Antigens from Histoplasma capsulatum and Blastomyces dermatitidis

Certain groups of fungi share chemical structures which makes difficult the isolation and differentiation of specific antigens by the usual methods of extraction and purification. Therefore, we have oriented our studies to the immunological and biochemical characterization of differences and similarities of molecular structures from fungi, etiologic agents of systemic mycoses, hoping to establish criteria for the utilization and handling of these antigens.A deproteinized polysaccharide-protein complex (D-PPC) was isolated from Histoplasma capsulatum and Blastomyces dermatitidis. The immunological studies with humoral tests indicate a variable cross reaction between antigens of both species. In immunodiffussion systems, the reaction was specific for each species using saline solution or phosphate buffer solution, while using an agarose veronal system, the cross reactions were very evident. In addition, differences in cross reactions were obtained with immunoelectrophoresis, haemagglutination and complement fixation microtest. This variation in cross reaction responses suggest that these antigens (D-PPC) share common structures but at the same time must have some different component owned by each one of the fungi which makes them more specific than crude antigens.     

Antigens and chemical composition of Blastomyces dermatitidis

Without these tools (skin-test and complement fixation antigens for epidemiological, diagnostic, and prognostic use) we are at least 50 years behind in our defining of the disease blastomycosis (63). This statement by Rippon et al. emphasizes the need for a well defined antigen or group of antigens from Blastomyces dermatitidis, not only for the two tests named above, but for any serological or immunological test. Several different preparations have been reviewed which are beginning to approach the quality necessary for such a sensitive and specific antigenic tool. All of these require further characterization and, in most instances, purification.One purified fraction isolated from blastomycin, the F fraction, has shown exceptional promise as a skin-test agent in guinea pigs but has not been further studied. A 10–30K molecular weight fraction of the yeast cytoplasm has shown good reactivity, and contains a protein shown by PAGE to be common to several skin-reactive preparations. This fraction has not been further purified.An ASWS preparation has been partially characterized and shown to be especially sensitive and specific in animals, though not yet in humans. Purification of this fraction by PAGE has uncovered a highly reactive protein which may be related to one isolated from the cytoplasm. Investigations using this purified component have not been attempted in humans.The A antigen has been well characterized regarding its applicability to human diagnosis. It has been partially purified and two of its components associated with its reactivity. It also has probably the best possibility as an immediate serologic tool. Even here, though, current preparations contain much extraneous material which could conceivably create cross-reactivity problems in the future.The ethanol-precipitate antigens which have shown such superior results in the past have not been employed recently, nor have they been extensively characterized. This fraction may contain the reactive A antigen or other antigens deserving of study. Hopefully, the use of this technique can be encouraged as one starting point for isolation procedures.The importance and isolation of enzymes and mannose containing antigens have probably not received adequate attention. An almost uniform identification of mannose in reactive preparations argues for purification procedures based on its presence.Finally, use of hybridoma technology to produce antibodies specific for antigens of B. dermatitidis promises to improve our understanding of the organism and to help isolate purer antigenic fractions.The search for antigens of importance in the immunology of B. dermatitidis should not be confined to any one or all of the antigens discussed above. A variety of components will likely be necessary for complete understanding of the disease and for diagnostic use. It has been observed that information of clinical value can be obtained from immunological reactions to several different antigens (35), thereby encouraging continuous efforts to obtain as many as possible.     

Blastomyces cerolytica (sp. n.) and its relation to Coccidioides

Summary In 1949 I discovered a yeast like fungus which was termedBlastomyces cerolytica. During the essay for exact determination of this fungus, it was found that it resemblesCoccidioides. Its yeast-like form is zymatic, malto-saccharasic. As organs of fructification aleurospores and chlamydospores were found, as well as asci containing hundreds af ascospores. It shows isogame heterothallism. It belongs to Taphrinales. A comparison between characteristics ofCoccidioides immitis andBlastomyces cerolytica is given. The diagnosis of this fungus concludes the investigation.

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Resumé En 1949 nous avons découvert un champignon levuriforme, lequel nous avons nomméBlastomyces cerolytique. Il nous a paru d'être très semblable au genreCoccidioides. Son état levuriforme se comporte comme une levure zymatique et malto-saccharasique. Les formes asexuelles sont des aleurospores et des chlamydospores et les formes sexuelles des asques géants remplis de centaines d'ascospores. Il paraît être isogame hétérothallique. Il appartient à des Taphrinales. Une comparaison était mise en vue des caractéristiques deCoccidioides immitis et deBlastomyces cerolytique. Nous donnons aussi une diagnose pour ce champignon.

