Evaluation of a western blot test in an outbreak of acute pulmonary histoplasmosis.

A western blot (WB) test was evaluated for detection of antibodies against native glycosylated and chemically deglycosylated M and H antigens of Histoplasma capsulatum in serum obtained from patients during the acute phase of pulmonary histoplasmosis that occurred during an outbreak. Of 275 serum samples tested by immunodiffusion and complement fixation (CF) samples from 40 patients affected during this outbreak and from 37 negative controls were tested by WB test. A group of patients whose sera were negative for CF antibodies and precipitins early in the acute stage of histoplasmosis but who all seroconverted during convalescence 6 weeks later were tested with the WB test. Antibodies against untreated H and M antigens were detected at a 1:100 dilution by WB test in 45% of the 20 acute-phase serum samples and in all 20 of the convalescent-phase specimens. The WB test's sensitivity for acute-phase specimens increased to 90% (18 of 20 specimens) when H and M antigens were treated by periodate oxidation to inactivate susceptible carbohydrate epitopes. When native glycosylated antigens were used in the WB test, positive reactions were observed in negative control serum specimens (3 of 37 specimens; 8%) and in serum specimens obtained from asymptomatic persons screened as part of the outbreak investigation (13 of 20 specimens; 65%). These positive reactions were also attributed to glycosidic epitopes since the specificity of the WB test increased from 78 to 100% when periodate-treated H and M antigens were used. WB test with deglycosylated H and M antigens of histoplasmin provides a rapid, sensitive, and specific test to diagnose acute pulmonary histoplasmosis before precipitins can be detected.

Inmunolocalización del complejo polisacárido-proteína en ultraestructuras de la fase micelial y levaduriforme de Histoplasma capsulatum / Immunolocalization of the polysaccharide-protein complex (PPCD-Histo) in the Histoplasma capsulatum mycelium and yeast phase ultrastructures

Introducción: El empleo de un antígeno parcialmente purificado denominado complejo polisacárido-proteína desproinizado de Histoplasma capsulatum (CPPD-Histo), utilizado para discriminar la histoplasmosis de diversas micosis pulmonares y otras enfermedades respiratorias en métodos inmunodiagnósticos de alta sensibilidad, ha sido motivo de estudio desde hace años por nuestro grupo de investigación: Objetivo: En este trabajo se planteó conocer la ubicación celular preferencial del antígeno CPPD-Histo, en las diferentes formas y estructuras de las fases micelial y levaduriforme del hongo. Material y métodos: El estudio se desarrolló mediante inmunolocalización con marcaje de oro coloidal para microscopia electrónica, usando anticuerpos primarios CPPD-Histo específicos. Resultados y discusión: La localización en microscopia electrónica mostró mayor concentración del CPPD-Histo en las zonas de mayor grosor de la capa externa de la pared celular de las proyecciones digitiformes de macroconidios, poco marcaje en pared celular de hifas, y una distribución dispersa de la marca en las levaduras. Conclusión: La pared celular de los macroconidios de la fase micelial del hongo es la estructura fúngica con mayor concentración del antígeno CPPD-Histo

Immunobiological activity of recombinant H antigen from Histoplasma capsulatum.

The H antigen from Histoplasma capsulatum is one of the known principal antigens of this fungus, but information regarding its identity and its participation in cellular immunity is largely unavailable. Therefore, we sought to determine both the nature of this antigen and the nature of its involvement in the cell-mediated immune response. The antigen was isolated from histoplasmin and digested with selected proteinases, and the cleavage products were subjected to reverse-phase high-performance liquid chromatography. Amino acid sequences of protein fragments were obtained by Edman degradation. A fragment of the gene encoding the H antigen was isolated by using degenerate primers in the PCR. This fragment was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence revealed homology to extracellular beta-glucosidases. A cDNA was generated by reverse transcription PCR and cloned into the expression vector pET 19b. Recombinant H antigen was isolated from inclusion bodies of Escherichia coli and tested for its ability to elicit and induce an in vitro cell-mediated immune response in BALB/c mice. Recombinant antigen stimulated splenocytes from mice immunized with viable yeast cells or with antigen suspended in adjuvant. Mice inoculated with H antigen were not protected against either a sublethal or a lethal inoculum of yeast cells. Thus, H antigen stimulates a cell-mediated immune response but does not induce a protective response to H. capsulatum.

Two-dimensional immunoelectrophoresis of histoplasmin and a purified polysaccharide-protein antigen of Histoplasma capsulatum.

Crude histoplasmin and a polysaccharide-protein complex (PPC-histo) antigens obtained from culture filtrates of Histoplasma capsulatum were analyzed by single and tandem two-dimensional immunoelectrophoresis (TD-IEP) using a rabbit hyperimmune anti-histoplasmin polyvalent serum. Single TD-IEP showed 14 arc precipitates for histoplasmin. Continuity of arcs 2, 6, and 7, and 9 and 10 was observed, suggesting a different polymeric configuration of the same antigen. This was also confirmed in tandem TD-IEP of histoplasmin with homologous (PPC-histo) and heterologous PPC's from Blastomyces dermatitidis, Paracoccidioides brasiliensis and Coccidioides immitis. Tandem TD-IEP of histoplasmin and PPC-histo displayed a similar antigenic pattern to histoplasmin alone, being arcs 1 and 3 more evident and apparently present only in histoplasmin and PPC-histo. Tandem TD-IEP showed common antigens among the other heterologous fungal purified antigens, and seems useful to observe the multiplicity of antigens present in fungal preparations and to identify those precipitates (arcs 1 and 3) that are predominant in the purified preparation.

Evaluation of cation exchange chromatography for the isolation of M glycoprotein from histoplasmin.

Cation exchange chromatography was evaluated to purify the M antigen from histoplasmin (HMIN). Two H and M antigen-containing fractions, soluble (S) and precipitate (PP), resulted from the initial 0.025 M, pH 3.5 citrate buffer dialysis step. The PP fraction contained 62% of the M antigen activity and was resolubilized. Both fractions were chromatographed on CM Sepharose CL-6B. Polysaccharide C antigen was abundant in the S fraction and most of it did not bind to CM Sepharose. M antigen-enriched fractions were eluted with 0.5 M NaCl. Re-chromatography of the relevant S fraction (S-II) and PP fraction (PP-II) by linear gradient fast protein liquid chromatography (FPLC) removed protein and C impurities. M antigen purified by FPLC from the PP-II fraction was depleted of other antigens when Western blots were probed with anti-M, anti-H and anti-C monoclonal antibodies (Mabs). M antigen was identified as a 94 kDa glycoprotein containing a specific-protein epitope and an epitope that reacted with a Mab against the polysaccharide C antigen. M antigen can be purified from HMIN by tandem cation exchange chromatography of the precipitable fraction on an open CM Sepharose CL-6B column followed by linear gradient FPLC.