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PubMed1992

Induction of stress protein synthesis in Histoplasma capsulatum by heat, low pH and hydrogen peroxide.

It is well known that some micro-organisms synthesize proteins when stressed by heat or other factors. The function of these proteins is not yet clear, but some of them are believed to be related to resistance against a hostile environment. Histoplasma capsulatum is an intracellular pathogenic fungus that multiplies inside macrophages and resists macrophage microbicidal mechanisms. To study the defense mechanisms of H. capsulatum and mimic the hostile environment the fungus may encounter during infection, we investigated protein synthesis by H. capsulatum (isolate G217B) when stressed by heat (40 degrees C), low pH (pH 4), or oxidative products (H2O2) using [35S]-methionine labelling. Analysis of cytosol proteins by SDS-PAGE and fluorography disclosed that H. capsulatum increased synthesis of six constitutive proteins and decreased synthesis of six proteins when stressed at 40 degrees C. When stressed by pH 4 or H2O2, H. capsulatum increased the synthesis of eight and five constitutive proteins, respectively, and decreased the synthesis of three proteins. Estimation of superoxide dismutase (SOD) and catalase enzymatic activity in cytosols from stressed H. capsulatum did not reveal an increase of these enzymatic activities compared to cytosols from non-stressed H. capsulatum. These results suggest that H. capsulatum increases the synthesis of some constitutive proteins when stressed by heat, low pH or H2O2, which might relate to pathogenicity, and are thus worthy of further study. These induced proteins are apparently different from SOD or catalase.

Catalase↗
PubMed2004

Susceptibility of the human pathogenic fungi Cryptococcus neoformans and Histoplasma capsulatum to gamma-radiation versus radioimmunotherapy with alpha- and beta-emitting radioisotopes.

UNLABELLED: Fungal diseases are difficult to treat in immunosuppressed patients and, consequently, new approaches to therapy are urgently needed. One novel strategy is to use radioimmunotherapy (RIT) with fungal-binding monoclonal antibodies (mAbs) labeled with radionuclides. However, many fungi manifest extreme resistance to gamma-radiation, such that the doses of several thousand gray are required for 90% cell killing, whereas for mammalian cells the lethal dose is only a few gray. We compared the susceptibility of human pathogenic fungi Cryptococcus neoformans (CN) and Histoplasma capsulatum (HC) to external gamma-radiation and to the organism-specific mAbs 18B7 and 9C7, respectively, conjugated to (213)Bi and (188)Re radionuclides. METHODS: CN and HC cells were irradiated with up to 8,000 Gy ((137)Cs source, 30 Gy/min). RIT of CN with (213)Bi- and (188)Re-labeled specific mAb and of HC with (188)Re-labeled specific mAb used 0-1.2 MBq per 10(5) microbial cells. After irradiation or RIT, the cells were plated for colony-forming units (CFUs). Cellular dosimetry calculations were performed, and the pathway of cell death after irradiation was evaluated by flow cytometry. RESULTS: Both CN and HC proved to be extremely resistant to gamma-radiation such that significant killing was observed only for doses of >4,000 Gy. In contrast, these cells were much more susceptible to killing by radiation delivered with a specific mAb, such that a 2-logarithm reduction in colony numbers was achieved by incubating them with (213)Bi- and (188)Re-labeled mAb 18B7 or with (188)Re-9C7 mAb. Dosimetry calculations showed that RIT was approximately 1,000-fold more efficient in killing CN and approximately 100-fold more efficient in killing HC than gamma-radiation. Both gamma-radiation and RIT caused cell death via an apoptotic-like pathway with a higher percentage of apoptosis observed in RIT-treated cells. CONCLUSION: Conjugating a radioactive isotope to a fungal-specific antibody converted an immunoglobulin with no antifungal activity into a microbicidal molecule. RIT of fungal cells using specific antibodies labeled with alpha- and beta-emitting radioisotopes was significantly more efficient in killing CN and HC than gamma-radiation when based on the mean absorbed dose to the cell. These results strongly support the concept of using RIT as an antimicrobial modality.

