Yeast Cells Of Histoplasma Capsulatum Research Articles

A quick and cost-effective method for diagnosing disseminated histoplasmosis in children

The examination of fecal mucus for detecting yeast cells of Histoplasma capsulatum has proved to be a useful tool for diagnosing disseminated histoplasmosis in paediatric patients in a study of 13 cases carried out in Ecuador.

Histoplasma capsulatum yeast cells attach and agglutinate human erythrocytes.

The ability of yeast cells of Histoplasma capsulatum to attach and agglutinate human erythrocytes has been described. This is the first report involving these yeasts in the hemagglutination phenomenon. Results revealed that the yeast cells were able to bind to erythrocytes irrespective of blood groups and to agglutinate them when a high density of yeast cells was used. Assays on the inhibition of yeast attachment to erythrocytes were also performed, using sugar-treated yeast cells. Results indicate that galactose (Gal), mainly the beta-anomer, specially inhibited yeast attachment. Disaccharides (Gal-derivatives) and glycosaminoglycans containing Gal residues, mainly chondroitin sulfate C, promote this type of inhibition. In addition, preliminary data of inhibition assays also involved a probable ionic strength driven mechanism mediated by sialic acid and heparan sulfate, suggesting that yeast binding to erythrocytes could be associated with negative charges of both molecules.

Activities of sordarins in murine histoplasmosis.

Sordarins are new antifungals which inhibit fungal protein synthesis by blocking elongation factor 2. Three compounds were evaluated in a murine model of histoplasmosis. Immune-competent mice were infected intravenously with 10(6) to 10(8) CFU of Histoplasma capsulatum yeast cells. Mice were treated either orally with sordarins or fluconazole from day 2 through 8 after infection or intraperitoneally with amphotericin B during the same period. Protection was measured by increased rates of survival for 30 days after infection or reduction of lung or kidney tissue counts 9 days after infection. All three of the antifungal drugs tested were protective compared with controls. Sordarins were effective at doses as low as 2 mg/kg of body weight/day. This novel class of drugs compared favorably with amphotericin B and fluconazole for the treatment of histoplasmosis.

Treatment of histoplasmosis with MK-991 (L-743,872).

BALB/c nu/+ immunocompetent and athymic (nu/nu) mice were infected intravenously with yeast cells of Histoplasma capsulatum. Mice were either given water (controls) intraperitoneally (i.p.) or given MK-991 i.p. once daily or twice daily. Protection was measured as prolonged survival or reduction in tissue counts. MK-991 was protective in immunocompetent mice, prolonging survival and reducing counts in spleen and livers at a dose as low as 0.05 mg/kg of body weight/day. MK-991 was modestly effective in athymic mice at a higher dose, 5 mg/kg/day. These studies suggest that MK-991 may be appropriate for clinical development in histoplasmosis.

Inhibition of growth of Histoplasma capsulatum yeast cells in human macrophages by the iron chelator VUF 8514 and comparison of VUF 8514 with deferoxamine.

Histoplasma capsulatum requires intracellular iron to survive and multiply within human and murine macrophages (M phi). Thus, iron chelators may be useful compounds in the treatment of histoplasmosis. In the present study we compared the efficacies of five different iron chelators with deferoxamine (DEF) for their capacity to inhibit the growth of H. capsulatum yeast cells in culture medium and within human M phi. Of the agents tested, only one, VUF 8514, a 2,2'-bipyridyl analog, was found to be effective. VUF 8514 inhibited the growth of yeast cells in tissue culture medium and within M phi in a dose-response fashion. In tissue culture medium, the 50% effective dose (ED50) of VUF 8514 was 30 nM and the ED50 of DEF was 1 mM. In human M phi, the ED50 of VUF 8514 was 520 nM and the ED50 of DEF was 4 mM. Thus, VUF 8514 was effective at a concentration 7.7 x 10(3)-fold lower than DEF in inhibiting the growth of yeast cells in M phi. Inhibition of the intracellular growth of yeast cells by VUF 8514 was reversed by holotransferrin and iron nitriloacetate, an iron compound that is soluble at neutral to alkaline pH. Thus, VUF 8514 inhibits the intracellular growth of yeast cells by acting as an iron chelator rather than through its capacity as a weak base. These data suggest that the hydroxamic acid siderophore of H. capsulatum yeast cells competes successfully for iron against some iron chelators but not others and that VUF 8514 may be a potential therapeutic agent for the treatment of histoplasmosis.

