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Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum.

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.

Animals↗

Cross-reactivity between antigens of Coccidioides immitis, Histoplasma capsulatum and Blastomyces dermatitidis in lymphocyte transformation assays.

The cross-reactivity of four Coccidioides antigens, three Histoplasma antigens, and two Blastomyces antigens were determined in lymphocyte transformation assays of 11 coccidioidin-reactive, histoplasmin-nonreactive subjects (group I), 13 coccidioidin-nonreactive, histoplasmin-reactive persons (group II), and 13 subjects who were skin test negative to both antigens (group III). Mycelial and yeast (or spherule)-phase antigens of the three fungi were included. Significant cross-reactivity was obtained with both coccidioidins, spherulin, and the alkali-soluble, water-soluble cell wall antigen of C. immitis, to the extent that the responses of histoplasmin-reactive persons were not statistically different (P > 0.05) from those of coccidioidin-reactive persons. In contrast, optimal dilutions of Histoplasma mycelial and yeast-phase lysates effectively distinguished (P < 0.01) responses of histoplasmin- and coccidioidin-reactive persons. The alkali-soluble cell wall antigen of H. capsulatum showed extensive cross-reactivity at most concentrations and was markedly stimulatory to lymphocytes of skin test-negative persons. Blastomycin elicited significant cross-reactions in histoplasmin-sensitive subjects and to a lesser extent in coccidioidin-sensitive subjects. The alkali-soluble cell wall antigen cross-reacted in cultures of histoplasmin-reactive persons but not in those of coccidioidin-reactive persons. All antigens effectively distinguished (P < 0.001) homologous responses of skin test-positive persons (groups I and II) from those of skin test-negative persons (group III). The extensive cross-reactivity in lymphocyte transformation assays in the absence of cross-reactivity in skin tests suggests that these two immune responses may be mediated by different T lymphocyte populations, may be elicited by different antigenic components, or both.

Antigens, Fungal↗

Evaluation of an ELISA for the detection of anti-Histoplasma ribosomal and antihistoplasmin antibodies in histoplasmosis.

We have developed an indirect sandwich enzyme-lined immunosorbent assay (ELISA) for the detection of antibodies to Histoplasma ribosomes and histoplasmin; we used this test for demonstration of these antibodies in sera from proven cases of histoplasmosis and other infections. Serum dilutions from five negative controls used in each experiment were normalized against 50 normal sera, and a factor of the mean absorbance was used to establish a positive reaction. Antiribosomal antibodies were detected in 97% of the known histoplasmosis patients with ELISA titers ranging from 1:100 to over 1:12,800. In contrast, antibodies to histoplasmin were detected in only 75% of these sera; titers ranged from 1:100 to 1:12,800. Cross-reactions with sera from other fungal infections (blastomycosis, coccidioidomycosis, paracoccidioidomycosis, cryptococcosis, candidiasis, and aspergillosis) were seen in 46% of the cases with ribosomes and 37% with histoplasmin. Fifty percent of the sera from tuberculosis patients gave positive reactions with ribosomes and 29% with histoplasmin. These results warrant further studies on the significance of antibodies to ribosomes and histoplasmin in immunity to histoplasmosis.

Antibodies, Fungal↗

[Preparation and study of a Paracoccidioides antigen needed for skin tests].

