Dermatitidis Yeast Research Articles

Induction and detection of cell-mediated reactions with different Blastomyces dermatitidis antigenic preparations.

Blastomyces dermatitidis yeast-phase antigens (killed cell, KWY; lysate, Lys; and filtrate, Fil) from canine isolate T-58 were compared with respect to the induction and detection of cellular immune responses in mice. The antigens exhibited good sensitivity and specificity when used to detect a delayed-type hypersensitivity (DTH) response in mice previously immunized with T-58 or Histoplasma capsulatum antigens. Greater reactivity was observed with the KWY and Lys antigens as DTH-inducing agents (immunogens) than with the Fil antigen. The antigens were also compared with regard to the induction and detection of a lymphoproliferative reaction using splenocytes from normal and sensitized mice, and optimal reactivity was demonstrated with the KWY and Fil antigen preparations both as immunogens (absorbance values 0.22-1.60 and 0.20-0.90, respectively) and as in vitro testing antigens (absorbance values 0.60-1.60 and 0.35-1.00 respectively) (P < 0.01). Peritoneal macrophages from mice sensitized with Fil and KWY antigens showed the greatest in vitro replication inhibition (RI) of B. dermatitidis yeast cells (RI values of 53% and 50% respectively) (P < 0.05). When mitogenic or antigenic lymphocytic supernatants were compared with respect to their ability to enhance the phagocytic activity of unelicited macrophages from normal mice to kill yeast cells, the T-cell mitogens (concanavalin A and phytohaemagglutinin) optimally activated the naive macrophages (45% and 44% RI respectively) compared with the B-cell mitogen (LPS) (23% RI) (P < 0.05). Similar results were obtained with the lymphocytic supernatants from mice immunized with KWY cells when activated using KWY or Fil antigens (46% and 40% RI respectively) (P < 0.05).

Genomic Cloning, Characterization, and Functional Analysis of the Major Surface Adhesin WI-1 on Blastomyces dermatitidis Yeasts

WI-1 is a 120-kDa surface protein adhesin on Blastomyces dermatitidis yeasts that binds CD18 and CD14 receptors on human macrophages. We isolated and analyzed a clone of genomic WI-1 to characterize this key adherence mechanism of the yeast. The 9.3-kilobase insert contains an open reading frame of 3438 nucleotides and no introns. The amino acid sequence of native WI-1 matches the deduced sequence of genomic WI-1 at positions 757-769, 901-913, and 1119-1138, demonstrating the cloned gene is authentic WI-1. The complete coding sequence has 30 highly conserved repeats of 24 amino acids arrayed in tandem in two noncontiguous regions of the protein. The repeat sequence is homologous to the Yersiniae adhesin invasin, the C terminus displays an epidermal growth factor-like domain, and the N terminus has a short hydrophobic sequence that may be a membrane-spanning domain. The tandem repeats are predicted to be at the exposed surface of the protein, thereby explaining the adhesive properties of WI-1. The WI-1 promoter contains a CAAT box (nucleotide positions 2287-2290), TATA box (2380-2385), and CT motif (2399-2508). Transcription is initiated within the CT motif at nucleotide 2431. A 5.5-kilobase subclone containing the full coding sequence of WI-1 was expressed as a histidine-tagged fusion protein in Escherichia coli. Recombinant WI-1 has the expected molecular mass of 120 kDa, is strongly recognized in Western blots by rabbit anti-WI-1 antiserum, and binds human macrophage receptors in the same manner as native WI-1. This work clarifies a key adherence mechanism of B. dermatitidis and will permit further analysis of WI-1-mediated attachment to host cells, receptors, and extracellular matrix.

The WI-1 antigen of Blastomyces dermatitidis yeasts mediates binding to human macrophage CD11b/CD18 (CR3) and CD14.

