RESUMO
Formation of inflammatory lesions, one of the pathologic consequences of infection with Trypanosoma cruzi, involves intricate cell-cell interactions in which cell adhesion molecules (CAMs) are involved. Sera from 56 Chagas' disease patients grouped according to disease severity were studied for the presence of soluble intercellular adhesion molecule-1 (s-ICAM-1), soluble endothelial selectin (s-E-selectin), soluble vascular cell adhesion molecule-1 (s-VCAM-1), soluble platelet selectin (s-P-selectin), and s-CD44 were studied to determine if they could be used alone or in different combinations as markers for specific diagnostic procedures. Comparisons were made between congenitally, acutely, and chronically infected patients and aged-matched, noninfected individuals, as well as between patients with chronic Chagas' disease grouped according to the severity of their heart-related pathology. No differences in levels of s-CAMs were detected between sera from children with congenital T. cruzi infection and sera from noninfected infants born from chagasic mothers. In contrast, titers of s-ICAM-1, s-VCAM-1, s-selectin, and s-CD44 but not s-P-selectin were significantly increased in sera from patients during the acute phase of infection with T. cruzi. Titers of s-VCAM-1 and s-P-selectin were increased in chronically infected patients. A positive association with disease severity in sera from patients with chronic disease was observed for the levels of s-P-selectin. In contrast, we found no association between clinical symptoms and levels of s-VCAM-1. Patients with chronic disease with severe cardiopathy also showed diminished levels of s-CD44 in comparison with healthy controls or patients with mild disease. The results are discussed in the context of pathology of Chagas' disease.
Assuntos
Moléculas de Adesão Celular/sangue , Doença de Chagas/patologia , Doença Aguda , Adolescente , Adulto , Idoso , Moléculas de Adesão Celular/química , Doença de Chagas/sangue , Doença de Chagas/congênito , Doença Crônica , Selectina E/sangue , Selectina E/química , Humanos , Receptores de Hialuronatos/sangue , Receptores de Hialuronatos/química , Lactente , Molécula 1 de Adesão Intercelular/sangue , Molécula 1 de Adesão Intercelular/química , Pessoa de Meia-Idade , Selectina-P/sangue , Selectina-P/química , Índice de Gravidade de Doença , Solubilidade , Molécula 1 de Adesão de Célula Vascular/sangue , Molécula 1 de Adesão de Célula Vascular/químicaRESUMO
Monoclonal antibodies (MoAbs) raised against Trypanosoma cruzi microsomal fraction (Mc) and cross-reactive with mammalian tissues were used to evaluate the ability of cross-reactive T. cruzi antigens to induce an immune response in Chagas' disease. Thus, we studied the ability of sera from Chagas' disease patients (CDP) with different degrees of cardiac dysfunction to block the immune recognition of these MoAb to the target antigen determining for each serum an inhibition index (II). By means of this approach we inferred that blocking of monoclonal antibody binding to T. cruzi microsomes by subjects' serum represents antibodies with the same reactivity. After serological and medical examinations, individuals were separated into the following groups: Chagas' disease patients without manifest cardiac involvement (CDP-0), CDP with suspected or borderline cardiac disease (CDP-1), CDP with moderate myocardial dysfunction (CDP-2), CDP with overt cardiac dysfunction (CDP-3) and controls including healthy subjects (HS) and patients with idiopathic myocarditis (IMP). The reactivity between MoAb 5F2 and its target antigen was significantly (p < 0.05) inhibited by sera from CDP irrespective of the clinical stage [CDP: n = 46, 50 +/- 20, mean II +/- SD: control: n = 16, 18 +/- 8]. Moreover, 5F2 was able to distinguish (p < 0.05) sera from CDP with mild disease (CDP clinical grade 0/1: n = 26, 34 +/- 18) from that of CDP with severe disease (CDP clinical grade 2/3: n = 20, 67 +/- 7). Moreover, the inhibitory capacity of sera from asymptomatic CDP (CDP-0) correlated with patients age (r = 0.66, p < 0.05). CDP-0 below or equal 40 years of age had results (n = 15, 25 +/- 13) comparable (p > 0.05) to that of controls while mean inhibition of CDP-0 over 40 years of age (n = 5, 60 +/- 5) was indistinguishable (p > 0.05) from that of patients with severe disease. Competitive assay with MoAb 5A9B11 also showed significant differences (p < 0.05) between sera from CDP (n = 46, 46 +/- 24) and controls (n = 13, 5 +/- 5). On the contrary, the differences observed between CDP with different cardiac involvement was not significant (mild: n = 26, 31 +/- 22; severe: n = 20, 66 +/- 11). However a thorough study of data from asymptomatic sera revealed the existence of two levels of reactivity, with low and high capacity to inhibit the reaction of 5A9B11 against Mc. On the contrary, CDP sera showed a blocking activity for 1A10C11 comparable to that of controls (CDP: n = 25, 19 +/- 9; control: n = 12, 14 +/- 6). Some cross-reactive MoAbs recognized epitopes partially composed of carbohydrates. Interestingly, 5F2 and 5A9B11 epitopes did not appear to have carbohydrates moieties. In summary, immunoinhibition assays revealed differences in the immune response of chronic chagasic patients against parasite epitopes. These results have opened the possibility to identify a prognosis marker of the disease suggesting the clinical utility of monitoring levels of these anti-Mc antibodies in patients with chronic Chagas' disease.