A cellular antigen from Paracoccidioides brasiliensis, obtained from the supernate of the disrupted yeast phase, was studied. It was prepared by a method similar to that for obtaining the spherulin from Coccidioides immitis. This antigen is composed of 55 mug/mg of polysaccharides, determined by the phenol method, and 7-8 mug/mg of protein, determined by Folin-Ciocalteu's technique. In vitro tests of antigenicity were done by immunodiffusion and complement fixation. The optimum dilution of the antigen was 0-5 mg/ml determined by microtiter complement fixation. The in vivo standardization of the cellular paracoccidioidin was performed on three groups of patients. The first was composed of 56 patients with mycologically proved paracoccidioidomycosis. The disease was active in 32 and clinically healed in 24 of this group. The second was composed of 15 cases of histoplasmosis with strong histoplasmin skin tests. The third group were 70 patients with no mycotic pulmonary diseases who had come from different zones in Argentina. Each individual was injected with 0-1 ml cellular paracoccidioidin (3 mg/ml), 0-1 ml of Fava Netto's antigen diluted 1/10, and 0-1 ml histoplasmin L48, diluted 1/100. The cellular paracoccidioidin skin test was positive in 44/56 (78.5%) patients with paracoccidioidomycosis. The Fava Netto's antigen gave 42 (75%) positive results and the histoplasmin skin tests gave 28 (50%) in the same group. Twenty-eight (50%) presented positive histoplasmin skin tests. The paracoccidioidin skin test was positive in 2 of the histoplasmosis group (13-3%) and the Fava Netto's antigen gave 3 positive results in the same group (20%). The histoplasmin skin test was positive in 32% of the individuals with no mycotic disease. Ten per cent of these exhibited positive reaction with both P. brasiliensis antigens. Of those paracoccidioidomycosis cases (Group 1) who had negative skin tests with 3 mg/ml of the cellular paracoccidioidin 7/10 gave positive skin tests when tested at a concentration of 8 mg/ml. Characteristic changes of cell medicated hypersensitivity were observed in the histopathological studies of the cellular paracoccidioidin skin test.

Antigens, Fungal↗

In situ localization of antigens of Histoplasma capsulatum using colloidal gold immune electron microscopy.

Histoplasma capsulatum contains multiple antigens, among them the H antigen and M antigen, which are useful in serologic testing for histoplasmosis. We prepared 7 mouse monoclonal antibodies (5 IgG, 2 IgM) to histoplasmin, and compared these with polyclonal histoplasmin antibodies raised in rabbits and mice. Both monoclonal and polyclonal antibodies were high titered by ELISA. Colloidal gold immune electron microscopy (CGIEM) showed that polyclonal antibodies to histoplasmin or H antigen bound at multiple sites in the cell wall, cytoplasm, and nucleus of Histoplasma yeast cells. In contrast, antibodies to M antigen selectively label the cell membrane and antibodies to alkali soluble cell wall antigen label only the cell wall. Polyclonal antibodies cross reacted extensively with other fungi, both by ELISA and CGIEM. Monoclonal antibodies stained only cytoplasmic epitopes, but also cross reacted with other fungi by electron microscopy. Only periodate treated H antigen elicited polyclonal antibodies which were more specific than those of untreated H antigen or histoplasmin.

Animals↗

Epidemiological study of sporotrichosis and histoplasmosis in captive Latin American wild mammals, São Paulo, Brazil.

Sporotrichosis and histoplasmosis are deep mycosis with a high incidence in human beings in Brazil. In domestic animals histoplasmosis has been described only in dogs, but the occurrence of sporotrichosis among domestic animals in Brazil has been described in dogs, cats, mules and asses. There is also a case of this disease reported in a chimpanzee (Pan troglodites). The purpose of this research was to perform an epidomiological study of these mycoses using delayed hypersensitivity tests (histoplasmin and sporotrichin) in Latin American wild mammals. This research was assayed using 96 healthy animals at Parque Zoológico de São Paulo, Brazil: Primates: 33 Cebus apella--weeping-capuchin and 16 Callithrix jacchus--marmoset; Procyonidae: 37 Nasua nasua--coatimundi and 10 Felidae (Panthera onca--jaguar; Felis pardalis--ocelot Felis wiedii--margay; Felis tigrina--wild cat). For intradermic tests, the following antigens were used: Sporothrix schenkii cell suspension (sporotrichin, histoplasmin-filtrate), Histoplasma capsulatum cell suspension (histoplasmin), and Histoplasma capsulatum (polysaccharide). The positivity to histoplasmin was 44.79% (Cebidae 15.15%; Callithricidae 6.25%; Procyonidae 86.49% and Felidae 50.00%, respectively). With respect to sporotrichin, 30.21% (Cebidae 6.06%, Callithricidae 0.0%; Procyonidae 64.86% and Felidae 30.00% respectively). The pattern of infection is similar to that shown by human beings and this may suggest that these animals could be involved in the epidemiologic chain of sporotrichosis and histoplasmosis, the second most prevalent human deep mycoses in Brazil. It is important to point out the absence of similar studies in Latin American wild animals.

