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Although antigen-reactive T lymphocytes play a central role in the host response to Histoplasma capsulatum, little is known of the nature of Histoplasma antigens recognized by these cells in vitro. Employing a murine T-cell line and two clones that are reactive with histoplasmin, we examined whether activation of T cells by histoplasmin required the presence of carbohydrate or protein moieties. The approach taken was to modify carbohydrate or protein molecules in histoplasmin by chemical or enzymatic digestion or by lectin adsorption. In parallel, antigen was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to correlate alterations in functional activity with changes in the electrophoretic appearance of histoplasmin. Treatment of histoplasmin with periodate (0.1 M, 0.05 M, and 0.01 M) or with the endoglycosidases N-glycanase and endoglycosidase H sharply diminished the capacity of histoplasmin to trigger responses by T cells. Reactivity of T cells to histoplasmin that had been adsorbed with lectins binding mannose, glucose, or galactose was reduced by greater than 70%; conversely, the responses by T cells to antigen that had been adsorbed with lectins specific for fucose, N-acetylgalactosamine, or N-acetylglucosamine ranged from 82 to 91% of that to control antigen. Proliferative responses by T cells to histoplasmin that had been digested with chymotrypsin, protease, or trypsin were 2 to 43% of control values. The electrophoretic appearance of histoplasmin was modified by some but not all of the treatments. Partially purified H and M antigens triggered proliferation of T cells. Thus, both carbohydrates and proteins must be present to induce optimal responses by T cells. A portion of the carbohydrates is N linked to proteins, and alpha-D-mannose (or alpha-D-glucose) and beta-D-galactose are the sugar ligands of carbohydrate-containing antigens.
We examined the pathways involved in presentation of native histoplasmin by adherent splenocytes (as a source of accessory cells) to JC1, a Histoplasma capsulatum-reactive murine T-cell line that is CD4+. JC1 did not respond to accessory cells that had been fixed with paraformaldehyde and then exposed to histoplasmin but did proliferate to antigen-pulsed cells that were subsequently fixed. Accessory cells that were coincubated with histoplasmin and sodium azide or 2-deoxy-D-glucose failed to induce proliferation of JC1. Moreover, accessory cells exposed to the lysosomotropic agents, chloroquine and ammonium chloride, were unable to present antigen. Monensin also inhibited presentation of histoplasmin if added to accessory cells concomitant with antigen. In contrast, accessory cells that had been pulsed with antigen for 2 hr and then exposed to each inhibitor for 2 hr stimulated proliferation of JC1. The antigen-presenting capacity of accessory cells that had been pulsed with histoplasmin for 2 hr was diminished considerably by subsequent treatment with phospholipase A2. Additional studies demonstrated that cerulenin, which depresses posttranslational lipid modification of proteins, abolished presentation of histoplasmin. The reactivity of JC1 was sharply reduced by anti-L3T4 (CD4) or by anti-I-Ab monoclonal antibody. The results not only indicate that presentation of histoplasmin requires active metabolic events within accessory cells, they also delineate the pathways involved in handling this antigen.
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.
Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.
The frequency of infection with the fungus Histoplasma, as judged by the results of skin tests with histoplasmin, varies widely from county to county across the USA and even over very short distances within some counties. Differences between various segments of the population, such as between Negroes and whites, living in one particular community are therefore difficult to interpret, as they might represent the effect of differences in ecological rather than in demographic factors within the community. Since the pertinent ecological differences could, however, be expected to balance out if a large number of communities were sampled, results of histoplasmin skin tests among Negro and white Navy recruits from 37 different communities are compared in the present report. The prevalence of reactors to histoplasmin ranges from zero to over 90% in residents of the different geographical areas, indicating wide variations in the ecological conditions affecting the growth and dissemination of the fungus, yet comparisons of results in Negroes and whites indicate that the factors influencing infection and sensitization of man appear to bear equally on both racial groups. Results with tuberculin PPD-S and the Battey PPD antigen in the same groups of recruits are included to supplement the results with histoplasmin.
A yeast phase histoplasmin, designated CYL, was studied in 156 humans living in areas of the United States endemic for histoplasmosis, coccidioidomycosis, or both fungal entities. Comparisons were made with Food and Drug Administration reference mycelial histoplasmin. Among persons without concurrent spherulin sensitivity, reactor rates and sizes were the same with both reagents. Some persons with spherulin sensitivity reacted only to the mycelial reagent. Histoplasmin-CYL skin tests did not induce significant changes in complement-fixing antibody in histoplasmin-reactive subjects.
Paracoccidioidin and histoplasmin intradermic tests were performed in 107 inhabitants in Arapiraca, Alagoas. The epidemiological survey was performed in both sexes and the ages varied from 2 to 71 years. Positive results with paracoccidioidin test were recorded in 11% and the histoplasmin test was positive in 14%. With the positivity of paracoccidioidin in the histoplasmin positive population the result shows a percentage of 33.33%, while with the positivity of histoplasmin in the paracoccidioidin positive population was 41%.