Assuntos
Anticorpos Antiprotozoários/imunologia , Especificidade de Anticorpos , Antígenos de Protozoários/imunologia , Doença de Chagas/imunologia , Epitopos/imunologia , Microssomos/imunologia , Trypanosoma cruzi/imunologia , Adolescente , Adulto , Idoso , Animais , Anticorpos Bloqueadores/imunologia , Carboidratos/imunologia , Cardiomiopatia Chagásica/sangue , Cardiomiopatia Chagásica/diagnóstico , Cardiomiopatia Chagásica/imunologia , Doença de Chagas/sangue , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miocardite/sangue , Miocardite/diagnóstico , Miocardite/imunologia , Oxirredução , Ácido Periódico/metabolismo , Trypanosoma cruzi/citologiaRESUMO
Chagas disease is associated with several immunological alterations. Although resistance against infection with Trypanosoma cruzi has been shown to be influenced by the immune system, its participation in the development of the disease remains unclear. In this regard, cytokines play a fundamental role since they are involved in the regulation of hemopoiesis, lymphopoiesis and affect the function of all cell types involved in an immune response. Interferon gamma (IFN-gamma) has been extensively involved as a protective lymphokine against T. cruzi. Macrophages activated by IFN-gamma result in the release of reactive oxygen metabolites (ROS) and nitric oxide (NO). On the other hand, interleukin 4 (IL-4), interleukin 10 (IL-10) and transforming growth factor beta (TGF-beta) are able to down-regulate the intracellular control of T. cruzi infection by IFN-gamma-activated macrophages, to inhibit NO release and to down-regulate the activity of the TH1 subset of cells (IFN-gamma producers). While TNF-alpha has been implicated in the resistance as well as in the generation of tissue damage, interleukin 6 (IL-6) and interleukin 1 (IL-1) are associated with a variety of alterations in endothelial cell function which may be responsible for the microvascular spasm seen in chagasic myocardiopathy. Several cytokines, including IFN-gamma, IL-1 alpha, IL-6 and TNF-alpha have been shown to modulate the expression of adhesion molecules which participate in inflammatory process by recruitment of lymphocytes into inflammatory sites, contributing to the progression of the local inflammatory reaction in chagasic cardiomyopathy. Thus, it has been shown that acute infection with different strains of T. cruzi induced enhanced expression of ICAM-1 not only on infiltrating leukocytes but also on sarcolemma of cardiocytes and paralleled the production of proinflammatory cytokines. Experimental infection with T. cruzi induces cytokine production which in time modulates the resistance against the parasite and probably the development of chronic Chagas disease. Therefore, it can be postulated that an alteration in quantity and/or quality of cytokine production may be the cause of chronic Chagas disease.