Three genetically related strains of Blastomyces dermatitidis (Bd) yeasts that differ in their expression of WI-1, an immunodominant cell wall Ag, were tested for their capacity to bind to human macrophages (M phi) in the absence of serum. These strains included American Type Culture Collection (ATCC, Rockville, MD) ATCC 26199, which is virulent for mice; an attenuated mutant strain ATCC 60915, which expresses 2.5-fold more WI-1 than strain 26199; and an avirulent mutant strain, ATCC 60916, which has sixfold more WI-1 than strain 26199. Attachment of both mutant strains to M phi was rapid and was maximum after 10 min at 37 degrees C. Attachment of strain 26199 to M phi was approximately 40% of that obtained with the mutants. Binding of Bd to M phi was temperature- and Mg(2+)-dependent, and heat-killed yeasts bound to M phi as well as viable yeasts. Experiments with receptor-specific mAbs demonstrated that 26199 yeasts bound predominantly to the LPS binding site on CD11b/CD18 (CR3). However, the mutants bound to M phi CD14 as well as CR3. Fab anti-WI-1 inhibited the binding of all strains to M phi by 69 to 78%. Latex microspheres coated with purified WI-1 or a 25-amino acid tandem repeat located within WI-1 also bound to M phi CR3 and CD14. These data demonstrate that: 1) WI-1 is a major ligand on Bd that mediates attachment of yeasts to human M phi; 2) the binding activity of WI-1 is located within the 25-amino acid tandem repeat; and 3) binding of Bd yeasts to M phi is mediated through the LPS binding site on CR3 and CD14.

Detection of antibodies and delayed hypersensitivity with Rotofor® preparative IEF fractions ofBlastomyces dermatitidisyeast phase lysate antigen

Blastomyces dermatitidis (dog isolate T-58) yeast phase lysate antigen was concentrated and separated by Rotofor preparative isoelectric focusing cell (Bio-Rad). The pH values of the fractions were determined and equilibrated to pH 7.2 and then analysed by enzyme-linked immunosorbent assay using horseradish peroxidase enzyme system against serum specimens from dogs with blastomycosis, histoplasmosis, aspergillosis, and coccidioidomycosis. The results showed a peak absorbance at pH 3.89-4.31 (fractions 4 and 5) with the blastomycosis serum specimens. This was a single sharp peak while the rest of the fractions were lower. In contrast the sera from dogs with histoplasmosis showed a peak absorbance at pH 5.54-5.97 (fractions 9 and 10), while the other mycoses showed patterns that did not resemble the blastomycosis or histoplasmosis specimens. Serum specimens from dogs with blastomycosis being treated with itraconazole were also assayed (pre-treatment and 1, 2, 3, and 12 months post-treatment sera). The characteristic peak for blastomycosis was observed and a decrease in the peak was seen as the treatment progressed. Fractions 3-12 were also used to detect delayed dermal hypersensitivity in hyperimmunized hairless guinea-pigs. Fraction 5 (pH 4.31) elicited the optimal response in B. dermatitidis-immunized animals, while no cross-reactivity was observed in guinea-pigs sensitized with Histoplasma capsulatum killed cells.

Altered expression of surface protein WI-1 in genetically related strains of Blastomyces dermatitidis that differ in virulence regulates recognition of yeasts by human macrophages.

The molecular basis for pathogenicity and virulence of the dimorphic fungus Blastomyces dermatitidis remains unknown. WI-1 is a major cell wall protein of B. dermatitidis yeasts and is a recognition target of both humoral and cell-mediated immunity. As an initial study to determine if WI-1 might be linked to virulence of B. dermatitidis, we quantified WI-1 expression on three genetically related strains that differ in their virulence for mice: wild-type virulent ATCC strain 26199, mutant ATCC strain 60915 (which is 10,000-fold reduced in virulence), and mutant ATCC strain 60916 (which is avirulent). Two principal alterations in WI-1 expression were observed in the mutants. First, the mutants express more WI-1 on their surface, as quantified by flow cytometry with monoclonal antibody to WI-1 and by radioimmunoassay, but the WI-1 on their cell wall is less extractable than that on the wild-type strain. Second, the mutants shed less WI-1 during culture and demonstrate impaired processing of shed WI-1. Surface alterations in WI-1 were accompanied by significant differences in the binding of the virulent and mutant strains to human monocyte-derived macrophages. Attachment of yeasts to macrophages paralleled and was proportional to the expression of WI-1. Compared with wild-type yeasts, both mutants bound to macrophages more rapidly and in two- to threefold-greater magnitude. Furthermore, about 75% of yeast binding to macrophages was inhibited by a Fab anti-WI-1 monoclonal antibody. These results suggest that altered WI-1 expression on attenuated and avirulent mutant B. dermatitidis yeasts greatly facilitates macrophage recognition and binding of yeasts and, in turn, may contribute to more rapid ingestion and killing in the host.

