Synergism of amphotericin B and 2-deoxyglucose against Histoplasma capsulatum yeasts in vitro.

The glucose analogue, 2-deoxyglucose (2DG), enhances both the fungistatic and the fungicidal action of amphotericin B in Fungizone (Squibb) against Histoplasma capsulatum yeasts in vitro. This synergistic effect is more pronounced when the test substances are incorporated in double-diffusion agar plates than in liquid medium. Minimum inhibitory concentrations for 2DG and amphotericin B in Fungizone have been established. The effects of components of Fungizone other than amphotericin B as clinically administered were also studied. Neither sodium desoxycholate nor phosphate buffer had any effect on the test organisms when used in recommended clinical concentrations. The 5% glucose infusion solution greatly enhanced the growth of the pathogen and markedly decreased the effectiveness of amphotericin B. H. capsulatum yeasts quickly became resistant to stepwise increases of Fungizone but not of 2DG. Susceptibility to amphotericin B and to 2DG increased with time within certain limits of exposure. The A (albino) phenotype of H. capsulatum is considerably more resistant to amphotericin B than the B (brown) phenotype, but there are no differences in susceptibilities to 2DG. The potential clinical applications of these studies are discussed, since experimental animals and man are reported to tolerate large amounts of 2DG. The incorporation of 2DG in the polyene antibiotic preparation would render it more effective at lower doses and would decrease clinical toxicity.     

Comparative study of the effect of thiabendazole and fungizone on Histoplasma capsulatum in macrophages.

The antifungal properties of Fungizone (amphotericin B intravenous solution) and thiabendazole on Histoplasma capsulatum within guinea pig macrophages were compared using the staining method and a newly developed plating method to determine the viability of intracellular H. capsulatum. The two methods were compared to determine the effectiveness of Fungizone and thiabendazole on H. capsulatum within macrophages. Fungizone was fungicidal for intracellular H. capsulatum, killing 99.9% of the yeasts at a concentration of 0.5 microgram/ml. There was some indication that non-viable intracellular yeasts were stained which could result in misinterpretation of the effectiveness of Fungizone using the staining method unless the yeasts are very closely examined for staining abnormalities. There was a good correlation between the two methods to demonstrate suppression of the multiplication of intracellular H. capsulatum in macrophages treated with 50 microgram/ml of thiabendazole. Thiabendazole was lethal for some intracellular H. capsulatum.     

Experimental histoplasmosis in mice treated with anti-murine interferon-gamma antibody and in interferon-gamma gene knockout mice

Histoplasma capsulatum is an important fungal pathogen in immunocompromised hosts, including AIDS patients. Experimental evidence suggests interferon-gamma (IFN) plays a role in host defense against H. capsulatum. In these studies we sought to demonstrate the importance of IFN in innate resistance to systemic histoplasmosis. The possible exacerbation of infection in BALB/c mice was assessed by administering 200 microg of hamster anti-IFN antibody prior to infection with H. capsulatum (2 x 10(6) yeasts, i.v.) and by comparing the severity of infection between BALB/c IFN gene knockout mice (GKO) and congenic control animals. In two separate studies, we found that anti-IFN treatment caused a dramatic loss of resistance to lethal infection and resulted in earlier mortality of IFN-depleted animals compared with normal IgG or no treatment (P<0.001). GKO mice were significantly (P<0.001) more susceptible to lethal infection than were control animals, and histological studies corroborated this. These studies clearly demonstrate that IFN is a vital part of the host's innate resistance to systemic infection with H. capsulatum and provide an additional rationale for studying IFN as an immunomodulatory therapeutic for the treatment of this disease.     

