ABSTRACT
Given that cardiovascular risk factors (CRF), such as smoking, alcoholism and hypertension, may contribute to the development of heart lesions, chronically Trypanosoma cruzi-infected individuals were studied to explore the relationship between the presence of such CRF, cardiomyopathy and antibodies that have been proposed to play a pathogenetic role in Chagas' disease. The targets of these antibodies were T. cruzi antigens such as cruzipain (Cz), a P ribosomal antigen (P2), and a component of myelin sheaths also present in T. cruzi (sulphatide). Individuals were classified into four groups on the basis of specific serology and presence of CRF: subjects with T. cruzi infection and CRF; those with positive serology and no CRF; seronegatives with CRF; and seronegatives without CRF, were analysed. Seronegatives or seropositives with CRF showed a greater occurrence of heart involvement (chest X-ray and/or electrocardiogram abnormalities). Seropositives with CRF displayed significantly higher levels of antisulphatide antibodies than the three remaining groups and higher levels of antibodies against Cz and P2 compared to the seropositives without CRF. Increased amounts of anti-P2 and antisulphatide antibodies were also found in seropositives with marked heart involvement. The presence of CRF is associated with a different profile of antibody responses and degree of cardiac effects.
Subject(s)
Cardiovascular Diseases/etiology , Chagas Cardiomyopathy/immunology , Chagas Disease/physiopathology , Animals , Cardiovascular Diseases/immunology , Chagas Disease/complications , Chronic Disease , Risk Factors , Trypanosoma cruzi/immunologyABSTRACT
Serological diagnosis of Trypanosoma cruzi infection is hampered by issues related to test specificity due to the cross-reactivity of most antigens with proteins of related parasites such as Leishmania spp. The recombinant calflagins are considered relevant antigens for the diagnosis of infection by Trypanosoma cruzi. In the present work, we describe two genes coding for putative calflagins in Leishmania major with the N-terminal moieties presenting high similarity with T. cruzi genes. This fact raised questions about their role in some cross-recognition of this antigen by sera from Leishmania spp.-infected individuals. The complete T. cruzi calflagin and two fragments of the protein, consisting of 146 amino acids of the N-terminal and 65 amino acids of the C-terminal regions, were expressed and evaluated against a panel of sera, which included well-characterized samples from T. cruzi, and Leishmania-infected patients. We were able to show that sera from Leishmania (Viannia) braziliensis-infected individuals recognized the recombinant full-length calflagin. Both the N-terminal and the complete protein presented the same high sensitivity (98.5% of sera from T. cruzi-infected patients was detected) but different specificities (94% and 98%, respectively, when evaluated against sera from people not infected by T. cruzi, including 15 sera from people infected with L. braziliensis). The C-terminal fragment presented low sensitivity (70%) but 100% specificity. We propose the use of these antigens in two sequential assays to optimize the serological diagnosis of T. cruzi infection in humans in geographic areas where Leishmania spp. infection is coendemic.
Subject(s)
Calcium-Binding Proteins/biosynthesis , Chagas Disease/diagnosis , Protozoan Proteins/biosynthesis , Trypanosoma cruzi/chemistry , Amino Acid Sequence , Animals , Antigens, Protozoan/biosynthesis , Antigens, Protozoan/chemistry , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/metabolism , Hemagglutination Tests/methods , Humans , Molecular Sequence Data , Protein Engineering , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Recombinant Proteins/biosynthesis , Sensitivity and Specificity , Sequence AlignmentABSTRACT
In the present work, we have determined the effect of expression vectors and their corresponding host bacteria on the antigenic performance of Trypanosoma cruzi P2beta (TcP2beta) full-length recombinant protein. The gene encoding the TcP2beta ribosomal protein was cloned in pMAL-c2 and pET-32a vectors that allow the expression of high levels of soluble fusion proteins. A panel of 32 positive and 32 negative sera was assayed with the purified proteins expressed using pMal-c2 (TcP2beta-MBP) and pET-32a (TcP2beta-TRX) vectors and with MBP and TRX purified from pMAL-c2 and pET-32a vectors, respectively. The antigenic behavior of each TcP2beta recombinant protein differed in the diagnostic performance in terms of DI(+) (93.7 for TcP2beta-MBP vs 100% for TcP2beta-TRX), in DI(-) (90.5 for TcP2beta-MBP vs 100% for TcP2beta-TRX) and in cross-reaction with negative sera. To determine if the higher reactivity of expressed pMAL-c2 protein was due to folding during protein expression or to a steric effect related to the protein adsorption at the titration plate, the reactivity of sera against soluble proteins was assessed by ELISA inhibition assays. As each soluble protein preserved its level of reactivity, we concluded that differences in reactivity were due to intrinsic characteristics of the proteins and not to differences in patterns of adsorption to the plates.
Subject(s)
Chagas Disease/diagnosis , Phosphoproteins , Protozoan Proteins , Trypanosoma cruzi/genetics , Animals , Blotting, Western , Carrier Proteins/genetics , Cloning, Molecular , Cross Reactions/immunology , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Gene Expression , Gene Library , Genetic Vectors/genetics , Histidine/genetics , Humans , Maltose-Binding Proteins , Phosphoproteins/genetics , Phosphoproteins/immunology , Polymerase Chain Reaction , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Ribosomal Proteins/genetics , Ribosomal Proteins/immunology , Sensitivity and Specificity , Serologic Tests , Thioredoxins/genetics , Trypanosoma cruzi/chemistry , Trypanosoma cruzi/immunologyABSTRACT
In the present work we propose a simple method for affinity purification of the 67-kDa lectin-like glycoprotein (LLGP-67) from Trypanosoma cruzi, the causative agent of Chagas' disease. The LLGP-67, which presents galactose binding activity and participates in the host cell recognition process, was previously purified by methods based on its interaction with galactose residues on erythrocytic membranes. We describe herein results showing that this protein can be purified from T. cruzi in a direct way using non-derivatized agarose as a chromatographic ligand. We also demonstrate the relevance of LLGP-67 as an antigen for human diagnosis of chagasic infection. Sensitivity and specificity for this antigen were calculated, being 98 and 98.11% respectively.
Subject(s)
Antigens, Protozoan/isolation & purification , Chagas Disease/diagnosis , Lectins/isolation & purification , Protozoan Proteins/isolation & purification , Trypanosoma cruzi/chemistry , Animals , Antigens, Protozoan/analysis , Antigens, Protozoan/immunology , Chagas Disease/parasitology , Chromatography, Affinity , Chromatography, Agarose , Enzyme-Linked Immunosorbent Assay , Female , Galactose/metabolism , Humans , Immune Sera , Lectins/analysis , Lectins/immunology , Protozoan Proteins/analysis , Protozoan Proteins/immunology , Rabbits , Trypanosoma cruzi/immunologyABSTRACT
Meeting of the Biotechnology Subcommittee Advisory Committee on Health Research in Buenos Aires, Argentina on April 16-19, 1996
Meeting of the Advisory Committee on Health Research, 31. Pan American Health Organization; Jul. 15-17, 1996
Subject(s)
Biotechnology , Health Services ResearchABSTRACT
Meeting of the Biotechnology Subcommittee Advisory Committee on Health Research in Buenos Aires, Argentina on April 16-19, 1996
Reunión del Comité Asesor de Investigaciones en Salud, 31. Organizacion Panamericana de la Salud; Jul. 15-17, 1996