Animals↗

Immunologic studies of patients with histoplasmosis.

Immunologic responses of patients with active histoplasmosis (Group I) were compared to the responses of healthy persons with positive histoplasmin skin-test reactions (Group II), and to those of healthy persons with negative histoplasmin skin-test reactions (Group III). Cellular immune responses were significantly depressed in patients. Seven (54 per cent) of 13 patients failed to respond to histoplasmin skin testing, and two (15 per cent) failed to respond to skin-test antigens unrelated to Histoplasma capsulatum. Lymphocyte transformation (LT) responses of patients were severely depressed in vitro (p less than 0.001) to Histoplasma antigens when compared to the responses of healthy, histoplasmin-reactive donors. Transformation responses to Candida antigen were not impaired in the patient population. Culturing lymphocytes of patients in serum obtained from healthy donors as opposed to autologous serum resulted in a significant (p less than 0.05) increase in LT responses to H. capsulatum. Responses to Candida antigen and to mitogens were not affected by the serum source. Serum-mediated suppression of LT responses correlated (p less than 0.01) with complement-fixing antibody titers to Histoplasma yeast-phase antigen and suggests that antibody, either alone or complexed with antigen, may suppress T-cell response in vitro in this disease.

Adolescent↗

[Investigation on the epidemiology of Histoplasma capsulatum infection in Nanjing district].

Two hundred and ninety-two permanent or temporary residents in Nanjing district were tested with histoplasmin (Histolyn-CYL, ALK/Berkeley Laboratories, USA) and PPD. Forty-nine (16.78%) subjects reacted to histoplasmin with 5.0-23.0 (9.5 +/- 4.2) mm induration. Positive reaction rate among people without pulmonary diseases (normal group) was 15.10% comparing to 17.74% to patients with pulmonary diseases. Positive reaction to PPD with 5-50 (14.2 +/- 4.7) mm induration was 56.16%, with 59.43% in normal group and 54.30% in patients with pulmonary diseases while 8.90% subjects reacted to both histoplasmin and PPD, 7.88% of the research subjects reacted to histoplasmin but not to PPD. Result suggested that there was herd infection of Histoplasma capsulatum in Nanjing district.

Adult↗

Paracoccidioidomycosis: an epidemiologic survey in a pediatric population from the Brazilian Amazon using skin tests.

Since Paracoccidioides brasiliensis and Histoplasma capsulatum are known to be present in similar environments, there have been many epidemiologic investigations regarding the prevalences of these two organisms. However, cross-reactivity can occur in paracoccidioidin and histoplasmin skin tests, and this usually results in the overestimation of the prevalence of P. brasiliensis. The prevalence of infection with P. brasiliensis was evaluated in a cross-sectional study of 298 asymptomatic school children in the Brazilian Amazon region (Mato Grosso State). In this investigation, the reactivity of children to two different P. brasiliensis antigen preparations, paracoccidioidin and a purified 43-kD glycoprotein (gp43), was compared with or without the co-administration of histoplasmin. In the group of individuals receiving paracoccidioidin who had a positive histoplasmin skin test result, the prevalence of exposure to P. brasiliensis was 44% (16 of 36). This reactivity to P. brasiliensis was significantly higher than that observed in other groups, which ranged from 4% to 6% (P < 5 x 10(-4) for each). Overall prevalence was 4.6% (95% confidence interval = 2.5-7.7%). These data suggest that gp43 provides a better estimate of exposure to P. brasiliensis when the co-administration of histoplasmin is desired.

Adolescent↗

Epidemiological investigation of Histoplasma capsulatum infection in China.