A skin test survey with paracoccidioidin and histoplasmin was conducted on 495 residents of Coari in the Brazilian state of Amazonas. Positive dermal reactions to paracoccidioidin and histoplasmin were seen, respectively, in 13.9% and 50.1% of the study subjects. Specific reaction to paracoccidioidin was observed in 1.6% of the individuals, and to histoplasmin in 37.8%. Cross-reactivity between the two antigens in persons who reacted positively to both skin tests was not totally reciprocal. There were no significant differences in dermal reactivity to either of the two antigens in either sex, or among different occupational groups. No precipitating antibody to paracoccidioidin or histoplasmin was detected in the sera of the skin test-positive individuals by the immunodiffusion test. Skin test positivity in the general population of Coari reaffirmed the concept of benign, self-limited infection in paracoccidioidomycosis and histoplasmosis. It also identified the equatorial forests of the Upper Amazon Basin as endemic zones of these two mycoses.
Previous experiments from this laboratory have shown that small numbers of lymphocytes could be stimulated by phytohemagglutinin to transform. Experiments in 15 subjects sensitized to histoplasmin demonstrated that antigenic stimulation with histoplasmin could be achieved with as few as 12.5 X 10(3) lymphocytes per 0.1 ml culture, (2,521 +/- 1.260) compared to unstimulated cultures (250 +/- 41), p less than 0.01, although false negative values (less than three fold increase in radionucleotide incorporation over unstimulated cultures) were obtained in 6 of 15 cases. Fourteen of 15 positive responders were correctly identified by culturing 25 X 10 (3) cells with histoplasmin for six days. The cell numbers required to give a blastogenic response with histoplasmin can be reduced to 25 X 10(3) cells in situations where cell availability is limited.
The epidemiology of histoplasmosis duboisii (African histoplasmosis) is not well understood. The present study was carried out to investigate the prevalence of skin sensitivity and to determine by immunodiffusion the presence of antibodies among humans to histoplasmin around a recently discovered natural focus of Histoplasma capsulatum var. duboisii in a bat cave in Ogbunike in the Anambra State of Nigeria. Out of the 40 subjects, all young adults aged 18-30 years, comprising cave guides, traders and farmers examined in the immediate vicinity of the cave, 14 (35.0%) gave a positive skin test. In another population of the same age group, comprising 620 persons, viz. traders, farmers, palm oil workers and some patients attending rural clinics, examined in other nearby areas in Anambra State, 55 (8.8%) reacted positively to histoplasmin. In the immunodiffusion tests, 2 (2.08%) of the 96 school children and 17 (9.4%) of the 181 young adults, including farmers, palm oil workers and traders tested amongst the population around the cave, demonstrated precipitating antibodies to histoplasmin in their sera. Only 5 (0.79%) of the 630 adults of the same age group with similar occupations examined from other areas in Anambra State had precipitating antibodies. Out of another 50 subjects examined, viz.; wood workers, traders, farmers, and school teachers in Nsukka in the Enugu State, two (4.0%) demonstrated antibodies. It is suggested that asymptomatic infections due to the duboisii variety of H. capsulatum may be common in the human population around the cave. A diligent search with the help of local hospitals and public health officials may reveal clinical cases of histoplasmosis duboisii with cutaneous and systemic lesions.
A cross-sectional epidemiological survey for paracoccidioidomycosis and histoplasmosis, including skin tests with paracoccidioidin and histoplasmin, physical examinations and X-rays, was conducted among three Tupí-Mondé Amerindian populations from Brazilian Amazonia. The study followed the diagnosis of an increasing number of cases of paracoccidioidomycosis among the Suruí in recent years. Positivity rates to paracoccidioidin and histoplasmin (> or = 5 mm of intradermal induration 24-48 h post-injection) were 43.8% and 78.7% for the Suruí, 6.4% and 5.8% for the Gavião and 14.9% and 80.5% for the Zoró, respectively. There was no significant difference in the results for males and females but marked differences were noted across age groups. The results of the univariate analysis were confirmed after adjustment for confounding variables by multiple logistic regression analysis: paracoccidioidin positivity was relatively high in the Suruí and histoplasmin positivity was relatively high in the Suruí and Zoró. The Suruí's greater exposure to Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis, is probably associated with their adoption of new subsistence practices. The epidemiology of this mycosis among the Tupí-Mondé appears to be related to the environmental and socio-economic changes taking place in Amazonia.