     

Electronmicroscopy of self-parasitism by Histoplasma capsulatum and Blastomyces dermatitidis

Intrahyphal as well as intrayeast hyphae were demonstrated by electron-microscopy in bothHistoplasma capsulatum andBlastomyces dermatitidis yeastlike and mycelial phase cells grown in agitated liquid media. These structures appeared to be rather common in cultures of yeastlike cells induced to convert to the mycelial phase. In many cases there was excellent preservation of ultrastructural detail of the parasitized cell which suggested that the cell may still be viable.

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Zusammenfassung InHistoplasma capsulatum so wie auch inBlastomyces dermatitidis wurden in Hyphen und Zellen der Hefe- so wie auch der Mycelphase intracellular Hyphen durch Elektronmikrokopie nachgewiesen, wenn sie in flüssigen Schüttelkulturen gezüchtet worden sind. Diese Strukturen waren in Zellen der Hefephase, die zur Überleitung in die Myzelphase angeregt worden sind, sehr häufig. In vielen Fällen war eine sehr gute Bewahrung der ultrastrukturalen Einzelheiten der parasitierten Zelle, die es nahegelegt hat, dass die Zelle noch immer lebendig ist.

     

Preparation and standardization of antigens of Histoplasma capsulatum and Coccidioides immitis

Methods have been developed for the separation and purification of various antigenically active cellular components ofH. capsulatum (34) andC. immitis (35), and chemical procedures have been employed in the characterization of these antigens. The concluding remarks illustrate the current trends in immunological studies ofH. capsulatum andC. immitis. Hopefully, these approaches will provide knowledge and insight into the basic biological properties ofH. capsulatum andC. immitis and should, in the future, culminate in the physicochemical characterization of their important antigens.     

Ultrastructural Changes During the Yeastlike to Mycelial-Phase Conversion of Blastomyces dermatitidis and Histoplasma capsulatum

Fine details of the sequential anatomical events occurring during yeast to mold morphogenesis of the dimorphic fungal pathogens Blastomyces dermatitidis and Histoplasma capsulatum as seen in ultrathin sections are described and illustrated by electron micrographs. Discrete intracytoplasmic membrane systems intimately associated with the plasma membrane were observed to be formed within 6 to 8 hr after induction of the conversion process. Within 12 to 18 hr, an intermediate or transitional cell with Woronin bodies at the septum was formed from the converting yeastlike cell. Both cells were noted to contain increased numbers of mitochondria. At approximately 48 hr from the initial induction of the conversion stimuli, the newly forming hyphal cells were observed to produce postconversional intracytoplasmic membrane systems seen normally in the ultrastructural organization of the fully established mycelial-phase cell. These membrane systems appear to be associated with normal septal formation. Although minor variations of time were observed in the occurrence of the sequential events, it is suggested that yeastlike to mycelial-phase conversion of these two fungal pathogens proceeds via a similar mechanism of ultrastructural reorganization.     

Fluorescent antibody techniques for identification of Coccidioides immitis and Histoplasma capsulatum

This is a review of practical uses of immunofluorescence in detection of the two fungi in host and environment and in identification of their cultures, as well as in serologic case finding. Reagents directed at the yeast phase ofHistoplasma capsulatum have been fairly successful in differentiating this species from others, the main difficulty being the tendency to cross-react withBlastomyces dermatitidis andH. duboisii. Conjugates for the mycelial phase ofH. capsulatum tend to cross-react withSepedonium andChrysosporium, but careful absorption may yield specific reagents. Anti-yeast-phase conjugates are a valuable adjunct to cultural and clinical methods when used to detect and identifyH. capsulatum in sputum and other clinical specimens. Conjugates specific for the spherules or tissue phase ofCoccidioides immitis have yielded false negative results when applied to clinical specimens. The fluorescent-inhibition procedure is useful for serologic case finding in histoplasmosis and the same technique has shown fairly good agreement in coccidioidomycosis with complement-fixation and tube-precipitin methods. Immunofluorescence reagents for the two species have been useful in screening surgical and autopsy specimens, animal tissues, and soils.Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).     

Serological Comparison of Spherules and Arthrospores of Coccidioides immitis

Spherule and arthrospore cellular preparations were sonic-treated and separated into their respective supernatant and sediment components. Complement-fixation tests with antispherule and antiarthrospore pooled rabbit sera revealed that the soluble antigens exhibited more serological activity than the sediment preparations. After autoclaving, an arthrospore cellular antigen exhibited increased activity with either antisera, whereas autoclaved spherules exhibited increased activity only with antispherule serum. Complement-fixation tests with coccicioidin and spherule culture supernatant preparations revealed quantitative or qualitative differences in antigenic determinants between these two morphological phases of Coccidioides immitis.     