Assuntos
Doença de Chagas/imunologia , Citocinas/fisiologia , Trypanosoma cruzi/imunologia , Animais , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/patologia , Doença de Chagas/patologia , Endotélio Vascular/patologia , Humanos , Imunidade Inata , Ativação de Macrófagos , Óxido Nítrico/biossíntese , Linfócitos T Auxiliares-Indutores/classificação , Linfócitos T Auxiliares-Indutores/imunologiaAssuntos
Anticorpos Antiprotozoários/biossíntese , Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Humanos , Tolerância Imunológica , Imunidade Celular , Interferon gama , Linfocinas/fisiologia , Camundongos , Fagócitos/parasitologia , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
Diferentes fracciones subcelulares de epimastigotes de T.cruzi fueron ensayadas en su capacidad de inducir protección o agresión en animales experimentales. La fracción flagelar (F) tuvo las mejores propiedades protectoras, sin efectos agresivos sobre los tejidos. Se prepararon varios anticuerpos monoclonales contra esta fracción. Dos de ellos, FCH-F8-1 y 4, mostraron capacidad de neutralizar la infectividad de tripomastigotes sanguíneos, de producir la lisis mediada por complemento de tripomastigotes de cultivo y de reconocer antígenos de la superfície de ambas formas epi y tripomastigotes. El anticuerpo FCH-F8-1, reconoce por inmunoprecipitación, una proteína de 85 kDa en tripomastigotes, mientras que en "blotting" reaccionó con una molécula de 43 kDa, en ambas formas del parásito. El otro anticuerpo, FCH-F8-4 reaccionó por esta última técnica, con varias proteínas de peso molecular entre 50 y 150 kDa, en epimastigotes y sólo con dos (15 y 48 kDa) en tripomastigotes. Ratones inmunizados con antígenos purificados por cromatografia de afinidad usando FCH-F8-4, fueron protegidos contra el desafio de formas infectantes. En una biblioteca de ADNc de epimastigotes de T. cruzi construída en el vector I gt11 se detectaron varios clones, tres con FCH-F8-4 y dos con FCH-F8-1. Dos clones, uno de cada grupo fueron estudiados, y (FCH-F8-1) 1 y (FCH-F8-4) 1. El tamaño de los insertos para ambos fue de 150 pares de bases y utilizados como sondas detectaron ARNm de epimastigotes de 3,5 y 5,0...
Assuntos
Camundongos , Animais , Anticorpos Monoclonais , Antígenos de Protozoários/isolamento & purificação , Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Vacinas Sintéticas/imunologia , Citotoxicidade Imunológica , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/imunologia , Mapeamento de PeptídeosRESUMO
Diferentes fracciones subcelulares de epimastigotes de T.cruzi fueron ensayadas en su capacidad de inducir protección o agresión en animales experimentales. La fracción flagelar (F) tuvo las mejores propiedades protectoras, sin efectos agresivos sobre los tejidos. Se prepararon varios anticuerpos monoclonales contra esta fracción. Dos de ellos, FCH-F8-1 y 4, mostraron capacidad de neutralizar la infectividad de tripomastigotes sanguíneos, de producir la lisis mediada por complemento de tripomastigotes de cultivo y de reconocer antígenos de la superfície de ambas formas epi y tripomastigotes. El anticuerpo FCH-F8-1, reconoce por inmunoprecipitación, una proteína de 85 kDa en tripomastigotes, mientras que en "blotting" reaccionó con una molécula de 43 kDa, en ambas formas del parásito. El otro anticuerpo, FCH-F8-4 reaccionó por esta última técnica, con varias proteínas de peso molecular entre 50 y 150 kDa, en epimastigotes y sólo con dos (15 y 48 kDa) en tripomastigotes. Ratones inmunizados con antígenos purificados por cromatografia de afinidad usando FCH-F8-4, fueron protegidos contra el desafio de formas infectantes. En una biblioteca de ADNc de epimastigotes de T. cruzi construída en el vector I gt11 se detectaron varios clones, tres con FCH-F8-4 y dos con FCH-F8-1. Dos clones, uno de cada grupo fueron estudiados, y (FCH-F8-1) 1 y (FCH-F8-4) 1. El tamaño de los insertos para ambos fue de 150 pares de bases y utilizados como sondas detectaron ARNm de epimastigotes de 3,5 y 5,0... (AU)
Assuntos
Camundongos , Animais , Doença de Chagas/imunologia , Vacinas Sintéticas/imunologia , Trypanosoma cruzi/imunologia , Antígenos de Protozoários/isolamento & purificação , Anticorpos Monoclonais/diagnóstico , Citotoxicidade Imunológica , Fragmentos de Peptídeos/imunologia , Mapeamento de Peptídeos , Camundongos Endogâmicos BALB CRESUMO
