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A cottony, light tan, filamentous fungus with pear-shaped microconidia and lacking tuberculated macroconidia was isolated from a bronchial lavage specimen. Subculture on several media at 37 degrees C failed to convert the fungus to a yeast form after several weeks; attempts at in vivo conversion in mice were also unsuccessful. Sera obtained several months apart showed M bands with Histoplasma capsulatum (HC) antigen by immunodiffusion and an increase in complement fixation titers with mycelial and yeast phase antigens of HC. Parallel identity was obtained on two occasions with exoantigen culture confirmation reagents for HC from Immuno-Mycologics as well as one of identity with Nolan reagents. Extracts from four Chrysosporium spp. strains had no identity reactions with HC with either kit. The fungus was identified as HC by the Accuprobe Histoplasma chemiluminescence-labeled DNA probe directed at ribosomal RNA, whereas all four Chrysosporium spp. isolates tested negative. DNA probes are a fast and accurate method to confirm the identity of aberrant fungal isolates.

Human neutrophils (PMN) demonstrated potent fungistatic activity against Histoplasma capsulatum (Hc) yeasts in a sensitive microassay that quantifies the growth of yeasts by the incorporation of [3H]leucine. At a PMN:yeast ratio of 1:2, PMN inhibited the growth of yeasts by 37%. Maximum inhibition of 85% to 95% was achieved at a PMN/yeast ratio of 10:1 to 50:1. Opsonization of the yeasts in fresh or heat-inactivated serum was required for PMN-mediated fungistasis, but ingestion of the yeasts was not required. Recognition and phagocytosis of opsonized yeasts was via PMN complement receptor (CR) type 1 (CR1), CR3, and FcRIII (CD16). PMN fungistatic activity was evident by 2 h, was maximum at 24 h, and persisted up to 5 d. In contrast, yeasts multiplied within monocytes to a greater extent than in culture medium alone. PMN from three patients with chronic granulomatous disease (CGD) inhibited the growth of Hc yeasts by an average of 97%, compared with 86% in three normal controls. Furthermore, preincubation of PMN with the lysosomotropic agent NH4Cl inhibited fungistatic activity in a concentration-dependent manner. Finally, experiments with subcellular fractions of PMN demonstrated that the principal component of the fungistatic activity of PMN was localized in the azurophil granules. These data demonstrate that human PMN possess potent fungistatic activity against Hc yeasts and further show that fungistasis is mediated by antimicrobial agents contained in the azurophil granules.

Recombinant cytokines and colony-stimulating factors (CSFs) were tested for their abilities to activate human monocytes/macrophages (M phi) to inhibit the intracellular growth of or kill Histoplasma capsulatum yeasts. None of the cytokines or CSFs or combinations of cytokines and CSFs activated M phi fungistatic activity when they were added to M phi monolayers concurrently with yeasts. In contrast, culture of monocytes for 7 days in the presence of interleukin 3, granulocyte-M phi CSF, or M phi CSF stimulated M phi fungistatic (but not fungicidal) activity against H. capsulatum yeasts in a concentration-dependent manner. Optimal activation of M phi by CSFs required 5 days of coculture, and the cultures had to be initiated with freshly isolated peripheral blood monocytes. Culture of monocytes with combinations of CSFs or addition of CSFs during the 24 h of coculture with the yeasts did not further enhance M phi fungistatic activity for H. capsulatum. Addition of gamma interferon or tumor necrosis factor alpha to CSF-activated M phi also did not enhance M phi fungistatic activity. These results suggest that interleukin 3, granulocyte-M phi CSF, and M phi CSF may play a role in the cell-mediated immune response to H. capsulatum by enhancing monocyte/M phi fungistatic activity.