Detection of constitutive molecules on Histoplasma capsulatum yeasts through single chain variable antibody fragments displayed in M13 phages

A nonimmune library, containing single chain variable fragments (scFv) of immunoglobulin human genes displayed on the surface of M13 filamentous phages, was used to recognize molecules exposed on Histoplasma capsulatum yeasts' surface, during their growth in synthetic medium. The scFv clones were checked in their consistency by Dot-ELISA using HRP/anti-M13 conjugate, and they were tested to recognize molecules on H. Capsulatum yeasts' surface by ELISA in plates. Three out of 80 scFv cones (C2, C6, and C52) reacted consistently with H. capsulatum molecules, and they recognized molecules from both H. capsulatum morphologic phases. However, C6 and C52 clones reacted better with molecules on the surface of whole yeasts, with molecules from the yeasts' cell-wall extract, and with molecules released to the supernatant of the yeast culture. Mycelial supernatants from other fungi, as well as from a Mycobacterium filtrate, were not recognized by scFv phage monoclones. Monoclones C2, C6, and C52 recognized yeast molecules irrespective of the H. capsulatum strains used; the C6 clone revealed a specific immunohistochemistry reaction when tested against homologous and heterologous fungal infected tissues. The scFv clones isolated will be a useful toll to define the role of their target molecules in the host-parasite relationship of histoplasmosis.     

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.     

Antibodies in Histoplasmosis

Production of precipitating and complement-fixing antibody in rabbits and other animals was induced by immunization with live yeast-phase cells of Histoplasma capsulatum. Results of studies of polysaccharide antigens from three strains of H. capsulatum, by quantitative complement-fixation with human and rabbit antisera, strongly suggest the presence of type specificity. The variations of titer during 11 weeks in one patient with histoplasmosis and the variations of titer among a group of patients with histoplasmosis were studied by use of quantitative complement-fixation tests.     

Retention of albino and brown phenotypes of Histoplasma capsulatum by liquid nitrogren refrigeration.

Pathogenic differences between albino (A) and brown (B) phenotypes of Histoplasma capsulatum for experimental animals, and serological differences between A- and B- derived histoplasmins or yeast-phase antigens, necessitate the retention of these phenotypes in culture for diagnoses and epidemiologic studies. Selected strains of A and B types were frozen in liquid nitrogen (LN) and stored for 6-9 months in the vapor phase of a LN refrigerator (-165 degrees C). The strains were tested by using Berliner's method for differentiation of these two phenotypes. The results have proved that the LN refrigeration can be used for long-term conservation to prevent the conversion of B type into the A type in vitro.     

The Histoplasma capsulatum vacuolar ATPase is required for iron homeostasis, intracellular replication in macrophages and virulence in a murine model of histoplasmosis

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mphi). To identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human Mphi. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron-replete medium, but not on iron-deficient media. On iron-deficient medium, the growth of the vma1 mutant was restored in the presence of wild-type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within Mphi was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28 degrees C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mphi, grow on iron-poor medium and grow as a mold at 28 degrees C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis, whereas the vma1/VMA1 strain was as pathogenic as WT yeasts. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostasis and in fungal dimorphism.     

African histoplasmosis: a review

African histoplasmosis caused by Histoplasma capsulatum var. duboisii is an important deep mycosis endemic in Central and West Africa and in the island of Madagascar. The disease is characterized by presence of granulomatous lesions in the skin, subcutaneous tissues and bones. Lungs and other internal organs are rarely involved. The natural reservoir of the etiological agent has only been recently discovered in a bat cave in Nigeria. The status of asymptomatic infection is not certain. Investigations on skin and serum reactivity have suggested frequent prevalence of asymptomatic infections due to H. capsulatum var. duboisii among the residents in the vicinity of the cave microfocus of the fungus. The exact portal of entry into the body is not known, but inhalation into the lungs and direct inoculation in the skin have been incriminated. Laboratory diagnosis is confirmed by in vitro conversion into large yeast forms (8-15 mum in diameter) and by the demonstration of these forms within giant cells of tissues of experimentally infected animals There are no major clean-cut physiological differences between the two varieties, viz. capsulatum and duboisii. The cell wall of H. capsulatum var duboisii contains a glucan with beta 1-4 linkages in addition to a galactomannan shared with H. capsulatum var. capsulatum. Like the var. capsulatum var. duboisii has marked proteinase and collagenase activities in both mycelial and yeast forms, suggesting a possible pathogenic role for these enzymes. Both varieties have a common exoantigen. The yeast form of H. capsulatum var. duboisii contains the antigen found in the serotype 1,4 of var. capsulatum. A monoclonal antibody test has been developed that can recognize some epitopes in H. capsulatum var. capsulatum but not in the var. duboisii. There is need to develop specific serological diagnosis for the disease. Also there should be greater international awareness about African histoplasmosis. Amphotericin B and several antimycotic azoles like ketoconazole, itraconazole and fluconazole have been successfully employed for treatment.     