Subcellular fractions of T. cruzi epimastigotes (Epi) were studied for their capability to induce protective or aggressive effects in animals. The flagellar fraction (F) showed the best immunoprotective properties without tissular aggression. Monoclonal antibodies were raised against F. Two of them, FCH-F8-1 and 4, were able to neutralize the infectivity of bloodstream forms, to mediate lysis by complement of cell culture derived[trypomastigotes (Tripo) and to recognize the surface of Tripo and Epi. FCH-F8-1 reacted with a 85 kDa protein from Tripo (assayed by immunoprecipitation) and with peptides of 43 kDa on Epi and Tripo (tested by immunoblotting). FCH-F8-4 recognized several proteins ranging from 50 to 150 kDa on Epi and two molecules of 15 and 48 kDa on Tripo. Mice immunized with antigens purified by affinity chromatography by using FCH-F8-4 were protected against the infection. Several recombinant clones were detected on a cDNA lambda gt11 expression library constructed from T. cruzi Epi (Tulahuén strain): three with FCH-F8-4 and two with FCH-F8-1. One clone recognized by each monoclonal antibody was studied gamma (FCH-F8-1) 1 and gamma (FCH-F8-4) 1. Both inserts were of 150 base pairs each; they detected a 3.5 and 5.0 kilobases Epi mRNA, respectively. Both inserts were sequenced, and the amino acid sequences were inferred. gamma (FCH-F8-4) 1 codified for a 19 aa peptide, PAFLGCSSRFSGSFSGVEP, and gamma (FCH-F8-1) 1 for a 29 aa peptide EFLERGRISCORHSYTSYTSCSDEHNVTPFC. The whole 19 aa peptide was synthesized. This peptide (SP4) inhibited the ELISA reactivity against the parasite of chronically infected and F immunized mouse sera.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anticorpos Monoclonais , Antígenos de Protozoários/imunologia , Doença de Chagas/imunologia , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/isolamento & purificação , Citotoxicidade Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/imunologia , Mapeamento de PeptídeosRESUMO
Subcellular fractions of T. cruzi epimastigotes (Epi) were studied for their capability to induce protective or aggressive effects in animals. The flagellar fraction (F) showed the best immunoprotective properties without tissular aggression. Monoclonal antibodies were raised against F. Two of them, FCH-F8-1 and 4, were able to neutralize the infectivity of bloodstream forms, to mediate lysis by complement of cell culture derived[trypomastigotes (Tripo) and to recognize the surface of Tripo and Epi. FCH-F8-1 reacted with a 85 kDa protein from Tripo (assayed by immunoprecipitation) and with peptides of 43 kDa on Epi and Tripo (tested by immunoblotting). FCH-F8-4 recognized several proteins ranging from 50 to 150 kDa on Epi and two molecules of 15 and 48 kDa on Tripo. Mice immunized with antigens purified by affinity chromatography by using FCH-F8-4 were protected against the infection. Several recombinant clones were detected on a cDNA lambda gt11 expression library constructed from T. cruzi Epi (Tulahuén strain): three with FCH-F8-4 and two with FCH-F8-1. One clone recognized by each monoclonal antibody was studied gamma (FCH-F8-1) 1 and gamma (FCH-F8-4) 1. Both inserts were of 150 base pairs each; they detected a 3.5 and 5.0 kilobases Epi mRNA, respectively. Both inserts were sequenced, and the amino acid sequences were inferred. gamma (FCH-F8-4) 1 codified for a 19 aa peptide, PAFLGCSSRFSGSFSGVEP, and gamma (FCH-F8-1) 1 for a 29 aa peptide EFLERGRISCORHSYTSYTSCSDEHNVTPFC. The whole 19 aa peptide was synthesized. This peptide (SP4) inhibited the ELISA reactivity against the parasite of chronically infected and F immunized mouse sera.(ABSTRACT TRUNCATED AT 250 WORDS)
RESUMO
The capacity of antibodies in serum from individuals with chronic Chagas' disease to react with antigens in different subcellular fractions of Trypanosoma cruzi varied according to the clinical status of the patients. Antibodies in serum of asymptomatic patients were directed mostly against antigens in the citosol of the parasite, whereas in overtly cardiopathic patients antibodies were directed mostly against antigens in the microsomal fractions.