Biomedical subjects

M Loyens

Publications and source records attributed to M Loyens.

31 records · Page 2Linked to original sources

PubMed1993

Characterization of a Leishmania antigen associated with cytoplasmic vesicles resembling endosomal-like structure.

In the present study we have used antibodies to Leishmania major promastigote antigens which were eluted from a glutathione-agarose column (LmGbp) and could identify several parasite components among different Leishmania species by using immunoprecipitation and Western blot techniques. The results also showed that some of LmGbp are present among the molecules released into the culture medium. Moreover, immunofluorescence assays clearly demonstrated that LmGbp are expressed by intracellular amastigotes. The electron micrographs of thawed cryosections of L. major-infected cells revealed that the antigens were associated with the membrane of the phagocytic vacuole. Moreover, the Western blot technique allowed us to identify, using other Leishmania species extracts and anti-LmGbp antibodies, a major polypeptide of an apparent molecular mass of 66 kDa. Immunofluorescence studies suggested that the 66 kDa polypeptide is associated with intracytoplasmic vesicles. Cryosections of Leishmania promastigotes improved the fine structure preservation of the organelles and enabled a number of features to be seen, particularly the structures considered as vesicles, which appeared as a complex tubulo-vesicular structure resembling mammalian cell endosomes and Leishmania organelles previously named 'megasomes'. Further studies using antibodies against the native 66 kDa protein will be needed to investigate the localization of the protein at the ultrastructural level and to follow its intracellular vesicular traffic.

Animals↗

PubMed1992

Cloning and sequencing of a 24-kDa Trypanosoma cruzi specific antigen released in association with membrane vesicles and defined by a monoclonal antibody.

In the present study we have used the Tcr7 monoclonal antibody (mAb) previously characterized as directed against Trypanosoma cruzi 24-25-kDa specific antigens, both are immunogenic in man and during experimental T cruzi infections. We have demonstrated that mAb Tcr7 was able to recognize two in vitro translation products of molecular weights of 24 and 25 kDa. This suggested the holoproteic nature of these two related antigens bearing at least a common epitope and allowed us to use Tcr7 for an immunoscreening of a lambda ZAPII T cruzi cDNA library. Indeed, we have obtained several positive clones and completely sequenced the largest one which encoded theoretically for a protein of 23.7 kDa. The sequence analysis revealed a nearly perfect homology between this clone and one already described by other investigators and was shown to express a major flagellar protein of T cruzi able to bind calcium. Using different overlapping peptides derived from the sequence of the cDNA clone, we have localized the immunoreactivity of mAb Tcr7 mainly on several primary sequences present in the N-terminal part of the sequence, suggesting that the mAb could recognize a discontinuous epitope. Moreover, the immunoelectron microscopy allowed us to show that the antigen(s) carrying the epitope reacting with mAb Tcr7 is (are) released in association with membrane vesicles which protruded from the parasite surface and the flagellar pocket. This new mechanism of antigen shedding is likely to be independent of phospholipase C-mediated release of GPI-anchored molecules.

Amino Acid Sequence↗

PubMed1992

Trypanosoma cruzi glutathione-binding proteins: immunogenicity during human and experimental Chagas' disease.

Following purification by affinity chromatography, three glutathione-binding proteins (TcGBP) of 45, 30, and 25 kDa were co-purified from Trypanosoma cruzi epimastigotes. Using 1-chloro-2,4 dinitrobenzene as substrate, a glutathione S-transferase activity of 70 nmol/min/mg of proteins was detected in the GSH binding fraction. An increased expression of TcGBP and total GST activity was observed upon incubation of parasites with phenobarbital, which is an inducer of GST synthesis. Immunofluorescence and electron microscopic experiments demonstrated that TcGBP were expressed by all developmental stages of the parasite, including infective forms. The expression of these proteins by intracellular dividing amastigotes could be in favour of a potential defensive role of these molecules against host attack. Results obtained by immunoprecipitation of in vitro translation products using anti-TcGBP antisera suggested that these three polypeptides are not glycosylated. In addition, antibodies directed against the TcGBP were found in a high proportion of T. cruzi-infected chronic chagasic patients' sera and in sera of chronically infected BALB/c mice. In contrast, acute chagasic patients' sera and acute-phase mouse sera were found to be poorly reactive with these proteins. Our results identify a new class of potential target antigens, which may be essential for the development of T. cruzi in its host. Their protective role in experimental models deserves to be investigated.