OBJECTIVE: To provide reliable information concerning the presence or the absence of Histoplasma capsulatum (H. capsulatum) infection in China, and data concerning this respect. METHODS: Three hundred normal people and 435 hospitalized patients, who lived in Hunan and Jiangsu provinces, and the Xinjiang Autonomous Region, were tested with yeast-phase histoplasmin (ALK/Berkerley Biologicals Laboratories, USA) and human pure protein derivative of tuberculin (PPD) on the volar surface of the forearm. Any reaction to the antigens over 5.0 mm in diameter of induration at 48-72 hours was considered positive. RESULTS: A total of 138 subjects (18.8%) in 735 patients reacted to histoplasmin with 5.0-45.0 (9.1 +/- 4.3) mm indurations. Significant differences of positive skin reaction rates in normal subjects were found in Hunan, Jiangsu and Xinjiang (8.9% vs 15.1% vs 2.1%). The overall positive rate of patients was 25.5%. Patients with tuberculosis [31.7% (78/246)] had a significantly higher positive skin reaction rate in comparison with those suffering from pneumonia [17.7% (11/62)], lung cancer [20.9% (9/43)], chronic obstructive pulmonary disease [17.3% (9/52)] and other diseases [12.5% (4/32)] (P < 0.01). Of 562 cases, 292 cases (52.0%) reacted to PPD with indurations of 5-50 (13.7 +/- 4.9) mm in diameter, 63 cases (11.2%) reacted to both histoplasmin and PPD, while 38 cases (6.9%) reacted to histoplasmin but not to PPD. CONCLUSIONS: The data suggest that there is H. capsulatum herd infection in China. The infection rate in Southeast China is higher than that in the Northwest, and the infection rate of patients with pulmonary tuberculosis is higher than that of normal persons and other pneumonopathy patients.

Adult↗

Immunomodulation by Histoplasma capsulatum products; polyclonal activation and mitogenic effects.

The presence of reactive spleen cells to sheep red blood cells (SRBC) in nonimmunized BALB/c mice injected with histoplasmin, the culture filtrate of Histoplasma capsulatum, was monitored for 21 days following inoculation. Polyclonal activation, as evidenced by a sharp increase in the number of anti-SRBC rosette-forming cells (RFC), as well as an enhanced response to heterologous non-cross-reactive erythrocytes from other species, was found in the spleens of these rodents on Days 11 to 13. Elimination of B-cell-derived RFC by the addition of complement indicated that the erythrocyte-binding cells consisted of both T- and B-lymphocytes. An immunosuppressive effect was detected if histoplasmin was injected 2 days before the antigen (SRBC), but could be reversed by injecting the filtrate 30 min prior to the antigen, as is found with polyclonal activators displaying immunosuppressive activity. Histoplasmin also had a mitogenic effect on lymphocyte obtained from the spleen, bone marrow, and thymus similar in magnitude to that produced by lipopolysaccharide (LPS) and concanaval in A. The biological significance of these findings is discussed.

Animals↗

Immunologic, genetic and social human risk factors associated to histoplasmosis: studies in the State of Guerrero, Mexico.

Immunologic and occupational aspects of the susceptible population exposed to Histoplasma capsulatum, the causative agent of histoplasmosis were analyzed in the Mexican State of Guerrero. Three areas were studied, Juxtlahuaca, Olinala, and Coyuca; in the first two, their populations refer contact with bat guano and/or avian excreta, which contain nutrients for fungal growth, while the Coyuca population referred no contact with the above mentioned excreta. Previous infection with H. capsulatum was determined by histoplasmin-skin test, and the response was higher in men than in women (93.87, 85.71, and 6.6% for men, and 78.94, 66.6, and 0% for women) in Juxtlahuaca, Olinala, and Coyuca, respectively. Labor activities related to a persistent contact with the fungus were considered as an occupational risk factor, histoplasmin-skin test reached 88.57 and 36.36% of positive response in individuals with high and low risk activities. A high percentage of histoplasmin responses was observed in subjects with constant contact with H. capsulatum, such as, cave-tourist guides, peasants, and game-cock handlers, and generally they developed the largest diameter of skin reactions. Genetic risk factor was determined by studying the gene frequency of the Major Histocompatibility Complex antigens in a sample of individuals and their degree relatives in Juxtlahuaca, Olinala, and Coyuca. Significant differences were found for HLA-B22 and B17 antigens in Juxtlahuaca, and for HLA-B22 in Olinala, in comparison to the usual gene frequency observed in the normal Mexican population. HLA results were important, considering that HLA-B22 was previously found to be possibly related to pulmonary histoplasmosis in Guerrero.