Changes in temperature and a variety of other stimuli coordinately induce transcription of a specific set of heat-shock genes in all organisms. In the human fungal pathogen Histoplasma capsulatum, a temperature shift from 25 to 37 degrees C acts not only as a signal that causes transcription of heat-shock genes, but also triggers a morphological mycelium- to yeast-phase transition. The temperature-induced morphological transition may be viewed as a heat-shock response followed by cellular adaptation to a higher temperature. We have found that by inducing thermotolerance, i.e., an initial incubation at 34 degrees C, the thermosensitive attenuated Downs strain of H. capsulatum can be made to resemble those of the more temperature-tolerant G222B strain with respect to mitochondrial ATPase activity and electron transport efficiency at elevated temperatures. Furthermore, if the heat-shock response is first elicited by preincubation at milder temperatures or stress, transcription of heat-shock mRNA in mycelial cells of Downs strain that shifted to 37 degrees C proceeds at rates comparable to those of the virulent strains.

Recombinant murine gamma interferon (rMuIFN-gamma) stimulates mouse peritoneal macrophages to inhibit the intracellular growth of the zoopathogenic fungus Histoplasma capsulatum. In some systems, the inhibition of growth of an intracellular parasite by rIFN-gamma has been related to nutritional constraints induced in the host cells by the lymphokine. Such an explanation might apply to H. capsulatum because the fungus is a functional methionine-cysteine (Met-Cys) auxotroph at 37 degrees C; its sulfite reductase is repressed at that temperature. For this reason, we set about to examine whether or not the antihistoplasma state induced in rMuIFN-gamma is due to a restriction in the availability of Met-Cys. Omission of Met-Cys from the medium in which macrophages were cultivated prevented H. capsulatum from growing within them. Addition of Met or Cys to the macrophage cultures did not antagonize the inhibitory effect induced in the cells by rMuIFN-gamma. Thus, there was no evidence from our work that rMuIFN-gamma evokes the antihistoplasma effect in mouse peritoneal macrophages by limiting the supply of Met-Cys to the fungus.

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that, under special conditions, converts from its more common mycelial form to a yeast-like form. Achieving this conversion, however, has been problematical for researchers. The present study tested conversion rates in ten Histoplasma capsulatum var. capsulatum strains using seven culture media, four of which were conventional and three novel. One of our novel media, MLGema, induced complete conversion of two strains within five days of incubation at 35 degrees C, and of all strains that eventually converted by the time of the second subculturing transfer, under defined experimental conditions. MLGema is also inexpensive and easy to produce.

We report the recovery of Histoplasma capsulatum from blood specimens cultured for Mycobacterium sp. in BACTEC 13A radiometric medium. H. capsulatum was recovered from six of eight blood specimens submitted for mycobacterial cultures from five human immunodeficiency virus-positive individuals. Initial positive metabolic signals occurred at a mean of 11 days, but no organisms were detected with acid-fast stains. The bottles remained positive, and after an additional incubation (mean, 8 days), yeast cells morphologically compatible with H. capsulatum were detected when aliquots were stained with acridine orange. Therefore, when radiometric mycobacterial blood cultures with persistent positive metabolic signals and negative acid-fast stains are encountered, acridine orange staining and subculturing for a variety of microorganisms, including fungi, e.g., H. capsulatum, should be considered.

The Yeast phase of Histoplasma capsulatum has stringent growth requirements. Transition from mycelium to yeast takes place only in the presence of cysteine and can be blocked by the -SH groups inhibitor p-chloromercury-phenylsulfonic acid (PCMS). Ultrastructural studies show lysis and degeneration of PCMS treated mycelium grown at 37 degrees C for 24 hours. Only 50% of PCMS treated mycelium appear degenerate when grown at 34 degrees C for 24 hours. The remaining cells have normal morphology with only slight changes in the cell wall structure. The effect of PCMS is permanent and hereditary. Mice injected with PCMS treated mycelium do not develop disease and are resistant to virulent strains of H. capsulatum when challenged.