Immunologic recognition of a 25-amino acid repeat arrayed in tandem on a major antigen of Blastomyces dermatitidis.

A 120-kD glycoprotein antigen abundantly expressed on Blastomyces dermatitidis yeasts is a target of cellular and humoral immune responses in human infection. To investigate the antigen and immune response more carefully at the molecular level, we screened an expression library from B. dermatitidis to identify clones that encode this antigen, designated WI-1. A 942-bp cDNA was isolated by immunologic screening with polyclonal, rabbit anti-WI-1 antiserum. Northern hybridization analysis showed that the cDNA hybridized to yeast message approximately equal to 3.9 kb. DNA and deduced protein sequence analysis of the clone demonstrated a 25-amino acid repeat arrayed in tandem, present in 4.5 copies near the 5' end, and rich in predicted antigenic epitopes. Further analysis showed strong homology in these tandem repeats with invasin, an adhesin of Yersiniae. Cloned cDNA was used to express a 30-kD fusion protein strongly recognized in western blots by rabbit anti-WI-1 antiserum, and by sera from all 35 blastomycosis patients studied. The fusion protein product of subcloned cDNA encoding only the tandem repeat also was strongly recognized in western blots by sera from the 35 blastomycosis patients, but not by sera from 10 histoplasmosis and 5 coccidioidomycosis patients. An antigen-inhibition radioimmunoassay showed that the tandem repeat alone completely eliminated rabbit and human anti-WI-1 antibody binding to radiolabeled native WI-1. From these results, we conclude that the 25-amino acid repeat of WI-1 displays an immunodominant B cell epitope, and that the carboxyl-terminus of the molecule exhibits an architecture that may promote adhesion of Blastomyces yeasts to host cells or extracellular matrix proteins and ultimately provide a clearer picture of the molecular pathogenesis of blastomycosis.

Therapeutic efficacy of a liposomal formulation of amphotericin B (AmBisome) against murine blastomycosis.

The efficacy of a liposomal amphotericin B preparation (AmBisome) and a deoxycholate suspension (Fungizone) were compared in a pulmonary model of murine blastomycosis. Male, four-week-old CD-1 mice were infected intranasally with 19,400 or 27,450 cfu of viable Blastomyces dermatitidis yeasts and intravenous therapy begun 4 days later. Groups of ten mice were given various dosages of Fungizone or AmBisome or were untreated. All treatments were given 3 times per week for 2 weeks. Deaths were recorded over 49 days and the number of viable yeasts in the lungs of survivors quantitated by viable counting. Therapy with 1.0 mg/kg/dose of Fungizone or 1.0, 3.0, 5.0, 7.5 or 20.0 mg/kg/dose of AmBisome were equivalent and prolonged survival over controls and lower dosages of each drug. No acute or chronic toxicities were observed with any regimen. Quantitation of residual numbers of yeasts in the lung showed that 70-100% of mice given 7.5 mg/kg or greater of AmBisome were free of infection and were superior to other regimens. At equivalent 1.0 mg/kg dosages Fungizone was superior to AmBisome. Although AmBisome was three-fold less active than Fungizone on a dosage basis, higher curative doses could be given that were not toxic. These data indicate that AmBisome should be further tested in other models and in clinical trials.

WI-1, a novel 120-kilodalton surface protein on Blastomyces dermatitidis yeast cells, is a target antigen of cell-mediated immunity in human blastomycosis.

