Hist-TcP2beta) are located in the central and hinge regions of the molecule. Nevertheless, mice immunized with Hist-TcP2beta were also able to elicit antibodies against the TcP2beta C terminus, a region which is highly conserved in both T. cruzi and mammal ribosomal P proteins. Strikingly, antibodies from infected animals recognized only the TcP2beta C terminus. By using these antisera with distinct profiles of epitope recognition, it could be shown that only C terminus-specific antibodies were able to increase the beating frequency of cardiomyocytes from neonatal rats in vitro by selective stimulation of the beta1-adrenergic receptor. Thus, antibodies against the TcP2beta C terminus elicited in the absence of infection are able to modulate a functional activity of host cells through a molecular mimicry mechanism.
Assuntos
Anticorpos Antiprotozoários/imunologia , Coração/fisiologia , Fosfoproteínas/imunologia , Sequência de Aminoácidos , Animais , Mapeamento de Epitopos , Imunização , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Fosfoproteínas/química , Estrutura Secundária de Proteína , Ratos , Ratos Wistar , Receptores Adrenérgicos beta 2/fisiologia , Proteínas RibossômicasRESUMO
An enzyme linked immunosorbent assay (ELISA) was developed for detecting IgM and IgG antibodies against Trypanosoma cruzi in blood bank donors from endemic or nonendemic areas. A crude extract of trypomastigotes from cultures was used as antigen. A total of 494 serum samples from patients with acute, congenital, or chronic form of Chagas' disease, and from healthy French individuals were studied. The sensitivity of the ELISA was determined with 89 serum samples from chagasic patients and was evaluated to 98.8%. The specificity was determined with 405 serum samples from French blood transfusion centers donors and evaluated to 98.3%. Two hundred and eighty-five serum samples from blood donors from Argentina and Brazil were also tested. Furthermore, in order to assess the absence of cross-reactivity with other protozoan infections, we studied 86 serum samples including (i) 32 individuals with cutaneous leishmaniasis living in a T. cruzi endemic region of Bolivia, and (ii) 54 patients from nonendemic area for Chagas' disease, 19 of them with kala-azar and 35 others with malaria.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Trypanosoma cruzi/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Doadores de Sangue , Doença de Chagas/epidemiologia , Doença de Chagas/imunologia , Doença de Chagas/prevenção & controle , Doenças Endêmicas , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Sensibilidade e EspecificidadeRESUMO
A large field study has been performed in the Cochabamba region of Bolivia with the aim of comparing the polymerase chain reaction (PCR) with other diagnostic methods for Chagas' disease. The amplification of Trypanosoma cruzi-specific kinetoplast DNA sequences in blood samples was compared with classical serological methods, specific IgM detection and direct parasite visualization for 268 school children in a single village where Chagas' disease transmission is active. Of 113 children positive by classical serology or buffy coat examination, 106 were detected by PCR (sensitivity: 93.8%). We did not observe any significant difference of PCR sensitivity between initial (IgM and/or buffy coat positive) and indeterminate stage (only IgG positive) patients. Among the remaining 155 children unconfirmed as chagasic (who were either only IgM positive, IgG-, IgM-, and buffy coat-negative) only 1 case was PCR positive. This case may be due to DNA contamination, or to a very recent infection not detected otherwise, or to specific immune depression. These results show that PCR is a very sensitive parasitological test for Chagas' disease in active transmission regions. The future follow-up of the possibly infected patients who were only IgM-positive should clarify the interest of PCR and IgM tests in the detection of starting infections.