Antibodies, Monoclonal↗
PubMed2004

[Intraspecific chromosomal variability in human pathogenic fungi, especially in Histoplasma capsulatum].

The ploidy, karyotype, and chromosome length polymorphism (CLP) of human pathogenic fungi were revised with emphasis on Histoplasma capsulatum, the causative agent of the systemic mycosis, histoplasmosis. Currently, different systems of gel electrophoresis are being used to determine fungal electrokaryotypes (EK). By renaturation kinetic and genomic reconstruction in H. capsulatum strains (G-186AS and Downs), estimated genome sizes of 23 and 32 Mb were determined for both strains, respectively. The haploid state was proposed for both strains, although aneuploidy was suggested for the Downs strain. Contour-clamped homogeneous electric field (CHEF), field inversion gel electrophoresis (FIGE), and Southern blot using different probes showed the presence of six to seven chromosomes in the Downs strain (low virulence), whereas four chromosomes were identified in the G-186B strain (high virulence). The use of these methods in the three major H. capsulatum reference strains (G-217B and Downs from the United States of America, G-186B from Panama) revealed distinct chromosome sizes, from 0.5 to 5.7 Mb, with CLP associated with chromosomes size and mobility. Recently, by CHEF, using 19 H. capsulatum isolates from Latin-America and the G-186B strain, five to seven chromosomes with 1.1 to 11.2 Mb molecular sizes were revealed, which again suggested CLP in H. capsulatum. However, to elucidate the EKs polymorphism in H. capsulatum and its relationship with the isolates phenotype more studies are needed to understand the mechanisms controlling ploidy variability.

Chromosomes, Fungal↗
PubMedDate not supplied

[In vitro sensitivity of Histoplasma capsulatum var. capsulatum to amphotericin B, ketoconazole, itroconazole and fluconazole].

The minimum inhibitory concentration (MIC) was determined by a micromethod of dilution in RPMI-1640 broth in order to know the sensitivity to amphotericin B, itraconazole, ketoconazole and fluconazole of 29 strains of Histoplasma capsulatum var capsulatum isolated in Cuba. Histoplasmosis is one of the main systemic mycosis at the world level and its incidence has increased during the last years, associated mainly with HIV infection and to the appareance of new epidemic outbreaks in different regions. The results obtained allowed to conclude that high values of IMC against fluconazole were found with a geometric mean of 55.5 ug/mL. For amphotericin B, ketoconazole and itraconazole, all the strains were inhibited at low concentrations with geometric means of 0.26, 0.17 and 0.125 ug/mL, respectively. The development for the first time in Cuba of a method to determine the IMC of the main antifungal drugs against H. capsulatum var capsulatum will make possible to establish the sensitivity patterns and to detect the appearance of resistance, which will contribute to know this important mycosis better.

Amphotericin B↗
PubMedDate not supplied

[First isolation of Histoplasma capsulatum from the urban bat Eumops bonariensis].

We report the first isolation of Histoplasma capsulatum var. capsulatum from a male bat Eumops bonariensis captured in Buenos Aires city in 2003. The pathogen was recovered from spleen and liver specimens, and was identified by its phenotypic characteristics. PCR with primers 1283, (GTG)5, (GACA)4 and M13 was used to compare both bat isolates with 17 human isolates, 12 from patients residing in Buenos Aires city, and 5 from other countries of the Americas. The profiles obtained with the four primers showed that both bat isolates were identical to each other and closer to Buenos Aires patients than to the other isolates (similarity percentage: 91-100% and 55-97%, respectively). The high genetic relationship between bat isolates and those from patients living in Buenos Aires suggests a common source of infection. This is the first record of E. bonariensis infected with H. capsulatum in the world, and the first isolation of the fungus in the Argentinean Chiroptera population. In the same way as these wild mammals act as reservoir and spread the fungus in the natural environment, infection in urban bats could well be associated with the increase in histoplasmosis clinical cases among immunosuppressed hosts in Buenos Aires city.

Americas↗
PubMedDate not supplied

[African histoplasmosis (Histoplasma capsulatum var. duboisii): a case report from Mali].