Effect of macrophage colony-stimulating factor (M-CSF) on macrophage morphology, phagocytosis, and intracellular multiplication of Histoplasma capsulatum.

M-CSF induced dramatic morphological changes in resident peritoneal macrophages (R-PM) in a time and dose dependent manner. Macrophages increased 2-3-fold in size and developed a dendritic morphology. Compared to macrophages cultured in medium alone M-CSF-treated macrophages were less phagocytic (67 versus 82%) for serum opsonized yeast cells of Histoplasma capsulatum. However, in M-CSF macrophages the intracellular multiplication of ingested yeast cells were significantly inhibited compared to growth in control macrophages. The fungistatic activity of M-CSF macrophages persisted for at least 48 h after infection.

Alterations in murine macrophage arachidonic acid metabolism following ingestion of nonviable Histoplasma capsulatum.

The effect of ingestion of heat-killed Histoplasma capsulatum yeast cells on the metabolism of arachidonic acid (20:4) to prostenoids and leukotrienes was examined in murine peritoneal macrophages (M phi s). H. capsulatum-containing M phi s exhibited a metabolite profile similar to that of zymosan-challenged phagocytes; however, there were differences with respect to the relative and total amounts of products produced. While proteose peptone-elicited M phi s exposed to H. capsulatum released quantitatively less prostaglandin E2 (PGE2) and leukotriene C4 than zymosan-treated M phi s, they metabolized a greater percentage of total product to prostenoids. In addition, whereas in vitro priming with gamma interferon increased both the PGE2 and leukotriene C4 contents of zymosan-stimulated M phi supernatants, similarly primed M phi s challenged with H. capsulatum selectively increased only PGE2 production. The immunosuppressive effect of a relative excess of prostenoids in H. capsulatum-containing M phi s may contribute to the overall disturbance in cell-mediated immunity characteristic of disseminated histoplasmosis.

Inhibition of murine macrophage protein kinase C activity by nonviable Histoplasma capsulatum.

Ingestion of Histoplasma capsulatum yeast cells inhibits the oxidative burst response of murine macrophages (M phi's). Since protein kinase C (PKC) is believed to be an integral part of the signal transduction pathway involved in the production of reactive oxygen intermediates, we investigated the relationship between PKC activity and oxidative burst inhibition in H. capsulatum-containing murine peritoneal M phi's. An inhibitory effect on both basal and phorbol myristate acetate-mobilized membrane PKC activities was observed in M phi's which had ingested H. capsulatum but not latex spheres. These results suggest that one way in which H. capsulatum may disrupt the oxidative burst is through a PCK-dependent mechanism.

Recognition of Histoplasma capsulatum yeast-cell antigens by human lymphocytes and human T-cell clones.

We have prepared a detergent extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells that is recognized by T cells from mice immunized with viable organisms or with the extract. A 62-kd antigen from this extract has been isolated and has been shown to be antigenic and to confer protective immunity in mice. In this study, we examined the in vitro proliferative response by human lymphocytes and human T-cell clones to both the extract from the cell wall and cell membrane and the 62-kd antigen, termed HIS-62. Seven healthy individuals were identified whose peripheral blood mononuclear cells responded to the cell wall and cell membrane extract from H. capsulatum yeast cells. Mononuclear cells from all seven individuals recognized histoplasmin. T-cell clones were generated from peripheral blood mononuclear cells from a single person using the extract from cell wall and cell membrane as the antigenic preparation. Nineteen clones were propagated; all expressed the surface phenotype CD3+, CD4+, T-cell receptor alpha/beta +. The clones were antigen-specific. Of 17 clones studied, none responded to extracts from Blastomyces dermatitidis or Coccidioides immitis or to tetanus toxoid. Sixteen of 19 clones recognized HIS-62 in vitro. Additional analysis revealed that cells from each of the seven individuals who responded to the extract mounted a proliferative response to HIS-62. Thus, the extract from the cell wall and cell membrane and HIS-62 are targets of the human cell-mediated immune response to H. capsulatum. Moreover, HIS-62 appears to be an immunodominant antigen.