Assuntos
Doença de Chagas/diagnóstico , Reação em Cadeia da Polimerase/métodos , Adolescente , Anticorpos Antiprotozoários/sangue , Bolívia/epidemiologia , Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , Criança , Pré-Escolar , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lactente , Leucócitos/parasitologia , Prevalência , População Rural , Sensibilidade e Especificidade , Testes SorológicosRESUMO
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.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Doença de Chagas/imunologia , Proteínas de Protozoários , Proteínas Ribossômicas/imunologia , Trypanosoma cruzi/imunologia , Doença Aguda , Animais , Argentina , Sequência de Bases , Doadores de Sangue , Brasil , Cardiomiopatia Chagásica/diagnóstico , Cardiomiopatia Chagásica/imunologia , Doença de Chagas/congênito , Doença de Chagas/epidemiologia , Doença Crônica , Diagnóstico Diferencial , Humanos , Isotipos de Imunoglobulinas/imunologia , Recém-Nascido , Dados de Sequência Molecular , Estudos SoroepidemiológicosRESUMO
The kinetic and isotypic pattern of hypergammaglobulinemia has been investigated in C3H/HeJ infected with Trypanosoma cruzi. Hypergammaglobulinemia appeared 14 days postinfection, increased until Day 28 postinfection, and persisted throughout the chronic phase (greater than 60 days of infection). The main isotype secreted was IgG2a, reaching 10-fold the control level. High titers of autoantibodies were found of IgM and IgG subclasses. Isotypic characterization of antibodies against myosin, myelin, and keratin, was performed and determined to be IgG2a subclass in the chronic stage of infection. Specific responses against T. cruzi took place 2 weeks postinfection when the parasitemia was high. Interestingly, parasite-specific response was maximal after 4 weeks of infection and plateaued during the chronic phase when parasites were rare. In contrast to the humoral polyclonal response in the chronic stage, showing a preferential IgG2a pattern, the anti-T. cruzi response consisted of all the different isotypes: IgM, IgG1, IgG3, IgG2a, and IgG2b, throughout the infection. Identical patterns of parasite antigens were recognized by IgG2a and IgG2b antibodies. Few different antigens were identified by the IgG3. Some antigens were recognized by several isotypes, others by only one isotype. With regard to the existence of antigenic cross-reactivities between host and parasite, we designed absorption experiments on parasite-specific immunoadsorbent showing that specific antibodies eluted from the column failed to recognize the natural antigens. These studies suggest that nonspecific and antiparasite-specific responses may be maintained by different regulatory pathways.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Doença de Chagas/imunologia , Imunoglobulina G/biossíntese , Trypanosoma cruzi/imunologia , Animais , Anticorpos Antiprotozoários/classificação , Especificidade de Anticorpos , Western Blotting , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/classificação , Imunoglobulina M/biossíntese , Camundongos , Camundongos Endogâmicos C3HRESUMO
Trypanosoma cruzi lambda gt 11 library from epimastogote derived mRNA was screened with human chagasic sera or sera from chronically infected mice. Strong reactive recombinants were detected with both sera. Two recombinant clones were studied in more detail and shown to be composed of the same 114-bp repetitive sequence coding for a 38 amino acid repetition. This repetition is the same size and shares greater than 60% homology with the reported T. brucei microtubule associated protein (MAP) p320. The insert of one of these clones, K1-7 (228 bp), was subcloned into pMSgt11 and the soluble recombinant polypeptide expressed. Antibodies against the K1-7 fusion polypeptide recognized a major 110-kDa band from cytoskeleton. Anti K1-7 monospecific antibodies detected several cytoskeletal proteins from 3T3 fibroblasts and bovine brain microtubule preparations. Reciprocally, anti-MAP1b monoclonal antibodies raised against bovine brain microtubule reacted with the K1-7 polypeptide on Western blots. The protein identified by K1-7 antibodies may be one of the parasite molecules associated to molecular mimicry.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Autoanticorpos/imunologia , Doença de Chagas/imunologia , Citoesqueleto/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Doença Crônica , Reações Cruzadas , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/imunologia , Sequências Repetitivas de Ácido NucleicoRESUMO
Experimental Chagas' disease--infection of mice with the protozoan parasite Trypanosoma cruzi--has been shown to increase the number of Ia-bearing cells in the spleen and the lymph nodes. The majority of these Ia-positive cells were Ig+ and included in the large cell fraction of lymphoid organs from T. cruzi-infected animals indicating that they were activated B cells. These data are consistent with the polyclonal B-cell activation occurring during acute and chronic T. cruzi infection. The levels of secreted natural antibodies, of both IgM and IgG isotypes, were significantly increased in the sera of the infected animals. The present communication demonstrates that in vivo anti-Ia treatment of C3H/HeJ mice infected with the CL strain of T. cruzi suppressed the polyclonal B-cell activation, affecting all the isotypes studied, including IgM, IgG2a and IgG2b, whose levels are predominantly increased during T. cruzi infection. In contrast to the decreased secretion of IgG autoantibodies, the levels of IgM autoantibodies were much less affected. The anti-Ia treatment totally abolished the specific anti-parasite response despite the fact that a pool of Ia-Ig positive cells remained after treatment.