We evaluated P388D1 macrophagelike cells as model host cells for studying the intracellular survival and strain-specific virulence of Histoplasma capsulatum. Previously characterized strains which were virulent for mice destroyed monolayers of these cells within a few days. In contrast, related avirulent "smooth" variants failed to do so even after 20 days, although they persisted within P388D1 cells for at least 7 days. On the basis of this observation, we developed a quantitative radiolabel assay to use as an initial screen for virulence. Another cell type lining the respiratory tract was then examined as a potential host for H. capsulatum. Hamster trachea epithelial (HTE) cells readily internalized a variety of strains lacking alpha-(1,3)-glucan in their cell walls; however, the tracheal cells were only rarely infected by organisms possessing this polysaccharide. We subsequently inoculated HTE cells with alpha-(1,3)-glucan-positive strains and enriched for the few yeasts infecting these cells. The progeny resembled smooth variants in terms of colony morphology, the absence of alpha-(1,3)-glucan in their cell walls, and their inability to kill macrophages. Did the HTE cells select for these variant yeasts from the parent inoculum or instigate a change from the parental phenotype? Following a 3-h uptake period, only 2% of the ingested yeasts lacked alpha-(1,3)-glucan. One day later, nearly half of the intracellular organisms lacked this polymer. This rapid conversion of a large proportion of the inoculum suggests some type of environmentally triggered change, perhaps analogous to phase variation seen in many other pathogens. Infection of epithelial cells or some other nonprofessional phagocyte during natural histoplasmosis might give rise to similar variants, thus establishing a reservoir of organisms capable of causing chronic or latent infections.

Human monocytes/macrophages (M psi) were infected with Histoplasma capsulatum yeast cells, and intracellular growth was quantified after 24 h of incubation in medium alone or in medium containing cytokines. Yeast cells multiplied within freshly isolated monocytes, cultured M psi, and alveolar M psi with intracellular generation times of 14.2 +/- 1.4, 18.5 +/- 2.1, and 19.9 +/- 1.9 h (mean +/- standard error of the mean), respectively. Monocytes and M psi inhibited the intracellular growth of yeast cells in response to cytokine supernatant; maximum inhibition was obtained when cytokines were added to cell monolayers immediately after infection. Opsonization of yeast cells in normal serum or in H. capsulatum-immune serum did not affect the intracellular generation time of yeast cells in either control M psi or cytokine-activated M psi.

Murine polymorphonuclear neutrophils (PMN) were examined for fungicidal and fungistatic activity against yeast cells of Histoplasma capsulatum. In a 16-h limiting dilution assay (LDA) (constant number of PMN versus decreasing numbers of yeast cells) where PMN to yeast cell ratios were high (0.4-1.5 x 10(5):1), murine PMN exhibited a limited fungicidal effect on H. capsulatum as measured by sterilization of cultures containing one to four yeast cells. On the other hand, inoculum colony forming units (c.f.u.) of H. capsulatum were not reduced by PMN in short-term (2 h) assays (PMN to yeast cell ratio, 500:1) even though H. capsulatum stimulated a brisk oxidative burst in PMN. In the same assays 84% of Candida albicans cells were killed. These findings indicate that H. capsulatum was resistant to killing by products of the oxidative burst generated by PMN and that it was slightly sensitive to PMN in 16-h LDA assays where a different microbicidal mechanism may be operative. In long-term co-culture assays, where the PMN to yeast cell ratio was 500:1, PMN consistently inhibited the replication of H. capsulatum after 24, 48, or 72 h of incubation. Inoculum c.f.u. failed to increase significantly in co-cultures, whereas in culture medium alone c.f.u. increased several-fold in 72 h. Based on these novel findings, we speculate that PMN might play a role in resistance to H. capsulatum due to their strong fungistatic activity and to a lesser extent their limited fungicidal action.

The ultrastructural changes which occur during the mycelium to yeast transition in Histoplasma capsulatum induced by a temperature shift from 25 degrees C to 34 degrees C are described and compared to those observed after a temperature shift from 25 degrees C to 37 degrees C. 24 hours after the temperature shift to 34 degrees C only 8% of the cells are lysed. However, many mitochondria have lost their characteristic elongated form and have become rounded. Vesicular cristae which are no longer oriented parallel to the long axis of the mitochondria are also observed. In contrast a temperature shift from 25 degrees C to 37 degrees C induces lysis of 70% of the cells; mitochondria are rarely observed in the remaining cells. These ultrastructural changes can be correlated with the uncoupling of oxidative phosphorylation and the production of heat shock proteins.