Protective efficacy of a 62-kilodalton antigen, HIS-62, from the cell wall and cell membrane of Histoplasma capsulatum yeast cells

We reported previously that a detergent extract of the cell wall and cell membrane of Histoplasma capsulatum yeast cells contains antigens recognized by T cells. In T-cell immunoblot analysis, a region encompassing 62 kDa was stimulatory for an H. capsulatum-reactive T-cell line and T-cell clones derived from C57BL/6 mice. In this study, we isolated a 62-kDa band, termed HIS-62, from electrophoresed cell wall and cell membrane of H. capsulatum yeast cells and examined its antigenicity and immunogenicity. C57BL/6, BALB/c, and CBA/J mice that were immunized with viable H. capsulatum yeast cells mounted a delayed-type hypersensitivity response to HIS-62 that was stronger than that of normal controls. Spleen cells from each strain of mouse immunized with viable yeast cells proliferated vigorously in response to HIS-62; conversely, splenocytes from control animals did not recognize this antigen. A T-cell line and 5 of 5 T-cell clones from C57BL/6 mice, 10 of 15 BALB/c T-cell hybridomas, and 8 of 12 CBA/J T-cell hybridomas recognized HIS-62. A cutaneous delayed-type hypersensitivity response to the antigen was apparent in each strain of mouse that was injected with 80 micrograms of HIS-62 mixed with Freund adjuvant. In addition, spleen cells from HIS-62-immunized mice proliferated in vitro in response to this antigen. Vaccination of each strain of mouse with 80 micrograms of HIS-62 conferred protection against a lethal intravenous challenge with H. capsulatum yeast cells. Thus, HIS-62 appears to be an important target of the cellular immune response to H. capsulatum and induces a protective immune response in mice.

Inhibition of intracellular growth of Histoplasma capsulatum yeast cells by cytokine-activated human monocytes and macrophages

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.

Antifungal activity of murine polymorphonuclear neutrophils againstHistoplasma capsulatum

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.

Antigenicity and immunogenicity of an extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells

In order to identify T-cell antigens from Histoplasma capsulatum yeast cells, we prepared a detergent extract of the cell wall and cell membrane of yeast-phase H. capsulatum G217B and analyzed its antigenicity and immunogenicity. Mice injected with viable H. capsulatum yeast cells or with 500 or 1,000 micrograms of the extract mounted a delayed-type hypersensitivity response to solubilized cell wall and cell membrane. Vaccination with this antigenic preparation conferred a protective immune response in mice that were challenged intravenously with H. capsulatum yeast cells. The extract induced in vitro proliferation by splenocytes from mice injected with either viable yeast cells or the soluble cell wall and cell membrane preparation. We also examined the profile of in vitro responses by a murine T-cell line and by cloned T cells to soluble cell wall and cell membrane by employing the technique of T-cell immunoblotting. Two prominent regions that stimulated the T-cell line and cloned T cells were identified. Fractions encompassing an area between 53 and 64 kDa caused proliferation by a T-cell line and five of six clones. Antigens recognized by the T-cell line and by three of six clones were contained in another area that extended from 69 to 82 kDa. The data demonstrate that this soluble extract from cell wall and cell membrane contains antigens recognized by T cells and mediates protective immunity. Moreover, T-cell immunoblotting provides a useful technique for mapping immunoreactive molecules from H. capsulatum yeast cells.