Animals↗

PubMed1992

Fibronectin cleavage fragments provide a growth factor-like activity for the differentiation of Trypanosoma cruzi trypomastigotes to amastigotes.

The morphological and biochemical events following Trypanosoma cruzi trypomastigote-fibronectin (Fn) interactions have been studied. Adhesion of trypomastigotes to Fn-coated surfaces is followed by Fn degradation. The proteolytic cleavage of Fn was demonstrated by qualitative and quantitative measurement of Fn degradation after its exposure to trypomastigotes as well as polyacrylamide gel analysis of Fn proteolysis by a parasite protease (s). The released Fn peptide fragments stimulated the transformation of trypomastigotes to amastigotes. The gelatin (45 kDa) and heparin (40 kDa) binding fragments were shown to be able to promote trypomastigote differentiation. In contrast, native Fn and the 120 kDa fragment (cell attachment domain) were inactive. Complementary investigations showed that the gelatin and heparin binding fragments stimulated parasite RNA synthesis and protein synthesis and phosphorylation but not DNA replication and increased parasite intracellular cAMP concentrations. These findings suggest that the proteolysis of Fn by parasite proteases, which occurs under physiological conditions, might facilitate invasion of target cells by trypomastigotes. The Fn peptides released during this process may act as "growth factor-like" substances.

Animals↗

PubMed1991

Trypanosoma cruzi: a carbohydrate epitope defined by a monoclonal antibody as a possible marker of the acute phase of human Chagas' disease.

An IgM monoclonal antibody (MAb) against a carbohydrate epitope present in Trypanosoma cruzi trypomastigote excretory-secretory antigens and expressed by different developmental stages of the parasite (epimastigote, trypomastigote and intracellular amastigote) was linked to a solid phase matrix and used as an antigen-capture antibody. Human serum complexes containing the epitope were then detected by using specific secondary antibodies against human immunoglobulin isotypes. Results of detection of IgM, IgG, and IgA serum complexes (SC) containing a T. cruzi polypeptide epitope showed that SC could be detected in 69% of the 13 Chagasic acute phase sera studied with IgG, in 84% with IgM, and in 75% with IgA. Only 16% (IgG-SC), 8% (IgM-SC), and 10% (IgA-SC) of chronic sera from 50 patients were positive. No patients with toxoplasmosis or rheumatoid factor were positive. Of the 11 leishmaniasis sera studied, four had IgG-SC, two had IgA-SC, and five had IgM-SC. Of the eight Yanomamo Indians infected by Onchocerca volvulus, three were found to have IgG-SC, two had IgM-SC, and two had IgA-SC. Thirteen sera from healthy individuals living in an endemic area were also studied. One subject had IgG IgM and IgA-SC. The results presented in this study show for the first time, the specific detection of IgM, IgG, and IgA immune complexes using a MAb against T. cruzi. The presence of the epitope in association with IgM antibodies in sera from patients with the acute phase of the disease suggests that this antigen(s) carrying the epitope that reacts with the MAb could be a marker(s) of active infection. In addition, the specificity of the serum complex capture assay allowed the detection of Chagas' disease in two different endemic areas (Argentina and Venezuela).

Acute Disease↗

PubMed1990

Trypanosoma cruzi: differential expression and distribution of an 85-kDa polypeptide epitope by in vitro developmental stages.