The history of 29-year-old male from Surinam with antibodies to HIV-1 and long-lasting fever, lymphadenopathy, pain in the right upper abdomen and a granulomatous hepatitis is described. The patient suffered from disseminated histoplasmosis, a fungal disease rare in The Netherlands, which is the indicator disease for the diagnosis of AIDS (CDC-IVCI). It is stressed that in seropositive patients coming from endemic areas, including Surinam, the possibility of this disease should be considered.

Histoplasma capsulatum isolates from three St. Louis area AIDS patients with disseminated histoplasmosis were found to be closely related to the temperature-sensitive, previously unique, Downs strain based on growth phenotype and restriction fragment length polymorphisms (RFLP) involving mitochondrial DNA, ribosomal DNA, and the yps-3 gene. H. capsulatum isolates from five non-AIDS patients in the St. Louis area with disseminated histoplasmosis or chronic pulmonary histoplasmosis had the growth phenotype and RFLP pattern characteristic of most strains isolated from other regions of the USA.

The ultrastructural morphology of the early phases of mycelium-yeast transition in Histoplasma capsulatum after a temperature shift from 25 degrees C to only 34 degrees C is described. Under this condition of lower temperature oxidative phosphorylation is not completely uncoupled and maximum production of heat shock proteins (hsp) occurs. 24 h after temperature shift more than 90% of the cells still appear vital. Alterations in the organization of the mitochondrial cristae are the only ultrastructural changes observed in these cells. In contrast, 70% of the cells degenerate 24 h after a temperature shift from 25 degrees C to 37 degrees C and in the remaining cells mitochondria are rarely observed. These observations are discussed in relation to the production of hsp, the uncoupling of oxidative phosphorylation and the virulence of different strains of H. capsulatum.

The exoantigen test has been successfully used in the last years for the immunological identification of pathogenic fungi. Twenty seven straing presumptively identified as Histoplasma capsulatum by morphologic methods, were studied by such test. The exoantigens obtained from each strain were studied by immunoprecipitation technique in front of histoplasmosis control sera experimentally obtained in rabbits. Reference strains of Histoplasma capsulatum. Histoplasma duboisii and Paracoccidioides brasiliensis were used. Of the strains studied, 96% was identified as H. capsulatum by the exoantigen test, a simple, rapid and highly specific technique greatly useful in the laboratories of Microbiology.

Human alveolar, peritoneal, and cultured macrophages were exposed in vitro to human recombinant interferon-gamma (rHuIFN-gamma) and were tested for their ability to inhibit intracellular replication of yeast-phase Histoplasma capsulatum. Exposure at various concentrations, and for different time periods, failed to activate the macrophages to inhibit multiplication of intracellular yeast. Macrophages were, however, activated by rHuIFN-gamma as shown by their ability to inhibit intracellular replication of Trypanosoma cruzi and by their enhanced production of superoxide when stimulated by phorbol myristate acetate. These data indicate that rHuIFN-gamma by itself does not activate human macrophages to inhibit intracellular proliferation of yeast-phase H. capsulatum.

Supernatants from concanavalin A-stimulated T-cell hybridomas activated macrophages to suppress the intracellular growth of Histoplasma capsulatum and to kill tumor cells. The supernatants had high interferon activity and were pH 2 sensitive and heat resistant. Gamma interferon is suggested to be among the active substances in the supernatants. Neither alpha interferon nor beta interferon activated macrophages for the Histoplasma growth inhibitory activity.