Functional analysis of Histoplasma capsulatum-reactive T-cell hybridomas

Activation of CD4+ T cells is a crucial step in the elimination of Histoplasma capsulatum yeast cells from tissues. However, only a limited amount of information exists concerning the immunobiology of H. capsulatum-reactive T cells that are CD4+. To facilitate the analysis of the functional activities of this T-cell subpopulation, we developed a panel of 10 murine T-cell hybridomas from splenocytes of immune C57BL/6 mice. All hybridomas reacted with monoclonal anti-CD4+ antibody and released interleukin-2 after stimulation with histoplasmin. Within 3 weeks, the reactivity of hybridomas to histoplasmin declined dramatically, yet the cells responded vigorously to yeast-phase preparations that were enriched for cytosol, cell wall, or cell membrane. Of 10 hybridomas studied, only one recognized heterologous fungal antigens. Responsiveness to yeast-phase antigens was restricted by I-Ab. We mapped determinants in cytosol and cell wall or cell membrane by the technique of one-dimensional T-cell immunoblotting. The patterns of responses of hybridomas to cytosol were nearly uniform. All hybridomas responded to two immunodominant regions in cytosol with masses ranging from less than or equal to 18 to 26 kilodaltons (kDa) and 35 to 39 kDa. All hybridomas tested responded to determinants in the cell wall or cell membrane preparation with masses of 35 to 39 kDa. These hybridomas provide a useful tool for defining yeast-phase antigens that trigger T-cell activation.

Effects of Histoplasma capsulatum on murine macrophage functions: inhibition of macrophage priming, oxidative burst, and antifungal activities.

Histoplasma capsulatum yeast cells fail to trigger an oxidative burst response in normal murine macrophages. The results of this study, in which an in vitro assay of macrophage antifungal effects was used, extend these findings. During 18 h of incubation, unprimed elicited murine macrophages inhibited H. capsulatum growth only when macrophages were present in great excess. Gamma interferon (IFN-gamma)-primed macrophages showed enhanced fungal growth inhibition but a similar requirement for an excess of phagocytes. Macrophages containing heat-killed H. capsulatum exhibited diminished antifungal effects toward viable H. capsulatum and Saccharomyces cerevisiae cells. Parallel experiments showed no comparable effect of ingested latex particles on macrophage antifungal activity. Using chemiluminescence as a measure of the oxidative burst, we found that macrophages primed in vitro with IFN-gamma alone failed to exhibit a significant response to triggering by H. capsulatum yeast cells unless a second priming agent (tumor necrosis factor alpha or bacterial lipopolysaccharide) was added to IFN-gamma. Furthermore, macrophage priming with single agents was blocked by the prior ingestion of heat-killed H. capsulatum. These studies provide evidence that ingestion of H. capsulatum yeast cells can induce a prompt and enduring deactivation of murine macrophages.

Stimulation of murine lymphocyte blastogenesis by mitogens in heat-killed Histoplasma capsulatum yeast cells.

In vitro blastogenesis by normal murine splenocytes from several mouse strains has been detected after exposure to heat-killed Histoplasma capsulatum yeast cells. Maximal lymphocyte stimulation induced by 10(4) heat-killed cells resulted in 20- to 45-fold increases in [3H]thymidine uptake by splenocytes when compared with responses by normal unstimulated lymphocytes. The kinetics for this response to heat-killed H. capsulatum cells has shown peak mitogenesis 3 days after culture. Examination of the mitogenic potential of soluble antigen preparations from H. capsulatum has revealed stimulation of lymphocyte blastogenesis with yeast cell sonicates and autolysates but not substances from autoclaved yeast cells. The levels of lymphocyte blastogenesis induced by sonicates or autolysates were comparable to mitogen responses stimulated by heat-killed cells. Preliminary biochemical characterization of the mitogenic factor(s) associated with yeast cell sonicates show two peaks of activity, at 178,000 and less than 12,000 Mr, which have a protein or glycoprotein nature. Finally, analysis of lymphocyte blastogenesis in cultures enriched for selected lymphocyte subpopulations has shown that T lymphocytes are preferentially stimulated by yeast cell mitogens.

