BAD1, an essential virulence factor of Blastomyces dermatitidis, suppresses host TNF-alpha production through TGF-beta-dependent and -independent mechanisms.
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We investigated how BAD1, an adhesin and virulence factor of Blastomyces dermatitidis, suppresses phagocyte proinflammatory responses. Wild-type yeast cocultured with murine neutrophils or macrophages prompted release of a soluble factor into conditioned supernatant that abolished TNF-alpha production in response to the fungus; isogenic, attenuated BAD1 knockout yeast did not have this effect. Phagocytes released 4- to 5-fold more TGF-beta in vitro in response to wild-type yeast vs BAD1 knockout yeast. Treatment of inhibitory, conditioned supernatant with anti-TGF-beta mAb neutralized detectable TGF-beta and restored phagocyte TNF-alpha production. Similarly, addition of anti-TGF-beta mAb into cultures of phagocytes and wild-type yeast reversed BAD1 inhibition of TNF-alpha production. Conversely, TGF-beta treatment of phagocytes cultured with knockout yeast suppressed TNF-alpha production. Hence, TGF-beta mediates BAD1 suppression of TNF-alpha by wild-type B. dermatitidis cultured in vitro with phagocytes. In contrast to these findings, neutralization of elevated TGF-beta levels during experimental pulmonary blastomycosis did not restore BAD1-suppressed TNF-alpha levels in the lung or ameliorate disease. Soluble BAD1 was found to accumulate in the alveoli of infected mice at levels that suppressed TNF-alpha production by phagocytes. However, in contrast to yeast cell surface BAD1, which induced TGF-beta, soluble BAD1 failed to do so and TNF-alpha suppression mediated by soluble BAD1 was unaffected by neutralization of TGF-beta. Thus, BAD1 of B. dermatitidis induces suppression of TNF-alpha and progressive infection by both TGF-beta-dependent and -independent mechanisms. (+info)
Rapid conversion of Histoplasma capsulatum, Blastomyces dermatitidis and sporothrix schenckii in tissue culture.
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A simple method for positive identification of Histoplasma capsulatum, Blastomyces dermatitidis, and Sporothrix schenckii is given. Primary tissue cultures of guinea pig peritoneal macrophage were inoculated with the mycelial phase of each organism and after 24 h the cells were stained and observed microscopically. The characteristic yeast phase could then be observed allowing for positive identification. (+info)
Delayed diagnosis of osseous blastomycosis in two patients following environmental exposure in nonendemic areas.
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Blastomycosis generally results from inhalation of Blastomyces dermatitidis conidia following exposure to contaminated soil in an endemic area. Primary infections commonly involve the lungs, although secondary dissemination to other body sites may occur. We describe 2 cases of osseous blastomycosis in people living outside the endemic areas. Both patients reported exposure to soilfollowing injury to the knee from occupational activities. Mold isolated from each case was identified as B dermatitidis by micromorphologic characteristics including yeast conversion testing and by a positive AccuProbe Blastomyces dermatitidis test (GenProbe, San Diego, CA). Retrospective review of histologic slides, initially reported as negative, identified rare poorly staining, broad-based budding yeast forms in each case. Both patients were treated successfully with itraconazole with no evidence of recurrent infection after 1 year These cases illustrate the importance of considering blastomycosis in the differential diagnosis of bony lesions, even though the patient may live outside an endemic area for B dermatitidis. (+info)
Rapid methods for identification of yeasts.
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Opportunistic infections by yeasts have been implicated as one of the major causes of complications in the compromised patient. Rapid recognition and identification of these yeasts is essential for patient management, but conventional liquid medium methods for completing identification tests are cumbersome and time consuming. Rapid tests have been devised based on modifications of methods commonly used in bacteriology. These rapid methods included tests for carbohydrate and nitrate assimilation, fermentation, and urease production. These were compared with several current methods for accuracy of results, for time to final identification, and for economy of time and reagents. In addition, the usual tests for pseudogerm tube formation, for production of hyphae or pseudohyphae, and for growth temperatures were included. The rapid tests achieved 96% or better accuracy compared with expected results, and 46 species of yeasts were identified in 1 to 2 days compared with the 10 to 14 days required by conventional liquid culture methods. (+info)
Requisite elements in vaccine immunity to Blastomyces dermatitidis: plasticity uncovers vaccine potential in immune-deficient hosts.
