Trypanosoma cruzi: identification of a galactose-binding protein that binds to cell surface of human erythrocytes and is involved in cell invasion by the parasite.

Trypanosoma cruzi must invade mammalian host cells to replicate and complete its life cycle. Almost all nucleated mammalian cells can be invaded by the parasite following a receptor-ligand recognition as an early prerequisite. In this work, we describe a 67-kDa lectin-like glycoprotein that binds to desialylated human erythrocyte membranes in a galactose-dependent way. This protein is present on the parasite surface in both infective and non-infective stages of T. cruzi. More interestingly, we demonstrate by lectin-immuno-histochemistry assays that the 67kDa protein is involved in the recognition of host-cell receptors in mouse cardiac tissue and human cardiac aortic endothelium and mammary artery tissue. Moreover, antibodies against the 67kDa glycoprotein inhibit in vitro host-cell invasion by 63%. These data suggest that the 67kDa glycoprotein in vivo is needed for host-cell invasion by T. cruzi.

Antibody maturation in Trypanosoma cruzi-infected rats.

The study of antibody avidity changes during infection has improved the understanding of the pathologic processes involved in several infectious diseases. In some infections, like toxoplasmosis, this information is being used for diagnostic purposes. Results of the evolution of antibody avidity for different specific antigens in Trypanosome cruzi-infected rats are presented. A Western blotting technique, combined with avidity analysis to identify antigens that elicit high-avidity antibodies, is suggested. In this system, antibodies showed high avidity values only during the chronic phase of infection and only in relation to antibodies against 21-, 33-, 41-, 42-, 56-, 58-, 66-, and 72-kDa antigens. Finally, a 97-kDa T. cruzi antigen, which was recognized by high-avidity antibodies and occurred in noninfected rats, was identified. These results allow us to evaluate the different antigens in chagasic infection. Our results show that with the correct choice of antigen it is possible to detect differences in maturation of antibodies and to discriminate, in an experimental model, between recent (acute) and chronic infections.

Inhibition of trypomastigotes' infectivity by a monoclonal antibody directed to a glycoconjugated fraction of epimastigotes of Trypanosoma cruzi.

In this work it was shown that the infectivity of trypomastigote forms of Trypanosoma cruzi was affected upon the interaction with the Monoclonal Antibody (McAb) 2E9, which was raised against a glycoconjugated fraction of membranes of epimastigotes (Tulahuen strain). Characterization of the epitope recognized by this McAb, as well as its effects on complement mediated lysis and host cell invasion are reported. Immunocytochemical analysis showed that the McAb was reactive with two macromolecules (41-58 kDa) present on Trypanosoma cruzi epimastigotes (Tulahuen and Y strain), while it recognized several trypomastigotes macromolecules, showing a more intense reactivity with a band of 80 kDa. By indirect immunofluorescence, it was found there were subpopulations of blood and tissue culture derived trypomastigotes which attach the antibody to varying degrees. Studies using chemical or enzymatically treated antigens suggested that the McAb 2E9 was directed against carbohydrate epitopes, which were identified as being--galactosyl residues. In addition, preliminary results are shown, suggesting that the epitope recognized by the McAb 2E9 is involved in adhesion/or internalization of trypomastigotes.

Anticarsia gemmatalis nuclear polyhedrosis virus replication in serum-free and serum-reduced insect cell cultures.

In order to develop a financially feasible process to produce Anticarsia gemmatalis Nuclear Polyhedrosis virus in cell culture, we developed a lipidic supplement to replace fetal calf serum in insect cell culture media. The supplement, prepared with an extract of lipids from hen egg yolk, allowed us to reduce the contents of serum in the culture medium from 10% to 1%. IPLB-Sf-21 cells could be kept along consecutive passages in serum-reduced medium. The replication of AgNPV in HEYLE-supplemented cultures was evaluated. Extracellular virions production was the same as in FCS-supplemented-cultures, but the production level of polyhedral inclusion bodies was significantly lowered in HEYLE-supplemented cultures. The reduced production of PIBs is related to a premature releasing of non-occluded particles as well as to a reduced synthesis of polyhedrin protein.

Expression of protozoan antigens by hybrids between Leishmania mexicana and myeloma SP2/0.

Hybrid cells were obtained between Leishmania mexicana promastigotes and mouse myeloma SP2/0 cells, and examined for expression of leishmanial antigens. A ratio of 1:10 of myeloma to T. cruzi cells was unsuccessful because of outgrowth of non-fused cells. With a ratio of 2:1 four waves of multiplication of 'chimeric' cells were observed over 45 days. The death of the hybrids after this period is explained by segregation of DNA and loss of chromosome material. Hybrid cells gave a positive reaction with antibodies in the sera from patients infected with Leishmania, as demonstrated by indirect immunofluorescence. Conversely, promastigote forms of Leishmania gave a negative reaction with the same antibodies, which recognize surface antigens of the amastigote stage of Leishmania. It is possible therefore that amastigote stage antigens are expressed on the surface of the 'chimeric' hybrid as a result of transformation of promastigotes following hybridization.

Peanut agglutinin affinity chromatography of Trypanosoma cruzi glycoproteins.

Glycoproteins from Trypanosoma cruzi epimastigotes have been extracted by diiodosalycilic acid and lithium salts, and phenol-water biphasic partition. Peanut agglutinin has been used in a one step preparative method for fractionating the total extract in order to separate the so-called galactose-terminal glycoproteins. The different fractions have been studied by SDS electrophoresis, ultracentrifugation and immunoelectrophoresis techniques. The experimental immunogenicity, antigenicity and specificity of the PNA affinity fractions has been evaluated.