Fine details of the sequential morphological events occurring during transition of microconidia (spores less than 5 micrometer in diameter) to the yeastlike phase of Histoplasma capsulatum as seen in ultrathin section are described and illustrated by electron micrographs. Masses of microconidia were obtained when the fungas was grown on a garden soil extract medium. Spores were incubated under in vitro environmental conditions conducive for phase transition (an enriched medium at 37 degrees C). Within 48 h of incubation, the microconidia either germinated to give rise to a short mycelium or the germ tube process became a yeast mother cell without further extension. The wall of the yeast mother cell was thin and smooth, and its cytoplasmic content was ultrastructurally complex, consisting of numerous lipid bodies, vacuoles, glycogen-like deposits, and membrane systems. Within 96 h, the mother cell underwent multipolar budding to form simultaneously linear hyphal and/or ovate yeastlike daughter cells. During the transition, new cell wall materials of the germ tube, the mother cell, and yeastlike daughter cells arose by blastic action from the innermost layer(s) of the wall of the precursor form. Lomasome-like vesicles were often seen in association with areas of new cell wall formation. After organellar migration into and septation of the daughter cells, the yeast mother cell's cytoplasmic content underwent marked degenerative changes.

Factors modulating host resistance to Histoplasma capsulatum are only partially understood. The role of iron-binding proteins in infectious diseases has been an area of recent in-depth investigation. The present study reaffirmed the necessity of iron for growth of H. capsulatum. Transferrin saturation was found to be of importance in withholding iron, and antigen-specific antibody had no added effect. Serums of patients with various clinical classes of histoplasmosis were found to exhibit abnormalities in iron metabolic parameters. However, based on transferrin saturation data, iron withholding by transferrin does not appear to be a significant host defense mechanism in vivo. Further studies presented herein suggest a protective effect of phagocytosis and sequestration by the macrophage-phagocyte system.

Isolation of Histoplasma farciminosum from five horses, showing typical signs of histoplasmosis farciminosi (epizootic lymphangitis) was successfully attempted. The mycelial form of H. farciminosum was isolated on Sabouraud dextrose agar enriched with 2.5% glycerol, brain heart infusion (BHI) agar enriched with 10% horse blood and PPLO dextrose glycerol agar. The last medium proved to be the most effective, both for primary isolation and subculturing of the fungus. It was found that on primary isolation, the lag phase of the mycelial form of the fungus was relatively long, involving 4-8 weeks at 25 degrees C. Colonies of the mycelial form of H. farciminosum appeared on subculture as a yellowish, light brown to deep brown, convoluted, waxy, cauliflower-like growth tending to form scant aerial growth. Conversion of the mycelial form to the yeast form of H. farciminosum was successful by subculturing either on BHI agar with 5% blood or on Pine's medium and incubating at 35-37 degrees C. Complete conversion to the yeast form was achieved only after 4-5 repeated serial transfers onto fresh media every 8 days. The yeast colonies were flat, raised, slightly or deeply wrinkled, white to light gray to grayish brown, and were pasty in consistency.

The highly variable mycelial phase cultures of Histoplasma capsulatum are generally described as brown to albino colonies with the albino type overtaking the brown in a unidirectional and mainly irreversible process. In this study it was found that cultures could be reversed from albino to brown type by manipulation of substrates. As the cultural morphology and stability were shown to be essentially substrate-dependent, the mycelial status of this organism was reassessed. It is proposed that albino and intermediate variants are usually artificially induced by conventional refined carbohydrate media, while crude guano substrates tend to promote cultures of a brown wild type.

Combinations of amphotericin B and ketoconazole had an additive effect in vitro against Histoplasma capsulatum and Cryptococcus neoformans; ketoconazole combined with flucytosine exerted an indifferent effect against C. neoformans. In vivo studies in athymic nude (nu/nu) mice and their heterozygous (nu/+) littermates demonstrated that treatment with the ketoconazole-amphotericin B combination resulted in longer survival of mice with cryptococcosis than did treatment with ketoconazole plus flucytosine. However, mice given ketoconazole plus amphotericin B did not survive significantly longer than those given amphotericin B alone (cryptococcosis) or ketoconazole alone (histoplasmosis). Combination chemotherapy with ketoconazole and amphotericin B may offer a modest therapeutic advantage over therapy with either drug alone.