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Understanding fundamental mechanisms of vaccine immunity will allow proper use and optimization of vaccines. Vaccination with a genetically engineered, live, attenuated strain of Blastomyces dermatitidis carrying a targeted deletion at the BAD1 locus confers sterilizing immunity against experimental lethal pulmonary infection. We found in this study that alphabeta T cells are requisite for durable vaccine immunity, whereas other T and B cells are dispensable. In immune-competent animals, CD4(+) T-cell derived cytokines TNF-alpha and IFN-gamma mediate vaccine immunity. Surprisingly, these factors are dispensable in immune-deficient animals, which rely on alternate mechanisms for robust vaccine immunity, yet still require O(2)(-) production rather than generation of NO. Our results clarify the cellular and molecular bases behind the first genetically engineered fungal vaccine. They also illustrate a sharp difference in vaccine mechanisms between immune-competent and immune-deficient hosts, which underscores the plasticity of residual immune elements in compromised hosts, and points to the feasibility of developing vaccines against invasive fungal infection in this fast growing patient population. (+info)
Nested PCR assays for detection of Blastomyces dermatitidis DNA in paraffin-embedded canine tissue.
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A Blastomyces dermatitidis nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family ONYGENACEAE: We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively. The B. dermatitidis PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy. Sequencing revealed that all PCR products were homologous to the B. dermatitidis WI-1 adhesin gene. No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma. The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively. Only six products were identified as being identical to B. dermatitidis 18S rDNA; they were exclusively obtained from specimens positive by the B. dermatitidis nested PCR. For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific H. capsulatum product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the B. dermatitidis PCR assay. In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of B. dermatitidis in canine tissue. (+info)
Preparative isotachophoretic separation of skin test antigens from blastomycin purified derivative.
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This investigation examined the theoretical and practical parameters requisite to preparatory istachophoretic separation of blastomycin purified derivative. It resulted in a relatively simple, two-step procedure for preparation of blastomycin antigens in milligram quantitites that exhibited sensitivity and specificity in experimentally infected guinea pigs. Analysis of the nine isotachophoretic fractions for skin test sensitivity and specificity provided some insight into the generally accepted unreliability of blastomycin when used for immunological evaluation. (+info)
Vaccine immunity to pathogenic fungi overcomes the requirement for CD4 help in exogenous antigen presentation to CD8+ T cells: implications for vaccine development in immune-deficient hosts.
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Systemic fungal infections with primary and opportunistic pathogens have become increasingly common and represent a growing health menace in patients with AIDS and other immune deficiencies. T lymphocyte immunity, in particular the CD4+ Th 1 cells, is considered the main defense against these pathogens, and their absence is associated with increased susceptibility. It would seem illogical then to propose vaccinating these vulnerable patients against fungal infections. We report here that CD4+ T cells are dispensable for vaccine-induced resistance against experimental fungal pulmonary infections with two agents, Blastomyces dermatitidis an extracellular pathogen, and Histoplasma capsulatum a facultative intracellular pathogen. In the absence of T helper cells, exogenous fungal antigens activated memory CD8+ cells in a major histocompatibility complex class I-restricted manner and CD8+ T cell-derived cytokines tumor necrosis factor alpha, interferon gamma, and granulocyte/macrophage colony-stimulating factor-mediated durable vaccine immunity. CD8+ T cells could also rely on alternate mechanisms for robust vaccine immunity, in the absence of some of these factors. Our results demonstrate an unexpected plasticity of immunity in compromised hosts at both the cellular and molecular level and point to the feasibility of developing vaccines against invasive fungal infections in patients with severe immune deficiencies, including those with few or no CD4+ T cells. (+info)