The expression by Trypanosoma cruzi developmental stages of an 85-kDa polypeptide epitope defined by the 155D3 monoclonal antibody (mAb) has been investigated. Immunoprecipitation revealed the presence of an 85-kDa antigen in the NP-40 soluble extract of parasites freshly released from infected fibroblasts; this antigen was not found in epimastigote and Leishmania infantum promastigote. Indirect immunofluorescence revealed that the mAb 155D3 failed to react with trypomastigotes, whereas extracellular amastigotes were heavily stained. Positive organisms displayed either surface or polar fluorescence. Since the same mAb immunoprecipitated the 85-kDa antigen in both radioactive iodine- and methionine-labeled trypomastigote detergent soluble extracts, the reactive epitope is likely to be hidden in a cryptic site in trypomastigotes. An alternative explanation for the negative immunofluorescence on trypomastigotes and the positive immunoprecipitation is the presence, in the extracts, of a small population of parasites already expressing the 155D3 epitope. Immunoelectron microscopy revealed that the target epitope is heterogenously distributed among the populations of differentiating parasites. Two types of immunogold labeling were observed: (a) mAb revealed a high amount of reactive material associated with the periphery of the parasites and (b) a label was observed on the inner surface of peripheral vacuoles that might correspond to cross sections of inflated flagellar pockets and in association with vesicles which were released by the parasites. The surface expression of the epitope recognized by the 155D3 mAb was followed by fluorescence-activated cell-sorting analysis. The results showed that the epitope is increasingly accessible during trypomastigote differentiation in vitro. Taken together, these results suggest that the epitope reacting with the 155D3 mAb is heavily expressed on extracellular amastigotes after the transformation process and, thus, appears to be developmentally regulated.

Animals↗

PubMed1990

Characterization of major surface and excretory-secretory immunogens of Trypanosoma cruzi trypomastigotes and identification of potential protective antigen.

The surface antigens of Trypanosoma cruzi trypomastigotes were identified by immunoprecipitation and were compared with metabolically labelled excretory-secretory products (ES) released by the parasites in vitro. A series of major immunogenic components in the ES antigens were revealed (160 kDa, 130 kDa and 80-110 kDa). The trypomastigote surface also bears the 130 kDa band and the 80-110 kDa complex. Competition experiments demonstrated the common antigenic structure of the ES and the surface antigens. Two-dimensional analysis of ES antigens immunoprecipitated by human Chagasic serum revealed several spots in the 80-110 kDa region with a wide range of isoelectric points (PI between 5.4 and 6.7). This reflects a charge heterogeneity of these polypeptides. The trypomastigote 85 kDa polypeptide was also identified in the ES antigens by using a monoclonal antibody against this antigen. Two-dimensional analysis of the 85 kDa proteins shed from the surface of trypomastigotes and immunoprecipitated by the monoclonal antibody 155D3 showed 2 major spots: a major part of the 85 kDa polypeptide was found at pH 6.5-6.6, whereas a substantial amount of the antigen was found at pH 5.7. An additional component with molecular weight of approximately 58 kDa and isoelectric points of 6.5 and 6.6, was also visualized. Detection of the 85 kDa polypeptide circulating in serum from patients with acute and chronic Chagas' disease was achieved using an enzyme-linked immunosorbent assay. In addition, the data obtained showed that a polyclonal antibody to the 85 kDa polypeptide could be used to passively induce a partial protection of Fischer rats against acute lethal infection. Thus, the antigens recognized by polyclonal antibody appear to play a role in the development of protective immunity against T. cruzi.

Animals↗

PubMed1989

Evaluation of a competitive antibody enzyme immunoassay for specific diagnosis of Chagas' disease.

A competitive enzyme immunoassay based on the use of a monoclonal antibody (MAb) specific for "component 5" of Trypanosoma cruzi was evaluated. The antigenicity and immunogenicity of this component has been observed in natural and experimental infections. The studies were conducted in an area of Bolivia where mixed infections with Leishmania braziliensis are frequent and present a problem in the accurate diagnosis of T. cruzi infections. The specificity and sensitivity of this assay as compared to the indirect immunofluorescence and ELISA tests were demonstrated. The present test has proved to be more specific than the immunofluorescence and ELISA tests.

Animals↗

PubMed1989

Trypanosoma cruzi: circulating polysaccharide factors excreted in vitro and in vivo.