We report a case of disseminated African histoplasmosis with lymph node and digestive involvement in a 19-year-old man living in the Kayes district of Mali. The patient, HIV-seronegative and not otherwise immunocompromised, presented voluminous cervical and axillary adenopathies as well as retrosternal and mesenteric tumor lesions. Direct examination of biopsy tissue showed numerous specimens of Histoplasma capsulatum var. duboisii. Because direct fungal techniques are the easiest and the most effective method of diagnostic investigation, no cultures were performed. Intolerance to therapy with amphotericin b and ketoconazole led its rapid replacement by surgical treatment: partial excision of the abdominal lesions led to partial remission of the symptoms.

Adult↗
PubMed1992

Digestion of Histoplasma capsulatum yeasts by human macrophages.

The strategies used by Histoplasma capsulatum yeasts to survive and multiply within human macrophages (M phi) are unknown. To better understand these strategies we studied the intracellular fate of viable vs heat-killed (HK) yeasts in human monocyte-derived M phi. Initial studies demonstrated that phagolysosome fusion was present in M phi ingesting either viable or HK yeasts. Viable yeasts multiplied within M phi phagolysosomes, whereas M phi completely digested intracellular FITC-labeled HK yeasts within 24 h after ingestion. This observation was confirmed by electron microscopy. M phi that had ingested colloidal gold-labeled HK yeasts contained gold particles but no visible yeasts at 24 h. Digestion of HK yeasts was evident as early as 4 h after phagocytosis, and was complete by 24 h. M phi digestion of HK yeasts was blocked completely when M phi were cultured for 24 h in the presence of chloroquine. In M phi simultaneously ingesting both viable and HK yeasts, viable yeasts multiplied, but HK yeasts were digested within the same cell. M phi that had ingested viable yeasts digested them completely when M phi were cultured for 24 h in the presence of cycloheximide or amphotericin B. Coculture of infected M phi with nystatin or ketoconazole resulted in inhibition of growth, but the yeasts were not digested. These data indicate that: 1), HK Hc yeasts are easily digested by preformed M phi lysosomal hydrolases; 2), viable Hc yeasts survive and multiply within M phi phagolysosomes, but the yeasts do not secrete a factor(s) that affects the ability of other phagolysosomes within the same M phi to digest killed yeasts; and 3), inhibition of yeast protein synthesis or cell wall biosynthesis is sufficient to render viable yeasts susceptible to digestion by human M phi.

Cells, Cultured↗
PubMed1991

Ultrastructural effects of PCMS on mycelial-yeast transition in Histoplasma capsulatum.

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.

4-Chloromercuribenzenesulfonate↗
PubMedDate not supplied

[Histoplasma capsulatum with aberrant morphology isolated in Argentina].

For the first time in Argentina, we describe a strain of Histoplasma capsulatum var. capsulatum with an aberrant morphology that was isolated from a single patient with AIDS. Mycelial phase cultures on agar Sabouraud at 25-28 degrees C showed white, glabrous, umbilicated and centrally radiated colonies. Unusual microscopic findings were the absence of typical conidia, the presence of terminal/intercalary chlamydoconidia with a diameter of 4 pm and of thickened hyphae. Fungal identification was confirmed by the detection of bands H and M species specific antigens in mycelial culture supernatants and reversion to the typical yeast phase on agar brain-heart-cysteine at 37 degrees C. The genomic DNA profile obtained by RAPD-PCR with primers 1281-1283 coincided with the predominant profile of H. capsulatum among isolates from Argentine patients.

AIDS-Related Opportunistic Infections↗
PubMed1991

[Pulmonary histoplasmosis due to Histoplasma capsulatum. A case].

The authors report a case of benign multinodular pulmonary histoplasmosis, occurring in a 65 year old woman coming back from Guatemala. The disease presented with both fever and cough. The diagnosis was made on a lung biopsy (by thoracotomy) that showed granulomas with giant cells, lymphocytes and central necrosis, and histoplasma capsulatum yeasts on Gomori Grocott coloration. The authors recall the main radiological forms of the disease, and the difficulties of the diagnosis. When not disseminated, histoplasmosis usually has a good prognosis and does not require any treatment.