The ultrastructural changes which occur during the first 24 h of mycelium to yeast transition have been studied in the dimorphic fungus Histoplasma capsulatum. A temperature shift controls mycelial to yeast transition. During the first 24 h respiratory rate, ATP and cytochrome concentration fall to very low levels. Ultrastructural observations showed that the plasma membrane became undulated and the cell wall lost its characteristic fibrous outer layer. At 8 h the ordered lamellar structure of the mitochondria was no longer apparent. 24 h after the temperature shift 70% of the cells were lysed. The remaining cells contained many cytoplasmic membrane structures; mitochondria were rarely observed. These changes are considered to be the morphological expression of the physiological events characteristic of stage one in mycelial to yeast transition.

During temperature-induced transition of the dimorphic pathogenic fungus Histoplasma capsulatum from the single yeast cell form to the multicellular mycelial form, there was an increase in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels as well as a striking accumulation of cAMP in the medium. cAMP levels also changed during the reverse mycelium-to-yeast transition.

Heat-shock proteins (hsp) were elicited when mycelia of the Downs strain and the more virulent G184A and G222B strains of Histoplasma capsulatum were shifted up to temperatures which induced the mycelial-to-yeast transition (34-40 degrees C). The classes of the major hsp which increased in synthesis in each strain were similar. However, the pattern of synthesis of these proteins at the different temperatures in Downs differed from those in the G184A and G222B strains: hsp synthesis in Downs peaked at 34 degrees C, whereas in G184A and G222B it was highest at 37 degrees C.

We have characterized balanced growth of yeast and hyphal cells of Histoplasma capsulatum and cells during transition from yeast to hyphae in a newly developed synthetic medium, R3B3. Homogeneous populations of yeast at 37 degrees C and hyphae at 25 degrees C grew in this medium with generation times of 10h and 19h, respectively. The growth rates were exponential, as demonstrated by the kinetics of net increase in dry weight. Identical rates of net increase were observed for ribonucleic acid (RNA) and protein, indicating conditions which approached steady state growth. In addition, this defined medium facilitated incorporation of radioactive precursors into RNA and protein and thus will allow for future detailed studies of macromolecular synthesis. When the incubation temperature of growing yeast cells was switched from 37 degrees C to 25 degrees C, the growth rate decreased as indicated in the generation time (19 h). Although the kinetic rates of RNA and protein synthesis also decreased, these rates were slightly faster relative to that observed for the increase in dry weight. During the later stage(s) of the yeast to hyphae transition a marked increase and subsequent decrease was observed for both RNA and protein. After this period of time, the synthesis of RNA and protein proceeded at the steady state rates usually observed in hyphal cultures.

Differences in the 30-day survival of Histoplasma capsulatum after intravenous injection indicated that the A/J strain of inbred mouse was more resistant to experimental infection than was the C57BL/6 strain. CFU from the spleens of infected animals increased during the first week after injection but gradually declined over the next 3 weeks. The CFU per gram of tissue in the C57BL/6 animals were 10- to 100-fold higher than were those in the A/J mice during the time between 7 and 28 days after infection. The units of gamma interferon (IFN-gamma) in supernatants of spleen cells stimulated with heat-killed yeast cells of H. capsulatum reached a peak at the time of the largest number of CFU per gram of tissue. The titers of IFN-gamma at days 3 to 5 were higher in the A/J mice than they were in the C57BL/6 mice, but from days 7 to 28, the titers of IFN-gamma were not correlated with the more efficient clearance of the fungus from the spleens of A/J mice. The L3T4+ spleen cells were shown to be active IFN-gamma producers. Treatment of Histoplasma-infected mice with anti-IFN-gamma antibody resulted in much larger tissue burdens of the fungus in the lungs and spleens of treated animals than in untreated animals. There was no marked difference in the result of treatment with anti-IFN-gamma antibody between A/J and C57BL/6 mice. Treatment of Histoplasma-infected mice with recombinant murine IFN-gamma did not alter the course of infection in either inbred strain of mouse.