A large body of experimental data has demonstrated the central role of T cells in acquired resistance to the dimorphic fungus Blastomyces dermatitidis. We examined the human T-cell response to WI-1, a 120-kDa B. dermatitidis yeast cell surface protein recently shown to be an immunodominant antigen of the B-cell response in infected humans. Peripheral blood lymphocytes from 10 blastomycosis patients studied proliferated in response to WI-1 (mean, 19,431 cpm) and to the standard, crude cell wall antigen, Blastomyces alkali- and water-soluble antigen (B-ASWS) (mean, 19,131 cpm); lymphocytes from 10 histoplasmosis patients and 10 normal control subjects did not respond to WI-1. WI-1 stimulation of patient lymphocytes and rechallenge with WI-1 or B-ASWS showed that the antigens share immunodominant epitopes. Of 100 WI-1-responsive T-cell clones derived from peripheral blood, 10 were studied in detail to assess the phenotype, function, and ligands recognized. The clones exhibit the CD3+ CD4+ phenotype of helper T cells; 2 of 10 clones (and 21% of antigen-stimulated peripheral blood lymphocytes) use the V beta 8 T-cell receptor gene element to respond to WI-1. All the clones proliferate in response to both WI-1 and B-ASWS but not other fungal antigens, and some mediate potent cytolytic effects on WI-1- and B-ASWS-labeled targets. WI-1 recognition requires antigen processing and presentation of epitopes in association with HLA-DR (to noncytolytic clones) and HLA-DP (to cytolytic clones). From these findings, we conclude that CD4+ T cells with regulatory and cytolytic properties are involved in the development of acquired resistance of B. dermatitidis, that the cells are directed against WI-1, and that the manner of display of WI-1 peptide epitopes in conjunction with major histocompatibility complex class II may influence the profile of the immune response.

Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and non-endemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti-Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.

Enzyme immunoassay detection of antibodies in canine blastomycosis using Blastomyces dermatitidis lysate antigens

A Blastomyces dermatitidis yeast phase lysate antigen (T-58) prepared in our laboratory was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in 201 serum specimens from dogs with blastomycosis. In addition 36 sera from non-infected dogs from an endemic area for blastomycosis and 36 sera from non-infected dogs residing in a blastomycosis non-endemic area were assayed. Sensitivity values ranged from 96.2% (53 pre-treatment sera) to 88.5% (148 post-treatment sera) with the specimens from dogs with confirmed blastomycosis. Minimal reactivity was experienced with the sera from non-infected dogs. Positive reactions were obtained with 8.3% of the endemic area sera and with 5.6% of the sera from the non-endemic area. Mean ELISA index values ranged from 7.66 with the pretreatment sera to 1.11 and 1.03 with the endemic area sera and non-endemic area specimens respectively.

Comparison of enzyme immunoassay and immunodiffusion for the detection of canine blastomycosis

Two Blastomyces dermatitidis commercial immunodiffusion antigens, Meridian Diagnostics and Nolan-Scott Laboratories, and two B. dermatitidis yeast phase lysate antigens, T-58 and K-Le, prepared in our laboratory were utilized in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in 61 serum specimens from dogs with blastomycosis. All antigens used in the ELISA were diluted to 50 ng of protein ml-1. Immunodiffusion (ID) tests were also performed on the sera using a Nolan-Scott ID antigen. Greater reactivity, based on the ELISA index value, was evidenced with the two yeast phase lysate antigens over the two commercial reagents in the ELISA. In addition the sensitivity of the ELISA with the T-58, K-Le and Meridian antigens was considerably greater than that of the ID test. The Nolan-Scott antigen, however, was able to detect more positive specimens when used in the ID procedure than in the ELISA. Therefore encouraging results were obtained with respect to the continued development and optimization of the ELISA using B. dermatitidis cell lysate antigens for the diagnosis of blastomycosis.

Isolation, purification, and radiolabeling of a novel 120-kD surface protein on Blastomyces dermatitidis yeasts to detect antibody in infected patients.

No well-defined Blastomyces-specific antigens are currently available. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify immunologically active molecules in the cell wall of B. dermatitidis. A major immunoreactive 120-kD protein (WI-1) was present in all five strains studied and comprised 5% of the protein in the cell wall extract obtained after freezing and thawing yeast cells. WI-1 was recognized by serum from all 10 patients with blastomycosis but by none of those from 5 patients with histoplasmosis. It was purified by electroelution, radiolabeled with 125I, and incorporated into a radioimmunoassay (RIA) for serodiagnosis of blastomycosis. Antibody to WI-1 was detected in 58 (85%) of 68 patients with blastomycosis (geometric mean titer, 1:2,981), in two (3%) of 73 patients with histoplasmosis, coccidioidomycosis, sporotrichosis, or candidiasis (titers, 1:86 and 1:91) and in none of 44 healthy persons. WI-1 was shown to be a surface molecule abundant on B. dermatitidis yeasts that were indirectly stained with serum from a rabbit immunized with WI-1. Approximately 0.93 pg of WI-1 or 4.7 x 10(6) WI-1 molecules were found on the surface of an individual yeast using an antigen-inhibition RIA; none was found on Histoplasma capsulatum or Candida albicans yeasts. We conclude that WI-1 is a novel, immunologically active surface molecule on the invasive form of B. dermatitidis and that WI-1 can be used to reliably detect antibody and study the immunopathogenesis of blastomycosis.