Until recently coccidioidin has been the only antigenic preparation available for detecting delayed dermal sensitivity induced by an experience with Coccidioides immitis. It is prepared from autolysates of the mycelial phase (saprophytic) of the fungus. A more sensitive reagent, spherulin, was developed in 1969 from the spherule phase (parasitic) of the organism. Use of spherulin showed that coccidioidin failed to detect approximately 30% of individuals specifically sensitive to C. immitis. However the potential of spherulin to detect cross-sensitivity induced by Histoplasma capsulatum was unknown. This information was considered to be germane because of the capacity of coccidioidin to detect a histoplasmal experience. Accordingly, both reagents as well as paracoccidioidin were compared simultaneously in 365 Columbian soldiers from areas endemic for histoplasmosis but not for coccidioidomycosis. At standard strength both preparations detected nonspecific responses in 0.8% to 3% of the histoplasmin negative and positive subgroups, respectively. At 10-times standard strength both preparations cross-detected histoplasmin sensitivity comparably; 5.1% to 7.1% of histoplasmin-positive subjects reacted with the coccidioidal antigens. No pattern of cross-reactivity was observed between paracoccidioidin sensitivity and sensitivity to the coccidioidal antigens.
Controlled yeast lysate (CYL) and controlled mycelial lysate (CML) histoplasmins were produced from Histoplasma capsulatum grown in a nutritionally lean, chemically defined medium. The lysates were assayed for skin-test activity in guinea pigs sensitized by infection with the homologous organism. In some studies, nonliving vaccine preparations were employed also. Inter-lot biological variation was minimal, and 20 lots of the CYL reagent elicited strong dermal reactions with high specificity. Further, CYL reagents were nonreactive in guinea pigs infected with Coccidioides immitis, whereas the commercial Food and Drug Administration preparations cross-reacted to some degree. The CML histoplasmins were generally less reactive than the CYL preparations and exhibited somewhat more inter-lot variation in sensitivity and specificity. No correlation between potency and protein:polysaccharide ratios were observed with either reagent. An intradermal test with the CYL reagent did not induce significant changes in the complement-fixing titer of sensitized guinea pigs. Such changes in sensitized animals were elicited by a skin test with commercial histoplasmin.
Numerous reports have indicated that a single histoplasmin skin test may stimulate humoral antibodies to Histoplasma capsulatum antigens in histoplasmin-hypersensitive individuals. Although these investigations concur that antibody elevations are evoked, they vary in the reported degree of incidence and response induced, and they cast doubt on the interpretation of serological tests in the diagnosis of histoplasmosis. Histoplasmin-hypersensitive subjects (114) were bled prior to administration of the skin test, 2 days later, at the time this test was read, and 15 and 30 days after testing. No significant antibody titers were observed at 2 days. At 15- and 30-day intervals, only 17 (15%) of the subjects demonstrated circulating antibodies. All 17 showed agar gel bands; 5 demonstrated no complement-fixation (CF) titers, 10 produced CF antibodies ranging from 1:8 to 1:16, and 2 demonstrated titers of 1:32. The data suggest that skin testing does not interfere significantly with antibody levels in sera drawn approximately 2 days after administration of antigen. However, since titers as high as 1:32 were obtained at later intervals, such reactions should be evaluated cautiously and only after consideration of clinical findings.
The present paper analyzes the histoplasmin electrophoretic profiles and the randomly amplified polymorphic DNA (RAPD) patterns of the fungus Histoplasma capsulatum isolated from Mexican patients with AIDS-associated histoplasmosis. Clinical isolates from Guatemala, Colombia, and Panama, as well as H. capsulatum isolates from different sources in nature, were also processed. All histoplasmin samples shared four antigenic fractions of 200, 49, 10.5, and 8.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to their percentage of relatedness, based on SDS-PAGE histoplasmin electrophoretic image analysis, H. capsulatum isolates were divided in two groups: group A contained all AIDS-associated isolates studied and two human reference strains from Mexican histoplasmosis patients without AIDS; group B included bat guano, infected bat, and cock excreta isolates from the State of Guerrero, Mexico, plus three human histoplasmosis strains from Guatemala, Panama, and Colombia. Polymorphic DNA patterns evaluated by RAPD-PCR showed three major bands of 4.4, 3.2, and 2.3 kb in most H. capsulatum isolates studied. Four groups were related by DNA polymorphisms: group I was formed by most of the AIDS-associated H. capsulatum isolates studied, one human histoplasmosis strain from Colombia, two human reference strains from Mexican patients without AIDS, and one human histoplasmosis strain from Guatemala. Group II consisted of only a single strain from Panama. Group III included three strains: one from a Mexican patient with AIDS and two isolated from nature in Guerrero (cock excreta and bat guano). The last, group IV, consisted of only one strain isolated from an infected bat, captured in Guerrero. A tight relationship between phenotypic and genotypic characterization was observed, and both analyses could be useful tools for typing H. capsulatum from different sources and geographic origins.