RESUMO
Several recombinant Trypanosoma cruzi proteins previously isolated were used as antigens to analyse antibody specificities present in sera from human infections. Some parasite proteins such as SAPA (Shed Acute Phase Antigen) are antigenic early after infection. Others, like antigens 1 and 30, are antigenic mainly during the chronic phase of the infection. To understand why different proteins are antigenic at different periods of infection, specificities of antibodies present in the sera of infected mice were compared with the antigens expressed by parasites collected directly from blood. Parasites collected during the acute parasitaemia peak expressed not only antigen SAPA, but also antigens 1 and 30. However, only antibodies against SAPA were frequently observed during the early period and also in the chronic phase of murine infection. Long-lasting antibodies against SAPA were detected regardless of the mouse and parasite strains used. Furthermore, all 8 recombinant clones detected in a T. cruzi expression library with pooled sera from acutely infected mice were homologous to the SAPA gene. These results show that even though parasites from the acute parasitaemia peak in mice may express simultaneously several proteins known to be antigenic, only antibodies against SAPA were consistently detected.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Doença de Chagas/parasitologia , Trypanosoma cruzi/imunologia , Doença Aguda , Animais , Anticorpos Antiprotozoários/imunologia , Especificidade de Anticorpos , Doença de Chagas/sangue , Doença de Chagas/imunologia , Doença Crônica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos NusRESUMO
A Trypanosoma cruzi antigen which is shed into the culture medium by the trypomastigote stage of the parasite and detected in blood of acutely infected mice was cloned and characterized. We designate this antigen shed acute phase antigen (SAPA). Five protein bands with apparent molecular masses ranging from 160 to 200 kDa were detected by immunoblotting of plasma from infected mice and in supernatants of cultured trypomastigotes upon reaction with antibodies against SAPA. A serum obtained from a patient acutely infected with Chagas' disease revealed a similar set of polypeptides in supernatants of cultured trypomastigotes when tested by immunoblotting. SAPA seems thus to be a major shed protein during the acute period of the disease. Twenty-six of 28 sera from human acute cases of Chagas' disease tested reacted with SAPA. Conversely, only 8-10% of sera from chronic cases of the disease contained detectable levels of antibody against SAPA. Sera from rabbits infected with six different parasite strains all contained antibodies against SAPA. Antibodies against SAPA are detectable 15 days after the manifestation of acute Chagas' disease symptoms in humans and 15 days post-infection in sera from mice and rabbits. The nucleotide sequence of a genomic clone encoding the 3' end of the SAPA gene revealed the presence of 14 tandemly arranged 12-amino acid-long repeats. A 39-amino acid-long region that is very hydrophobic precedes the stop codon. Due to its early appearance it might be possible to design diagnostic assays which are based on SAPA for identification of recently infected cases of Chagas' disease.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/análise , Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Doença Aguda , Adulto , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/genética , Sequência de Bases , Criança , DNA/genética , Feminino , Humanos , Immunoblotting , Masculino , Camundongos , Dados de Sequência Molecular , Coelhos , Sequências Repetitivas de Ácido Nucleico , Trypanosoma cruzi/genética , Células VeroRESUMO
Se presenta la experiencia acumulada entre noviembre de 1984 a enero de 1987 en revascularizaciones de 9 extremidades inferiores con puentes al tobillo. La indicación quirúrgica fué por salvamento de extremidad y la técnica quirúrgica empleada fué la de safena in situ, con un 100% de utilización de la vena. Tanto el origen del puente desde la arteria poplítea como su salida doble parecen ser buenas alternativas en casos bien calificados. Se enfatiza la necesidad de un adecuado estudio angiográfico pre o intraoperatoria para la planificación quirúrgica. En la serie no hubo mortalidad operatoria y bajos índices de complicaciones graves. El salvamento obtenido fué de 7 extremidades y su fracazo no significó elevar el nivel de amputación primario. Los puentes al tobillo para salvamento de extremidades parece ser una alternativa técnica promisoria, por lo que deben ser intentados en los pacientes con enfermedad distal y pie potencialmente recuperable
Assuntos
Pessoa de Meia-Idade , Humanos , Masculino , Feminino , Extremidades/cirurgia , Angiografia , Artéria Poplítea/cirurgia , Veia SafenaRESUMO
Chromosomal DNA from Trypanosoma cruzi, the agent of the American trypanosomiasis (Chagas' disease), was used for construction of a DNA library, employing the expression vector lambda gt11. Nine clones encoding different parasite antigens were isolated from this library by screening with an antiserum from a Chagasic patient. Nucleotide sequence analysis showed that seven out of the nine isolated clones code for antigens which contain tandemly repeated amino acid sequence motifs. Each of the seven antigens contains a unique repeat, ranging in length between 5 and 68 amino acids. The length of the repeats is highly conserved within each clone. Fusion proteins, expressed from two of the clones, reacted with a large proportion of sera collected from Chagasic patients in Argentina, Brazil and Chile. These clones appear thus to encode antigens which are shared between different strains of T. cruzi. Immunofluorescence experiments with live parasites showed that three of the antigens were detectable on the surface of trypanosomes.
Assuntos
Antígenos de Protozoários/genética , Sequências Repetitivas de Ácido Nucleico , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Animais , Doença de Chagas/imunologia , DNA , Imunofluorescência , Hibridização de Ácido Nucleico , Proteínas Recombinantes de Fusão/imunologia , Trypanosoma cruzi/genéticaRESUMO
A genomic DNA library from Trypanosoma cruzi, the agent of Chagas' disease, was constructed in the gt11 lambda vector and was screened with serum from a Chagasic patient. Out of 53 positive clones, 23 plaques were purified to homogeneity and 10 different groups were defined by cross-hybridization experiments and by reaction of antibodies selected with products from each recombinant clone. Native T. cruzi proteins of molecular mass ranging from 85 to larger than 205 kDa that share antigenic determinants with products of the recombinant clones were observed in Western blots of parasite extracts. Some of the native proteins were detected in the trypomastigote stage of the parasite, while others were present in epimastigotes as well. The latter result was confirmed for some recombinant clones by hybridization of the cloned DNA with Northern blots of parasite RNA. Clones from each group reacted differently with nine sera from rabbits infected with several T. cruzi strains as well as with eight sera from human patients. Clone 7 was detected by all rabbit sera but not by three human sera. Conversely, clones 1, 2 and 30 were detected by all human sera but failed to be detected by most rabbit sera. We conclude that several proteins from T. cruzi are antigenically active during infection and that some of them differ in their ability to generate antibodies in rabbit or human infections.