Growth factor(s) present in a spent liquid medium after culture of the yeast form of Histoplasma capsulatum enhanced both yeast and mycelial growth of nine isolates tested. Hydroxamic acid extracted from the culture fluid displayed growth factor activity.

Ribonucleic acid (RNA) polymerases of Histoplasma capsulatum (yeast phase) were fractionated by phosphocellulose chromatography and partially characterized. Three distinct, active fractions were seen. The major RNA polymerase species was inhibited strongly by alpha-amanitin, whereas the other two were resistant. When either slightly purified (HSE) extract or the major active component was assayed at 37 C, the incorporation of tritiated uridine monophosphate into RNA stopped after 10 to 15 min. In contrast, the synthesis continued for at least 1 h at 23 C. The other two RNA polymerase species exhibited higher rates of incorporation when tested at 37 C, and continued to synthesize RNA even after 60 min. However, by that time the levels of incorporation at 23 C were higher than at 37 C for all three enzymes. The temperature sensitivity was not affected by changing substrate concentration or employing either native or denatured calf thymus deoxyribonucleic acid as a template. These results are compared with the data obtained with RNA polymerases from different fungi and other organisms. A possible involvement of RNA polymerase(s) in morphological differentiation of H. capsulatum is discussed.

Controlled attempts to release viable conidia from cultures of Blastomyces dermatitidis and Histoplasma capsulatum with air samplers were unsuccessful. Air velocity was not fount to be a limiting factor since the conidia remained attached after conidiophores had been violently shaken by air currents in an observation chamber. Wetting these same conidiophores readily released the conidia. It is inferred that in nature conidia may be liberated by exposure to water and then dispersed by air currents.

Within 48h following the induction of mycelial to yeast-like phase conversion of Histoplasma farcininosum, randomly occurring hyphal cells were observed to contain multiple nuclei and markedly increased numbers of mitochondria. Yeast-like cells arose as buds from swollen tips of terminal hyphae, as sessile buds along the hyphae, and as buds from chlamydospores. Yeast-like cells were characterized by the presence of numerous buds over the surface of the mother cell. Bud scars were evident in the cell wall of the mother cell following abscission of the bud cell. Little similarity was noted between the fine structure of yeast-like H. farciminosum and that reported for H. capsulatum. The yeast-like cells of H. frciminosum underwent rapid transformation to the mycelial phase at 25 degrees C. The hyphal cell wall originated from the inner layer of cell wall of the yeast-like form. The cytoplasm of the hyphal cell usually contained a single nucleus, scattered mitochondria and occasional lipid storage bodies. Occasionally, Woronin bodies were observed at the septal pore.

We have analyzed a step in cysteine biosynthesis in several strains of the pathogenic dimorphic fungus, Histoplasma capsulatum. Mycelial cells of all strains tested are prototrophic. However, the yeast phase cells of most stains do not grow in the absence of -SH-containing compounds due to the apparent lack of an active form of sulfite reductase, a crucial enzyme in the cysteine biosynthetic pathway. In contrast, the yeast phase cells of one strain (Downs) have been found to have an active sulfite reductase and can grow in the absence of cysteine if serine is added. A different metabolic block must thus exist in this strain. Sulfite reductase in the yeast form of Downs strain is completely repressed by growth on cysteine while the mycelial form seems to be constitutive. The yeast and mycelial phase extracts were analyzed on polyacrylamide gels. A distinct protein band appeared in extracts prepared from the yeast cells incubated in minimal or serine-containing media, but not in extracts from mycelia or from cysteine-grown yeast cells.

