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.

     

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.     

Investigation on dimorphism of Blastomyces dermatitidis by agar-implantation method

By the agar-implantation developed by the authors the process of conversion on Blastomyces dermatitidis from mycelial phase to yeast phase was observed.First of all slide cultures of the fungus were prepared at room temperature, Upon confirmation of good hyphal growth, a cover glass was removed and a part of medium was cut out in a square of about 3 mm a side.After mice were laparotomied, each agar block cut out was implanted in the peritoneal cavity of mouse. The mice implanted with the agar blocks were killed, two each, every day for 14 days, and thereafter at intervals of a week for 2 months. Therefore, the implanted agar blocks were all recovered. They were examined directly by a light microscope with histopathological and electron microscopic examinations carried out at the same time.Within the peritoneal cavity of mouse, the intercalary and terminal chlamydospores were formed from hyphae. These subsequently swelled to become yeastlike cells and proliferated thereafter by budding.     

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.     

Ultrastructural aspects of cytoplasmic ribosomes from Histoplasma capsulatum and Blastomyces dermatitidis as revealed by heavy metal staining

Cytoplasmic ribosomes were isolated and purified from sonicates of the mycelial and yeastlike growth forms of the pathogenic dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Similar ribosomal fractions were prepared from Neurospora crassa and Saccharomyces cerevisiae. These latter organisms were selected as typical filamentous and yeastlike monophasic fungi, and their ribosomes were used as reference standards. High resolution electron microscopy permitted a comparison of both positively and negatively-stained ribosomes to those dehydrated without heavy metal salt. Such studies revealed statistically significant differences in physical dimensions. Cautious interpretations of substructural detail of the various ribosomal preparations suggested both interphasic and interspecies differences.     

Antigenic Relationships of 14 Treponemes Demonstrated by Immunofluorescence

Immunofluorescence was used as an aid in the antigenic grouping of 14 cultivable treponemes. Antisera were prepared versus each treponemal strain, and the antiglobulins were conjugated with fluorescein isothiocyanate. A common antigen-antibody system, detected in the strains studied, was removed by absorption of each conjugate with Reiter or Borrelia vincentii treponemes. Thus, five categories based on shared group-specific antigens were revealed. Serogroup I: Reiter, English Reiter, Kazan, Kazan numbers 2, 4, 5, and 8. Serogroup II: Nichols and Noguchi. Serogroup III: three oral treponemes, MRB, FM, and N-39. Serogroup IV: B. vincentii. Serogroup V: Treponema zuelzerae. The five serogroups apparently are related by an immunofluorescent common antigen.     

Calcium binding by the essential virulence factor BAD-1 of Blastomyces dermatitidis

BAD-1 (Blastomyces adhesin 1), a 120-kDa protein of Blastomyces dermatitidis, functions as an adhesin, immune modulator, and essential virulence factor. Structurally, BAD-1 is composed of a short N-terminal region, a core of 30 tandem repeats critical for virulence, and a C-terminal epidermal growth factor domain that binds the protein to yeast cell surface chitin. Each of the 30 acidic residue-rich tandem repeats contains a sequence that resembles the calcium-binding loop of the EF-hand domain found in many calcium-binding proteins. Here, we investigated the binding of calcium by BAD-1 and its biological significance. Yeast washed with double distilled H2O released surface-bound BAD-1, but EGTA washes were an order of magnitude more efficient, suggesting an interaction between BAD-1 and calcium. Immobilized BAD-1 was stained with ruthenium red dye, an indicator of calcium-binding proteins. In equilibrium dialysis, BAD-1 bound 45Ca2+ with an affinity of 0.41 x 10(-5) m and a capacity of 27 calcium/mol. Mass spectrometry confirmed this capacity. Elevated [Ca2+] diminished BAD-1 solubility. Upon deletion of its C-terminal epidermal growth factor-like domain, BAD-1 resisted aggregation by elevated [Ca2+] but retained its affinity and capacity for calcium. Removing 20 copies of the tandem repeat, however, sharply reduced the capacity of BAD-1 for calcium. Growth of the bad-1 null yeast was inhibited by 5 mm EGTA, and re-expression of BAD-1 in trans or the addition of exogenous purified BAD-1 restored growth. Thus, BAD-1 is a high capacity calcium-binding protein. This property contributes to the structure and function of BAD-1, as well as to B. dermatitidis acquisition of calcium from the environment.     

In Vitro and In Vivo Activity of Hamycin Against Blastomyces dermatitidis

Clinical responses of patients with blastomycosis to treatment with hamycin have been variable. An explanation for this was sought in a series of studies in which in vitro and in vivo susceptibilities to hamycin of five strains of Blastomyces dermatitidis were compared. Minimal inhibitory concentrations of hamycin for the five strains indicated uniformly high levels of in vitro susceptibility (0.008 to 0.016 μg/ml). In vivo activity was measured in infected mice treated intraperitoneally for a period of 28 days with doses of the drug ranging from 0.001 to 0.030 mg per mouse. Significant differences in response to treatment among the five strains were noted (P < 0.001), and protective doses were found to vary from 0.001 to >0.030 mg per mouse per day. Further observations of infected mice after treatment revealed marked rates of relapsing infection, and several strains caused death. Persistent inapparent infections were also detected on culture of selected organs. Toxicity due to hamycin alone was not observed. These results suggest that variations in clinical responses to hamycin therapy in treatment of blastomycosis reflect differences in pathogenesis and host response in vivo to the infecting organism rather than differences in susceptibility of B. dermatitidis to hamycin.     

