The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris-HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal D-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68kD and 180kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for beta-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues.
Non-adherent cells from PPD+ tuberculosis patients (TBP PPD+) and from healthy individuals treated with whole tuberculosis anergic immune sera or with its protein A-Sepharose IgG fraction, or with sera fraction separated by PPD-Sepharose chromatography, were submitted to immunofluorescence assays. Anti-human IgG or IgM FITC-conjugate were used to reveal the assays, and results were expressed by a fluorescence percentage or fluorescence index. The presence of IgG over the surface of PPD+ non-adherent cells was detected. High fluorescence percentages were observed only in those PPD+ cells treated with whole anergic serum or with its IgG fraction. Positive fluorescence index values were obtained only in those PPD+ cells treated with anergic serum, meanwhile fluorescence index was always negative when non-bound fractions from PPD-Sepharose were used. Results suggest that non-adherent population are the cell targets for the serum inhibitory factor, which previously has been detected to inhibit antigen response in PPD reactive cells and, point out the specific behavior of this factor, since it was eliminate by PPD-Sepharose chromatography. The IgG nature of the factor was demonstrated by SDS-PAGE and immunoelectrophoresis.
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