Deoxyribonucleic acid base composition of the yeastlike and mycelial phases of Histoplasma capsulatum and Blastomyces dermatitidis

The base composition in moles percent guanine plus cytosine (%GC) of both nuclear and mitochondrial deoxyribonucleic acid (DNA) isolated from the yeastlike and mycelial phases of the dimorphic fungal pathogens Histoplasma capsulatum and Blastomyces dermatitidis was determined by techniques of thermal denaturation and CsCl buoyant density gradient equilibrium centrifugation. The mean observed values for GC content of nuclear DNA from H. capsulatum and B. dermatitidis were 47.3 and 48.2%, respectively. What is speculated to be mitochondrial DNA was found to be 34.0% for H. capsulatum and 34.3% for B. dermatitidis. Thermal denaturation curves for Blastomyces DNA indicated a bimodality in thermal denaturation profiles, thereby suggesting a significant mitochondrial DNA contamination. Mitochondrial DNA appeared to represent a smaller percentage of the total DNA prepared from Histoplasma, and was not observed consistently to affect%GC values as determined by thermal denaturation profiles. On the basis of the now known perfect stage of B. dermatitidis (Ajellomyces dermatitidis) as a member of the family Gymnoascaceae, the close approximation of%GC content of nuclear DNA of this fungal organism with that of H. capsulatum suggests possible phylogenetic relationship. It is suggested that the just reported, but as yet unclassified, perfect stage of H. capsulatum may be found to be phylogenetically a primitive form of the Gymnoascaceae.     

EFFECT OF A SPERMATOGONIAL CHALONE ON THE GROWING RAT TESTIS

Immature rats were used in an experiment to test the possible influence of a spermatogonial chalone on the expanding spermatogonial population in their developing testes. An extract from adult rat testes was injected intraperitoneally into 33-day-old rats and control animals were injected with an equal amount of saline. Two groups of normal adult rats similarly injected with the testicular extract and saline solutions served as additional controls. Following these injections, all animals were administered a dose of ³H-thymidine 10 hr before sacrifice. An analysis of the labeling indices of the various types of spermatogonia revealed that in young rats injected with testicular extract the percentage of labeled type A spermatogonia was significantly lower than in control animals. In contrast, the labeling indices of Intermediate and type B spermatogonia were similar in the two groups of young rats. In the two groups of adult animals, there was no difference in the labeling indices of type A or of other types of spermatogonia. These data indicated that the saline extract of adult testes contained a substance, a spermatogonial chalone, inhibiting specifically the proliferation of some type A spermatogonia. The results also support the concept that a spermatogonial chalone may intervene, through its action on the spermatogonial stem cell population, to arrest the growth of the seminiferous tubules as the animal reaches maturity.     