Animals↗

Histoplasmosis and Paracoccidioidomycosis in northwestern Argentina III. Epidemiological survey in Vipos, La Toma, and Choromoro - Trancas, Tucumán, Argentina.

The present work was undertaken to obtain epidemiological data on the extent and distribution of Histoplasma capsulatum var. capsulatum and Paracoccidiodes brasiliensis diseases in the Vipos, La Toma and Choromoro areas. Skin test surveys with histoplasmin and paracoccidioidin were carried out in the permanent human population of those localities. Mycological sputum studies and serological tests were performed to skin test reactors to determine if there were signs or symptoms of active mycotic disease. La Toma and Choromoro are highly prevalent areas of histoplasmosis (>30% the histoplasmin positive individuals) whereas Vipos can be relatively considered a highly prevalent area (between 20-30% the histoplasmin reactors) according to the normally accepted range used to define an endemic disease [2]. Early Histoplasma capsulatum infection (<10 years old) is reported for Vipos and Choromoro. La Toma has the highest rate of previous exposure to P. brasiliensis detected in the studied area (10.2%). Vipos residents are not infected with P. brasiliensis.

Adolescent↗

ELISA for early diagnosis of histoplasmosis.

An ELISA was developed and evaluated as a method for detecting antibodies against glycosylated and deglycosylated histoplasmin (HMIN). Sera from patients with histoplasmosis, paracoccidioidomycosis, sporotrichosis, coccidioidomycosis, aspergillosis, cryptococcosis and healthy donors were tested by ELISA against purified, deglycosylated histoplasmin (ptHMIN) and compared with purified, native (i.e. glycosylated) histoplasmin (pHMIN). Although cross-reactivity was not abolished when ptHMIN was used in the test, it was reduced (pHMIN ELISA 93 % versus ptHMIN ELISA 96 %). However, there were statistically significant differences between the sensitivities of these two methods for the detection of antibodies (pHMIN ELISA 57 % versus ptHMIN ELISA 92 %; P < 0.001) and between the efficiency of the methods (pHMIN ELISA 83 % versus ptHMIN ELISA 95 %; P < 0.001). These parameters compare better than previously published data relating to the use of treated HMIN in diagnostic ELISAs. Some of the reactivities of serum samples were compared by immunoblotting using deglycosylated HMIN and by immunodiffusion using the crude antigen. The results demonstrated that cross-reactions with heterologous sera in both ELISAs could also be observed in immunoblotting and arose from shared protein epitopes. These data suggest that ELISA using deglycosylated HMIN is a very sensitive diagnostic method and, by using commercially available antigen, it can be easily standardized and performed faster than previous Western blot-based tests using the same antigen. It provides a useful adjunct to existing methods of diagnosis that could be applied even in situations where laboratory facilities were relatively limited.

Antibodies, Fungal↗

The distribution of coccidioidomycosis in Southern California.

In coccidioidin skin test surveys among persons of high school age in Saugus, Canoga Park, Banning and Palm Springs areas the average incidence of positive reaction was 15 per cent. Although considerably less than the 68 per cent incidence reported among high school students of Kern County, it is high enough to indicate pockets of relatively high endemicity in Southern California below the San Joaquin Valley. Histoplasmin tests were performed on most of the persons tested with coccidioidin in this survey. The over-all incidence of positive reaction in the group was 7.6 per cent. Most of the subjects with positive reaction to histoplasmin gave a history of having previously lived in some area in the central United States where histoplasmosis is known to be endemic.A few subjects who had positive reaction to coccidioidin tests and who had lived in areas known to be endemic for coccidioidomycosis but not for histoplasmosis, also had positive reaction to histoplasmin. However, the induration produced was always smaller than that caused by the coccidioidin reaction, and there was minimal confusion in interpreting the tests.