恒河猴STLV-1血清抗体流行病学研究

目的调查我国恒河猴STLV-1流行病学特征.方法用免疫荧光法和蛋白印迹法对537只野捕恒河猴,365只繁殖猴和44只D型逆转录病毒抗体阳性猴作了STLV-1血清抗体检查.结果 10岁以上恒河猴的STLV-1抗体阳性率(15.5%)高于10岁以下的恒河猴(6.2%);雌猴的STLV-1抗体阳性率(17.5%)高于雄猴(5%);D型逆转录病毒抗体阳性猴群的STLV-1抗体阳性率(34.1%)高于野捕猴群(8.8%).结论在我国的野生和饲养恒河猴群中广泛存在STVL-1血清抗体阳性动物,其抗体阳性率与动物的年龄和性别有关.     

TALEN-Mediated Gene Mutagenesis in Rhesus and Cynomolgus Monkeys

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Non-specific incorporation of nucleic acid precursors in Blastomyces dermatitidis and Histoplasma capsulatum

Incorporation of thymidine, thymidine monophosphate (TMP), thymidine triphosphate (TTP), uridine and orotic acid into DNA, RNA and protein in Blastomyces dermatitidis and Histoplasma capsulatum was studied utilizing a specific acid hydrolysis technique developed for these fungi. Thymidine was incorporated to the greatest extent (approximately 0.5 % of added label) followed by uridine, orotic acid, TMP and TTP. In Blastomyces, uridine and orotic acid labeled primarily RNA. TMP and TTP labeled RNA, DNA and protein at nearly the same level. In Histoplasma RNA was labeled poorly by any of these precursors. TMP and TTP labeled DNA predominately and protein to a slightly lower level. Deoxyadenosine or uridine media supplements of 250 g/ml did not enhance incorporation. All precursors tested were found to be nonspecific in that RNA, DNA and protein were labeled. All data indicate that neither RNA nor DNA synthesis can be specifically measured in whole cells or acid precipitates by any of these precursors. Specific radiometric monitoring with these isotopes therefore requires the separation of these macromolecules.     

Inhibition of Histoplasma capsulatum and Blastomyces dermatitidis by Pseudomonas aeruginosa in vitro

Summary It was accidentally discovered thatPseudomonas aeruginosa inhibited the growth ofHistoplasma capsulatum when both of the organisms were isolated from a series of sputum specimens obtained from a single patient. Experiments were performed to determine if there is any inhibitory effect on other systemic fungi.Three fractions were obtained from a broth culture ofP. aeruginosa and their antibiotic effects tested against various systemic fungi and bacteria.Reviewed in the Veterans Administration and published with the approval of the Chief Medical Director. The statements and conclusions published by the author are the results of his own study and do not necessarily reflect the opinion of the Veterans Administration.     

Play Between Adult Male and Infant Rhesus Monkeys

Play among four pairs of adult male and infant rhesus monkeysis described and compared to mother-infant and peer play. Datawere collected while the adult males reared the infants fora period of 7 months in the absence of mothers and peers. Typesof play were categorized as solitary, parallel, and interactive.Sex differences in play patterns are described, as are antecedentand consequent events. Adult male-infant play was found to beof much greater intensity and mean duration than mother-infantplay.     

RT-SHIV感染中国恒河猴及体内传代

目的了解RT-SHIV感染中国恒河猴的感染特点,研究RT-SHIV在中国恒河猴中传代特点;建立RT-SHIV中国恒河猴动物模型,为评价HIV-1药物有效性提供动物平台。方法选择4只健康恒河猴,其中两只动物经上肢静脉感染RT-SHIV病毒,感染急性期采取外周血分离CD8-PBMC,扩增病毒,将新制备的病毒静脉感染另外两只中国恒河猴,通过监测血浆病毒载量,CD4＋/CD8＋比值,CD4＋T淋巴细胞和B淋巴细胞的绝对数,了解实验猴的感染状态,同时分析病毒RT基因变异情况。结果 4只动物均获得系统性感染,且传代动物急性期表现更为强烈,RT基因在感染和传代的过程中共观察到3个氨基酸的改变。结论本研究为RT-SHIV中国恒河猴模型的建立提供了基础信息。     

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.