Efficacy of Itraconazole in Blastomycosis in a Murine Model and Comparison with Ketoconazole

Mice were infected intranasally with B. dermatitidis yeasts, and after infection treated orally. Itraconazole 50-150 mg/kg/day was protective (prolonged survival) against lethal infection, although the infection was not sterilized. Itraconazole was approximately 3 times as potent as ketoconazole. These results suggest advantages for itraconazole therapy clinically.

In vivo activation of peripheral blood polymorphonuclear neutrophils by gamma interferon results in enhanced fungal killing

The effect of in vivo administration of murine recombinant gamma interferon (IFN) on the fungicidal activity of murine peripheral blood polymorphonuclear neutrophils (PB-PMNs) was studied. Mice were injected intramuscularly with 250, 2,500, 25,000 or 250,000 U of IFN 5 h before collection of peripheral blood. Purified PB-PMNs were cocultured in vitro with Blastomyces dermatitidis yeast cells for 2 h. PB-PMNs from untreated mice killed 44.5 +/- 12.5% of the fungal inoculum, whereas PB-PMNs from mice treated with 25,000 or 250,000 U of IFN showed significantly enhanced in vitro killing (68.0 +/- 9.4% [P less than 0.005] and 72.3 +/- 1.1% [P less than 0.001], respectively). Treatment with 250 or 2,500 U of IFN or 25,000 U of heated (100 degrees C, 15 min) IFN had no effect. The IFN-induced activation of PB-PMNs was transitory. Significant enhancement of PB-PMN killing activity occurred 1, 2, or 5 h after in vivo IFN administration, but no enhancement was observed 16 or 24 h after IFN treatment. Enhanced fungicidal activity by PB-PMNs from mice treated for 5 h with 25,000 U of IFN correlated with an increased release of superoxide anion (O2-) in vitro after stimulation of PB-PMNs with phorbol ester; normal PB-PMNs and IFN-activated PB-PMNs, respectively, produced 2.2 +/- 2.5 and 23.5 +/- 4.8 nmol of O2- per 10(6) PB-PMNs per 30 min (P less than 0.005). The exogenous addition of compounds that antagonize or inhibit the formation of oxygen radicals (superoxide dismutase, catalase, dimethyl sulfoxide, or sodium azide) significantly inhibited fungal killing by both normal and IFN-activated PB-PMNs. In addition to the enhanced microbicidal activity and superoxide generation demonstrated in vitro with constant cell numbers, there was a transient leukocytosis (particularly neutrophilia) in peripheral blood at doses of IFN and at times after IFN administration where enhanced activity was also demonstrated. In summary, our results indicate that PB-PMNs can be activated in vivo for enhanced killing of a fungal target. The enhanced killing capacity of IFN-activated PB-PMNs is due at least in part to the enhancement of oxidative killing mechanisms.

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.

Immunomodulation by Blastomyces dermatitidis: functional activity of murine peritoneal macrophages.

Cell-mediated immunity plays the dominant role in the immune response of mice to Blastomyces dermatitidis infections. Since macrophages play an important role in cell-mediated immunity, the interactions between sensitized murine peritoneal macrophages and the yeast phase of B. dermatitidis were investigated. Scanning electron microscopy showed that the sensitized macrophages readily phagocytized B. dermatitidis yeast cells. In addition, there appeared to be activation of metabolic pathways within the sensitized macrophages, as indicated by increased chemiluminescence activity during phagocytosis. Sensitized macrophages were significantly better at controlling intracellular proliferation of the yeast cells when compared to nonsensitized cells. This was determined by disruption of macrophages and plating for viable yeasts. Scanning electron microscope observations offered further substantiation. Experiments with Candida albicans indicated that B. dermatitidis non-specifically activated macrophages. At 2 h postphagocytosis, 30% fewer C. albicans in B. dermatitidis-activated macrophages were able to form germ tubes. These studies demonstrated the multiple potential of activated macrophages with regard to their functional activity.