California↗

[Study of yeast phase Histoplasma capsulatum antigens used for skin tests].

The results of skin tests with two antigens of the yeast phase of Histoplasma capsulatum are presented. Both antigens were able to fix complement and form precipitating bands in the presence of sera from patients with active histoplasmosis. Their sensitivity was lower than the metabolic antigen usually employed in serology. Results of skin tests obtained with both yeast phase antigens and a standard histoplasmin coincided in animals infected with H. capsulatum. Only one cross reaction was observed in animals inoculated with Paracoccidioides brasiliensis. Skin tests in humans were conducted on patients with and without mycotic disease. The percentages of positive reactions in patients with histoplasmosis were not significantly different between control histoplasmin and whole cell extracts. In the patients with non mycotic diseases the frequency of positive tests varied between 28.8% and 32%, which agrees with previous statistical data for the general population of the area. Equally the positive percentage of 55% and 68% in patients with paracoccidioidomycosis coincided with the results of previous studies. The histological patterns of these skin tests showed that they were produced by cell mediated hypersensitivity. The sensitivity of a whole yeast cell extract was similar to histoplasmin L48, but its preparation was quicker and easier to perform and it had no foreign substances from the culture medium, so we think that it would be easier to standardize it chemically.

Animals↗

Decreased suppressor cell activity in disseminated granulomatous infections.

The effect of granulomatous infections upon the activity of a T lymphocyte subclass in human peripheral blood that can be induced by concanavalin A (Con A) to function in a suppressor mode was studied. Peripheral blood lymphocytes (PBL) from eleven patients with disseminated mycotic or mycobacterial infections or from controls were preincubated with and without Con A, washed and cultured with allogeneic PBL freshly drawn from healthy donors sensitive to histoplasmin. DNA synthesis was then measured in co-cultures stimulated by Con A, histoplasmin, or by the mixed lymphocyte culture (MLC) reaction alone. As compared with cells preincubated without Con A, the Con A-pretreated cells were significantly less effective in suppressing the responses of normal PBL to histoplasmin (P < 0.01), and in a one-way MLC reaction (P < 0.05). The Con A-induced suppressor activity of PBL from nine patients with localized granulomatous infections did not differ significantly from that exerted by PBL of normal controls in two of the three co-culture systems employed. These studies suggest that either dysfunction or a reduction of the Con A-inducible T-suppressor cell subpopulation in peripheral blood is frequent among patients was disseminated granulomatous infections.

Adolescent↗

Immunology of histoplasmosis: humoral and cellular activity from a polysaccharide-protein complex and its deproteinized fraction in experimentally immunized mice.

In a previous publication it was reported that a polysaccharide-protein complex (PPC), sensitive to beta-glucosidase, was isolated from Histoplasma capsulatum. This complex was strongly reactive in an agar gel diffusion assay with sera from patients with histoplasmosis, but was unreactive with sera from patients with coccidioidomycosis. Here, the studies with human sera have been expanded and attempts were made to determine the response of mice immunized with nonviable H. capsulatum or Coccidioides immitis to PPC or its deproteinized fraction (D-PPC) using more sensitive tests for antibody and including also test for cell-mediated immunity. Histoplasmin and coccidioidin were compared with PPC or its deproteinized fraction (D-PPC) in all assays. In a counterimmunoelectrophoresis (CIE) assay, PPC and D-PPC reacted only with sera from patients with histoplasmosis, whereas cross reactions were noted with histoplasmin and coccidioidin using heterologous sera. Cross-reaction were observed with all four antigen preparatins and both types of antisera using a microcomplement fixation assay. The assay for macrophage migration inhibitory factor (MIF) was also relatively nonspecific, in that inhibition occurred with cells from animals sensitized with Histoplasma or Coccidioides using both homologous and heterologous antigens. In the footpad assay, histoplasmin and coccidioidin were highly cross-reactive in animals sensitized with the heterologous fungus, but the PPC and D-PPC from H. capsulatum elicited significant reactions only in animals sensitized with Histoplasma.

Animals↗