The passive transfer of spleen cells from 1 month old mice into adult syngeneic mice, abrogates their resistance to histoplasmal infection. This suppressive state was detected in two cell populations, one non-adherent and another adherent with radioresistant characteristics. The transferred spleen cells were treated by different anti-sera: anti-theta, anti-adherent cells (produced in rabbits) and monoclonal anti-Thy 1.2 respectively. The irradiated and non-irradiated adult recipient mice were infected with Histoplasma yeasts utilizing the Lethal Dose50 for 1 month old mice. The infection course was determined by death percentage, the histoplasmosis murine signs and the number of the fungal colony forming units (CFU) from the infected spleens. The results of the anti-sera treatment suggest that non-adherent as well as adherent cells participate in the suppressive phenomena. A lower number of CFU was identified in infected animals which received cells treated with anti-Thy 1.2 anti-sera.

Cycloheximide and chloramphenicol in combination have a greater effect on yeast cells of Histoplasma capsulatum than on the mycelial phase of this fungus. In contrast, clotrimazole was found to be more effective against mycelia. Miconazole produced a pronounced effect against both phases wheras tolnaftate was only slightly active. Sulfadiazine and griseofulvin were completely inactive against both phases. The differential sensitivities of the 2 phases of H. capsulatum to various antibiotics can be useful in studying the transition of the dimorphic fungus from 1 phase to the other.

Plasma membrane vesicles from the yeast and mycelial phases of Histoplasma capsulatum have been purified and characterized. The method of purification involved differential centrifugation of ballistically fractured cells followed by sedimentation through discontinuous sucrose density gradient and equilibrium centrifugation. Purity of the preparation was assessed by electron microscopy. The protein composition of the membrane preparations from the yeast and mycelial phases of the fungus was analyzed by polyacrylamide gels. A comparison of the two morphologic phases revealed quantitative and qualitative differences in the expressions of several membrane-specific proteins. Physical differences in the appearance of the membranes were also observed by electron micrography of membrane preparations. Alteration in membrane fluidity may be one of the many causes for differences in the appearance of membrane vesicles in the two phases.

We have formulated two defined media which yield excellent plating efficiency of the yeast phase of Histoplasma capsulatum. Neither requires the addition of conditioned medium or purified siderophores, although the simpler of the two media includes hemin as a source of solubilized iron. Agarose, rather than conventional agar, is the solidifying agent for both media. Plating efficiency usually exceeds 90% with both North American and Central American isolates of H. capsulatum.

An apparatus to trap and quantitate 14CO2 is described. When used to measure antibiotic effects on Histoplasma capsulatum and Mycobacterium bovis (BCG), there was an inverse quantitative relationship between 14CO2 evolved and antibiotic concentration. The technique should prove useful for analyses that require trapping and quantitation of 14CO2, including antimicrobial sensitivities of slow-growing organisms.

Mice immunized subcutaneously with 10(6) viable yeast cells of Histoplasma capsulatum exhibited host protection to a challenge infection and also exhibited concomitant manifestations of cellular immunity to a culture filtrate antigen (yeast extract dialysate (YED)-histoplasmin). Protective immunity was determined by quantitative culture of organisms from infected spleens on a solid medium containing a growth factor produced by the organism. As early as 4 days following immunization, host protection was observed in mice. Positive footpad swelling, indicative of delayed-type hypersensitivity, also was apparent at the early time period (4-7 days) after immunization. By days 14 and 21 following immunization, the mice exhibited maximum footpad responses and maximum splenic clearance of yeast cells. As an additional correlate of cellular immunity, YED-histoplasmin was coupled with chromic chloride to murine erythrocytes (M-RBC) and mixed with immune or nonimmune splenic lymphocytes to detect antigen-specific rosette forming cells (RFC's). Only splenic lymphocytes from mice immunized with H. capsulatum formed antigen-specific RFC's (greater than or equal to 0.2%). Pretreatment of splenic lymphocytes with antitheta (theta) serum plus complement greatly reduced the numbers of RFC's, illustrating their T cell nature. Host protection and cell-mediated immune responses decreased substantially by day 28 following immunization.