Immunoadjuvant Effects of &lt;italic&gt;Blastomyces dermatitidis&lt;/italic&gt; Against EL 4 Lymphoma in C57BL/6J Mice&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN4"&gt;4&lt;/xref&gt;

The ability of thimerosal-killed Blastomyces dermatitidis yeast cells, which greatly enhance the cell-mediated immune response in C57BL/6J mice, to act as an immunopotentiator against EL 4 lymphoma was investigated. Mice treated with yeast cells were protected from as many as 10(4) tumor cells. Complete suppression of tumor growth was observed in treated animals that received ip injections of 10(2) or 10(3) tumor cells. The mice, however, were not immune to further EL 4 lymphoma cell challenge. The lack of tumor-specific immunity indicated nonspecific suppression of tumor growth probably by macrophages. Ten days after treatment, the peritoneal macrophages from mice that showed complete tumor suppression were tested for their ability to prevent in vitro tumor cell proliferation. These macrophages demonstrated 90% inhibition of [3H]thymidine incorporation by EL 4 tumor cells at a 100:1 effector-to-target cell ratio. Macrophages from B. dermatitidis-treated animals exhibited a twofold increase in specific lysis of EL 4 at 10 and 15 days compared to resident macrophages. Spleen and lymph node cells from protected animals showed no cytotoxic activity against EL 4 in a 51Cr release assay. Treatment of tumor-bearing mice with a single dose of B. dermatitidis was effective only if administered within 24 hours of tumor establishment. These results demonstrated that nonviable B. dermatitidis prohibits the growth of EL 4 under conditions where Corynebacterium parvum fails to do so.

Cell-mediated immunoprotection in blastomycosis.

Delayed hypersensitivity can be induced in C57BL/6J mice by two subcutaneous injections of Merthiolate-killed Blastomyces dermatitidis yeast cells in Freund incomplete adjuvant. Development of delayed hypersensitivity peaked at the day 18 post-primary antigen-emulsion injection, as determined by footpad sensitivity tests. Mice rendered hypersensitive to B. dermatitidis were protected from the lethal effect of a blastomyces infection. The protection effects were shown both in mortality tests and in data from organ cultures as expressed by indices of resistance. Data from this study show that there is a close parallel relationship between host resistance and the prevailing level of delayed hypersensitivity.

Isolation of a purified skin test antigen from Blastomyces dermatitidis yeast-phase cell wall.

Preparative polyacrylamide gel electrophoresis was used to isolate individual components of an alkaline-soluble-water-soluble fraction of the cell wall of Blastomyces dermatitidis yeast phase. One component isolated demonstrated exceptional specificity and reactivity when tested on guinea pigs infected with B. dermatitidis. This component displayed no cross-reactivity when tested on animals infected with Histoplasma capsulatum. The significance of isolation of a purified, specific antigen is discussed.

Passive transfer of delayed hypersensitivity to Blastomyces dermatitidis between mice.

This study further characterized the delayed hypersensitivity state induced in animals by Blastomyces dermatitidis exposure. Passive transfer of delayed hypersensitivity by transfer of cells and inhibition of migration of peritoneal exudate cells were studied, using sensitized mice of two inbred strains. Donor mice were subcutaneously inoculated with viable B. dermatitidis yeast cells. After 15 days, spleen cells or serum from these animals were injected intravenously into normal recipients of the same strain. After 24 h these mice were footpad tested with killed B. dermatitidis yeast cell antigen. Mice receiving spleen cells from sensitized animals had a significant increase in footpad thickness 24 to 48 h after testing. Those receiving only serum remained negative. Migration of peritoneal exudate cells from blastomyces-sensitive donor mice was inhibited by presence of blastomycin but not by mycobacterial antigen. Neither blastomyces-sensitive nor control animals reacted to footpad or migration inhibition testing with mycobacterial antigen.