Angiogenesis and its hormonal control in the corpus luteum of the pregnant rat

Beginning on Day 8 of pregnancy (Day 1 = sperm in vaginal smear), rats were injected i.p. with [3H] thymidine (TDR), killed 3 h later, and corpora lutea (CL) were dissected and saved for determining radioactivity in the acid-insoluble fraction or for autoradiography to determine labeling index (LI) of luteal and endothelial cells. An approximate doubling in DNA content in CL occurred between Days 13 and 14, with a high level maintained through Day 23. This was reflected in an abrupt increase in [3H] TDR incorporation on Day 13, with the peak reached on Day 14 and a subsequent decline to baseline values on Day 18. Autoradiography revealed that the LI of luteal endothelial cells rose from 2.1% on Day 12 to 10.0% on Day 14, and the LI of luteal cells correspondingly increased from 0.3% to 2.3%. Hypophysectomy (H) on Day 12 resulted, by Day 14, in no change in serum progesterone (P4) and TDR incorporation and LI of endothelial cells. However, after H and hysterectomy (HS) on Day 12, by Day 14, animals had low values for LI of endothelial and luteal cells, [3H] TDR incorporation and serum P4. After H + HS at Day 12, animals injected daily with estradiol cyclopentylpropionate (200 micrograms/day) on Days 12-14 had serum P4, [3H] TDR incorporation and LI of endothelial cells comparable to intact controls but not to luteal cells. However, similar treatment with testosterone cypionate (200 micrograms/day) or P4 (10 mg/day) did not maintain [3H] TDR incorporation or LI of either cell type, although serum P4 and estradiol levels were restored to normal values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monosaccharide and Chitin Content of Cell Walls of Histoplasma capsulatum and Blastomyces dermatitidis

Cell walls of Histoplasma capsulatum and Blastomyces dermatitidis, obtained by mechanical breakage of yeast- and mycelial-phase cultures, were lipid-extracted and then fractionated with ethylenediamine. Unextracted cell walls, lipid-extracted cell walls, and the three fractions resulting from ethylenediamine treatment were examined for monosaccharide and chitin content. The yeast-phase cell walls of five strains of H. capsulatum fell into two categories, designated chemotypes I and II, one of which, chemotype II, was similar to yeast-phase cell walls derived from three strains of B. dermatitidis. H. capsulatum chemotype I cell walls were characterized by lower content of material soluble in ethylenediamine, higher chitin content, and lower monosaccharide content than H. capsulatum chemotype II or B. dermatitidis cell walls. Approximately 80% of the monosaccharides of chemotype I cell walls was combined in forms susceptible to attack by mild acid hydrolysis, compared with about 50% of the monosaccharides of chemotype II and B. dermatitidis. H. capsulatum and B. dermatitidis yeast-phase cell walls could be distinguished, however, by their susceptibility to attack by a crude enzyme system derived from a Streptomyces sp. incubated with chitin as the only carbon source. Both glucose and acetylglucosamine were released from H. capsulatum cell walls, regardless of chemotype, during enzymatic hydrolysis, whereas only acetylglucosamine was released from B. dermatitidis yeast-phase cell walls. Mycelial-phase cell walls of H. capsulatum and B. dermatitidis were characterized by lower content of material soluble in ethylenediamine, higher proportions of mannose, and lower chitin content than their respective yeast phases. Glucose and acetylglucosamine were both released from all mycelial-phase cell walls, whether H. capsulatum or B. dermatitidis, by the crude enzyme system.     

Dimorphism in Histoplasma capsulatum: a model for the study of cell differentiation in pathogenic fungi.

Several fungi can assume either a filamentous or a unicellular morphology in response to changes in environmental conditions. This process, known as dimorphism, is a characteristic of several pathogenic fungi, e.g., Histoplasma capsulatum, Blastomyces dermatitidis, and Paracoccidioides brasiliensis, and appears to be directly related to adaptation from a saprobic to a parasitic existence. H. capsulatum is the most extensively studied of the dimorphic fungi, with a parasitic phase consisting of yeast cells and a saprobic mycelial phase. In culture, the transition of H. capsulatum from one phase to the other can be triggered reversibly by shifting the temperature of incubation between 25 degrees C (mycelia) and 37 degrees C (yeast phase). Mycelia are found in soil and never in infected tissue, in contrast to the yeast phase, which is the only form present in patients. The temperature-induced phase transition and the events in establishment of the disease state are very likely to be intimately related. Furthermore, the temperature-induced phase transition implies that each growth phase is an adaptation to two critically different environments. A fundamental question concerning dimorphism is the nature of the signal(s) that responds to temperature shifts. So far, both the responding cell component(s) and the mechanism(s) remain unclear. This review describes the work done in the last several years at the biochemical and molecular levels on the mechanisms involved in the mycelium to yeast phase transition and speculates on possible models of regulation of morphogenesis in dimorphic pathogenic fungi.     