Chitin synthetase from the yeast and mycelial phases of Blastomyces dermatitidis

Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 ° C; pH optimum was 7–7.5; Mg⁺² optimum was 5–10 mM.The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment.     

用免疫荧光单克隆抗体对脊髓灰质炎病毒抗原表位的分析

用间接免疫荧光与中和试验筛选出来的抗脊髓灰质炎2型与3型不同毒株的12个单克隆抗体,其中3个仅有免疫荧光活性,9个具有中和与免疫荧光活性。用免疫荧光活性的单克隆抗体进行试验,发现它们在识别特异性抗原表位方面与中和性单克隆抗体相似,显示出株特异的、几个毒株共同特异的或型特异的抗原表位。根据表位分布关系及特征,可以用来鉴别型内毒株的特征、毒株的抗原分析、抗原变异的研究以及疫苗相关病例的鉴别。所得结果与中和性单克抗隆抗体及T1-寡核苷酸指纹图谱分析一致。而免疫荧光单克隆抗体识别抗原表位的活性范围比中和性单克隆抗体更广。另外还发现某些兼有荧光与中和两活性的同一个单克隆抗体,用不同方法(IF与NT)进行试验时,与相同毒株出现不同表位反应,这点是值得引起注意需待进一步证实的重要问题。     

Blastomyces dermatitidis Antibody and Antigen Detection: Comparison of Four Lysate Antigens and Antibodies Prepared from Human Isolates from a Blastomycosis Outbreak

Mycopathologia - Blastomycosis is a systemic fungal disease of humans and other animals produced by the thermally dimorphic fungal organism, Blastomyces dermatitidis. Recent studies have focused on...     

鸡传染性支气管炎病毒广西分离毒株抗原相关性的研究

本研究将26个传染性支气管炎病毒(Infectious bronchitis virus,IBV)广西分离毒株以及参考株M41和常用疫苗株H120、Ma5和4/91共30个毒株,分别与根据这些毒株S1基因高变区Ⅰ的基因分型结果而选取的属于3个不同亚群的7个代表性分离毒株和常用疫苗株H120、Ma5和4/91制备的共10个单因子血清,在鸡胚气管环培养(TOC)上进行病毒中和试验,然后根据中和试验结果对1985～2008年间课题组所分离的26个IBV广西地方流行毒株与3个常用疫苗株H120、Ma5和4/91以及参考毒株M41的抗原相关性及其血清型进行分析。结果显示,30个试验的毒株分属7个不同的血清型,其中26个分离株有两个优势血清型(占总分离株的68%),分别是血清1型(包含13个毒株)和血清2型(包含5个毒株)。此外,我们还将分离毒株的血清分型结果与重要抗原基因(包括S1、N、M和3′UTR)的分型结果之间的关系进行了比较,发现它们之间的分型结果不尽相同。研究结果表明,近年来广西存在多个血清型IBV的流行而且不同时期流行的优势血清型不同,分离毒株之间以及分离毒株与疫苗毒株之间的抗原相关性也存在着差异。     

Cell Wall Composition of the Yeastlike and Mycelial Forms of Blastomyces dermatitidis

The thermally induced changes in the cell wall polysaccharides of Blastomyces dermatitidis strain BD64, which produces a yeastlike form (Y form) at 37 C and a mycelial form (M form) at 20 C, were examined. The cell walls of the Y and M forms contained 36 and 51% of hexoses, respectively. The M-form cell wall contained glucose, galactose, and mannose in a molar ratio of 1:0.1:0.2. The Y-form cell wall contained mainly glucose and a very small amount of galactose and mannose. The glucans of the cell wall of the Y form consisted of about 95% alpha-glucan and 5% beta-glucan, whereas those of the M-form cell wall consisted of about 60% alpha-glucan and 40% beta-glucan.     

Live Blastomyces dermatitidis yeast-induced responses of immune and nonimmune human mononuclear cells

Peripheral blood mononuclear cells from humans with treated blastomycosis or from normal persons were cultured with live Blastomyces dermatitidis yeast. There was no inhibition of growth of the fungus in this suspension culture technique but morphologic and functional differences of the human cells were great between the two groups. Lymphocyte stimulation by live Blastomyces yeast was found in the patient group but not in the normal donors. These events add to the observations that cellular immunity is expressed in blastomycosis.     

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.     

Lack of a Close Relationship Between Three Strains of Human Rhinoviruses as Determined by Their RNA Sequences

The possible genomic homologies between three serotypes of human rhinoviruses (HRV 1A, HRV 2, and HRV 14) were investigated. First we confirmed that these viruses were unrelated by the criterion of the absence of common antigenic determinants on the surfaces of the native virions, as detected by cross-neutralization of complementfixation. RNA-RNA hybridization was then examined with purified, highly radioactive, double-stranded, replicative-form RNA and excess single-stranded virion RNA. Single-stranded RNA showed 100% homology with the minus strand from the replicative-form RNA of the same type of virus. HRV 1A, HRV 2, and HRV 14 showed low intertypic homologies; these were not significantly greater than those found between the rhinoviruses and polivirus, which were used as a negative control. The immunological relationship and the RNA homology between HRV 1A and HRV 1B were also examined by the above techniques. It was confirmed that HRV 1A and HRV 1B share some surface determinants and it was also found that HRV 1B RNA shares 70% homology with HRV 1A RNA.     

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.