Aged↗
PubMed1991

Antifungal activity of murine polymorphonuclear neutrophils against Histoplasma 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.

Animals↗
PubMed1990

The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion.

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.

CD18 Antigens↗
PubMed1990

Evidence that the M antigen of Histoplasma capsulatum var. capsulatum is a catalase which exhibits cross-reactivity with other dimorphic fungi.

A panel of monoclonal antibodies (Mabs) was raised against histoplasmin, the antigen derived from the mycelial phase of Histoplasma capsulatum var. capsulatum which contains the diagnostically useful H and M antigens. A number of Mabs were obtained which recognized a 70-75 kD component of an antigenic preparation of H. capsulatum var. capsulatum by Western blotting. When reacted with histoplasmin by Western blotting the Mabs recognized a similar 70-75 kD band, together with a series of higher molecular mass bands at approximately 130, 190 and 230 kD, a pattern which correlates strongly with both the published relative molecular mass (Mr) of the M antigen and the known subunit structure of the enzyme catalase. These Mabs were also shown to recognize a commercial preparation of Aspergillus niger catalase by ELISA. Other dimorphic fungi were also reactive with these Mabs by Western blotting, indicating the presence of common epitopes on the catalase molecules of these species.

Antibodies, Monoclonal↗
PubMed1990

[Ultrastructural study of the yeast-mycelium transition in Histoplasma capsulatum. II. Changes at 34 degrees C].

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.

Fungal Proteins↗
PubMed1990

[Ultrastructural study of the mycelium-yeast transition in Histoplasma capsulatum. I. Changes at 37 degrees C].

Ultrastructural changes observed during the first 24 hours of mycelium to yeast transition in the dimorphic fungus Histoplasma capsulatum are reported. During this period the plasma membrane becomes undulated and the cell wall loses its characteristic fibrous outer layer. At 8 h the ordered lamellar structure of the mitochondria is no longer apparent. 24 h after the temperature shift 70% of the cells are lysed. The remaining cells contain many cytoplasmic membrane structures; mitochondria are rarely observed. These morphological changes are probably correlated with the physiological events characteristic of mycelial to yeast transition.

Cell Wall↗
PubMed1988

Quantitative plating of Histoplasma capsulatum without addition of conditioned medium or siderophores.

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.

Culture Media↗
PubMed1988

Identification of Histoplasma organisms in circulating eosinophils of a dog.

Disseminated histoplasmosis was diagnosed in a 10-year-old dog that had chronic diarrhea, weight loss, fever, and anemia. The diagnosis was based on detection of Histoplasma organisms in circulating neutrophils, monocytes, and eosinophils. The dog had severe histoplasmal fungemia, which may have been caused by treatment with prednisolone.

Animals↗
PubMed1988

Immunoidentification of Histoplasma capsulatum and Blastomyces dermatitidis with commercial exoantigen reagents. Effect of culture age.

We have studied the effect of the length of incubation on the reliability of commercial exoantigen test reagents (Nolan/Scott Biological Laboratories Inc, and Immuno-Mycologics Inc). Antigen extracts, tested in each commercial system, were prepared from duplicate sets of cultures of Histoplasma capsulatum (12 isolates) and of Blastomyces dermatitidis (11 isolates) after one to six weeks of growth. For isolates of H capsulatum, antigens necessary for immunoidentification were detected in most cultures in two weeks and in all cultures after four weeks of incubation for both Nolan and Immuno-Mycologics reagents, and continued to be detected for at least six weeks. Positive results could usually be obtained from exoantigen tests for H capsulatum before dimorphism could be demonstrated by conversion of the mold to yeast. Diagnostically significant antigen was not uniformly present in all cultures of B dermatitidis during the test period, but ten of 11 isolates were positive at some time during the six weeks using the Nolan reagents; antigen of only one of 11 isolates was detected by the Immuno-Mycologic reagents. Results of exoantigen tests for B dermatitidis were usually not available until after the identity had been confirmed by conversion of the mold to yeast phase on cottonseed agar.

Antigens, Fungal↗