Evaluation of Systemic Antifungal Agents in X-Irradiated Mice

The effect of X irradiation on the survival time of animals experimentally infected with pathogenic fungi was studied, and the activity of antifungal agents in pre-irradiated hosts was evaluated. A 24-hr preinfection dose of X irradiation decreased the survival time of mice infected with Cryptococcus neoformans and Histoplasma capsulatum to a greater extent than Candida albicans or Blastomyces dermatitidis infections. Exposure to 400 r caused a significant reduction in the variation (S(2)) survival time of C. albicans or H. capsulatum mouse infections. A single 100-mg/kg dose of 5-fluorocytosine or amphotericin B administered within 24 hr postinfection significantly extended the survival time of mice infected with C. albicans. Delayed treatment with amphotericin B was effective against C. neoformans infections. Four 50-mg/kg doses of 5-fluorocytosine were more effective than a single 200-mg/kg dose against C. neoformans infections. A single dose of amphotericin B provided significant protection when administered 48 hr postinfection against B. dermatitidis in preirradiated mice. A single dose of saramycetin 48 hr postinfection was highly effective against H. capsulatum mouse infections. A 100-mg/kg dose of amphotericin B was only effective against this fungal pathogen when administered within 8 hr postinfection. In vivo activity of the antifungal agents studied was detected within 8 to 14 days. The relative in vivo activity of several antifungal agents indicated the importance of considering their individual pharmacological properties for optimum effectiveness. The experimental model used in this study should be useful for the detection and for the preclinical evaluation of new antifungal agents.     

Basophil-sensitizing antibody response to herpes simplex viruses in rabbits.

Antibodies to herpes simplex virus type 1 and type 2 were detected in the sera of rabbits by release of histamine from basophils sensitized in vitro with the sera. The time course of the appearance of the antibodies, the dose-response curve of the release of histamine in relation to antigen concentration, the sedimentation characteristics of the antibodies in sucrose gradients, and the ability to destroy the sensitizing capacity of the sera with heat suggest that the antibodies being assessed were of the IgE class. These antibodies were induced in animals injected intradermally, intramuscularly, and i.p. with live virus. The antibodies were detected 1 week after primary injection and a similar time course of antibody appearance was observed after a second or third injection. The same cross-reactivity between type 1 and type 2 virus observed with IgG antibody was also observed with IgE antibody.     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

Development and Use of a Polyvalent Conjugate to Differentiate Histoplasma capsulatum and Histoplasma duboisii from Other Pathogens

Previous investigations have demonstrated the existence of five Histoplasma capsulatum serotypes. Available specific fluorescent-antibody reagents stain only four of the five serotypes. Antibodies produced against the most complete H. capsulatum serotype were labeled with fluorescein isothiocyanate to develop a reagent specific for H. capsulatum that was reactive with all the known serotypes. The unadsorbed reagent not only stained all the H. capsulatum serotypes, but it also stained cultures of Blastomyces dermatitidis, H. duboisii, several Candida species, and a variety of other fungi. Adsorption of the conjugate with antigens of C. albicans produced a reagent that intensely stained only H. capsulatum, H. duboisii, and B. dermatitidis. Differentiation of B. dermatitidis from the Histoplasma species was accomplished by application of a B. dermatitidis specific fluorescent antibody to antigens positive with the H. capsulatum reagent. At present, differentiation of H. capsulatum from H. duboisii may be accomplished only by animal inoculation. Our data substantiate the antigenic relationships hypothesized earlier, and they indicate that H. capsulatum shares at least two antigens with the other fungi that were studied.