An antigen factor (EF), thermostable and soluble in trichloroacetic acid was detected in the supernatant fluid of epimastigote cultures of Trypanosoma cruzi and in the sera of patients with acute Chagas disease. An hyperimmune antiserum to this antigenic factor was obtained in rabbits. The EF was revealed on the fibroblast surface membranes of rats infected with trypomastigotes, using the indirect immunofluorescence technique. The presence of EF in the sera of patients with acute Chagas disease as well as in the supernatant of epimastigotes culture at logarithmic phase, leads to its association with a process of parasite proliferation. Being EF a component of the parasite, its origin both in vitro and in vivo could be the result of an excretion-secretion of parasite or simply a result of the parasite's death. It can be postulated that the same as in other protozoic infection, EF could be used by T. cruzi in the process of cell penetration.

Adult↗

PubMed1986

Specific and sensitive immunological diagnosis of Chagas' disease by competitive antibody enzyme immunoassay using a Trypanosoma cruzi-specific monoclonal antibody.

Coexistence of Chagas' disease with leishmaniasis and T. rangeli infection in endemic areas and cross-reactivity between corresponding etiological agents can confuse the immunodiagnosis of Chagas' disease. A discriminative serological test could therefore represent a major advance in specific immunodiagnosis. A competitive antibody enzyme immunoassay against a component 5-enriched preparation, using a T. cruzi species-specific monoclonal antibody has allowed development of a specific serodiagnosis of Chagas' disease with high sensitivity (96.6% in undetermined and chronic phases of infection). This test can differentiate Chagas' disease from other cross-reacting parasitic diseases in areas where concomitant infections are unknown or suspected.

Animals↗

PubMed1984

Different monoclonal antibodies against the component 5 specific for Trypanosoma cruzi.

Three murine monoclonal antibodies (I-35/67, II-190/30, III-160/18) produced by immunization against total epimastigote extract or component 5-enriched fractions of Trypanosoma cruzi (Tehuantepec strain) have been demonstrated to recognize the component 5 specific for T. cruzi. By immunofluorescence studies, one of these monoclonal antibodies (I-35/67) was shown to bind to the epimastigote cell surface, and the two others (II-190/30 III-160/18) were preferentially directed against internal subcellular organelles. Immunoprecipitation using these monoclonal antibodies followed by SDS-PAGE analysis, has led to identification of four molecules with apparent molecular weights of 72 Kd, 51 Kd, 43 Kd, and 24 Kd in T. cruzi epimastigotes. Potential uses of these monoclonal antibodies in serological tests and immunochemical analysis of target antigens are discussed.

Animals↗

PubMed1982

[Production of monoclonal antibody anti-antigen 5 for Trypanosoma cruzi].

Monoclonal antibodies against Trypanosoma cruzi have been produced by fusion of Sp 2/0 myeloma cells and splenic lymphocytes from BALB/c Mice immunized with a T. cruzi antigenic fraction. Antigenic studies of these monoclonal antibodies have led to the identification of IgG 1 antibody against the component 5 specific for T. cruzi (cellular clone II 190/151). These monoclonal antibodies will give rise not only to new diagnostic and epidemiological perspectives but will also aid in achieving a better understanding of the biological role of this antigen.

Animals↗

PubMed1980

Circulating immune complexes and anti-IgG antibodies in mucocutaneous leishmaniasis.

Circulating immune complexes (CIC), anti-IgG, anti-DNA, and anti-collagen autoantibodies (Ab) were investigated in sera from patients with South American leishmaniasis. No significant levels of anti-DNA or anti-collagen autoantibodies were observed. Only a few patients with cutaneous leishmaniasis or with only one mucocutaneous lesion showed values for CIC and anti-IgG Ab higher than those in the control group. In contrast, both CIC and anti-IgG Ab were demonstrated in most patients with several lesions due to mucocutaneous leishmaniasis (MMC). Moreover, a close correlation was noticed between the detection of CIC and anti-IgG Ab in MMC patients. This relationship suggested that part of the detected CIC could have been formed by IgG-anti-IgG complexes. The involvement of these immunopathologically active substances in the clinical evolution of mucocutaneous leishmaniasis is discussed.

Antigen-Antibody Complex↗