Determination of viability ofHistoplasma capsulatumyeast cells grownin vitro: comparison between dye and colony count methods

The viability of Histoplasma capsulatum yeast cells grown under different conditions was determined by dye tests with Eosin-Y and Janus Green B and by colony counts of cells plated on brain-heart infusion agar supplemented with histoplasma growth factor and bovine serum albumin (BHI-SAG). The test samples included cells grown on brain-heart infusion agar at 37 degrees C for 2-7 days, cells grown in glucose-cysteine broth medium for 1-31 days, and cells grown on brain-heart infusion agar for 3 days at 37 degrees C and then irradiated by ultraviolet light. The colony count indicated that the viability of the yeast cells grown on brain-heart infusion agar for 2 or 3 days varied between 68 and 100% depending on the isolates. The viability, however, dropped from 16 to 29% by day 7. The results of dye tests showed 78 to 99% dye-negative cells among the 2- and 3-day-old cultures while the number of dye-negative cells dropped to 32-36% on day 7. The colony count with the cells grown in the broth culture showed 100% viability until day 7 and dropped significantly by day 9. The results of dye tests showed no correlation with the colony count findings. The survival curve of ultraviolet-irradiated cells determined by colony count showed that irradiation at 180 erg mm-2 killed more than 50% of cells; fewer than 10% of cells survived 360 erg mm-2. The results of the dye test showed no difference between the irradiated and control populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical staining of Histoplasma capsulatum at the electron microscopic level

Rabbits were immunized with histoplasmin emulsified in Freund's complete adjuvant. Antibody raised in these rabbits was exposed to Histoplasma capsulatum yeast cells, either in tissue culture medium, or after in vitro or in vivo phagocytosis by mouse macrophages. The sites of antibody binding were identified using an immunoperoxidase technique. At least two sites of antibody binding were identified, one to the fungal cell wall and the other to the outer cell membrane. Within 6 h after phagocytosis by macrophages, fungal cell walls appeared roughened, with what appeared to be cell wall antigen released into the phagolysosome, appearing associated with the phagolysosome membrane, and possibly adjacent macrophage cytoplasm. Similar staining of fungal antigen was noted in alveolar macrophages which had ingested Histoplasma capsulatum after a respiratory challenge. This method may be useful in detailing the host/pathogen interactions which occur in human pulmonary histoplasmosis.

Detection of an antigenic cell wall layer inHistoplasma capsulatum

Histoplasma capsulatum yeast cells have been studied by immunoelectron microscopy using rabbit polyclonal antisera and a biotin-avidin-peroxidase detection system. An antigenic surface layer has been visualized in the cell wall of immunostained organisms. This layer was not seen in samples prepared by standard electron microscopic methods or in negative controls used with the immunocytochemical technique. Without immunostaining the cell wall of Histoplasma appeared almost transparent. In contrast, after immunoperoxidase staining the cell wall was conspicuous, bounded by the darkly stained outer layer. This electron dense layer, appeared to be a reservoir of surface antigens that were recognized by anti-Histoplasma antibodies.

Protective effect of poly-2-vinylpyridine-N-oxide on susceptibility of silica-treated mice to experimental histoplasmosis.

We have studied the ability of poly-2-vinylpyridine-N-oxide (PVNO), a lysosomal stabilizing agent, to abrogate the cytotoxic effects of silica on macrophages. Male C3H/HeN mice were pretreated with PVNO and inoculated intravenously with silica particles. At 24 h after silica injection, silica-treated and -untreated mice were challenged intravenously with varying doses of live yeast cells of Histoplasma capsulatum. All mice receiving silica died when challenged with 5 X 10(5) yeast cells of Histoplasma sp. compared with no deaths in PVNO-pretreated animals and 10% mortality in controls not receiving PVNO or silica. When animals were given 2.5 X 10(5) yeast cells (a sublethal dose), the protective effect of PVNO was seen by a reduction in splenomegaly and viable Histoplasma sp. present in the spleen. Furthermore, PVNO alone showed a significant protective effect (P less than 0.05) against a lethal challenge with Histoplasma sp. Prior treatment with PVNO also protected mouse peritoneal macrophages from the cytotoxic effects of silica particles in vitro. These results indicate that PVNO abrogates the cytotoxicity of silica particles on macrophages and also increases the resistance of mice to histoplasmosis.