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D Kruse
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Coccidioides immitis fractions which are antigenic for immune T lymphocytes.
The principal mechanism of resistance to coccidioidomycosis in experimental animals has been reported to be T-cell-mediated immunity. We have generated a Coccidioides immitis antigen-specific murine T-cell line to identify specific macromolecules capable of eliciting an immune mouse T-cell proliferative response. The murine T cells were stimulated in vitro with a soluble conidial wall fraction (SCWF), which has been previously characterized by humoral and cellular immunoassays. The SCWF was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane, and the stained blot was cut into seven pieces based on the molecular size of the SCWF components. The nitrocellulose membrane strips were converted into antigen-bearing particles and tested in a T-cell proliferation assay. Antigenic components of the SCWF in the molecular size range of 43 to 66 kDa were identified as the most immunoreactive. In a parallel study, we used a cDNA expression library derived from mRNA of the mycelial phase of C. immitis, which was constructed in lambda gt11 to identify clones that encoded T-cell-reactive fusion proteins (FPs). The cDNA library was screened by using anti-SCWF rabbit serum, and the FPs expressed in Escherichia coli were isolated and tested for T-cell response in the same manner as the SCWF components. The nucleotide sequence of a 0.2-kb cDNA insert encoding a protein which elicited vigorous T-cell response was determined. The isolated cDNA insert hybridized to a single 1.9-kb mRNA band in a Northern blot of the total RNA fraction of the mycelial phase of C. immitis. Antibody with affinity for the T-cell-reactive FP was isolated from anti-SCWF rabbit serum by solid-phase immunoadsorption. The FP-specific antibody reacted with a 47-kDa polypeptide in Western blots (immunoblots) of the SCWF. The same antibody preparation was used for immunoelectron microscopy to show that the FP was localized in the walls of arthroconidia and spherules of C. immitis. Attempts to clone and sequence the entire gene which encodes the T-cell-reactive protein are under way. The results of this study should lead to the determination of the complete structure of an important T-cell-stimulating antigen of C. immitis.
Amino Acid Sequence↗
Antigen complex of Coccidioides immitis which elicits a precipitin antibody response in patients.
The occurrence in patients of elevated levels of immunoglobulin M (IgM) precipitin antibody to Coccidioides immitis antigens, which are commonly detected by the immunodiffusion-tube precipitin (TP) assay, is suggestive of primary nondisseminating coccidioidomycosis. We previously demonstrated that the concanavalin A-bound mycelial culture filtrate plus lysate preparation is a source of at least two TP antibody-reactive antigens (TP-Ags), which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 120- and 110-kDa fractions. Evidence is presented here that the crude filtrate plus lysate preparation contains additional lectin-bound, TP antibody-reactive fractions as well as a component which elicits a complement fixation antibody response in patients. The 120- and 110-kDa fractions were isolated from the antigen complex and further characterized in this paper. Both TP-Ags are glycoproteins and have been shown by immunoelectron microscopy to be colocalized within cytoplasmic vesicles and the wall of spherules. Deglycosylation of these TP-Ags by sodium periodate treatment resulted in a loss in patients of 82 to 95% of IgM adsorption to the antigens as detected by the enzyme-linked immunosorbent assay (ELISA). Comparison of their carbohydrate compositions revealed that mannose and glucose are the predominant monosaccharides of both TP-Ags but only the 120-kDa fraction contained 3-O-methylmannose, a sugar which appears to be unique to C. immitis among the systemic fungal pathogens. We previously showed that 3-O-methylmannose is at least partly responsible for the reactivity of IgM antibody with the 120-kDa TP-Ag. Good correlation was shown between results of immunodiffusion-TP assays and ELISAs of IgM response to both the 120- and 110-kDa fractions by using 70 serum samples from patients with proved coccidioidomycosis. However, only 2.8% (3 of 109) of the serum samples from patients with other mycoses and nonmycotic infections showed IgM adsorption to the 120-kDa TP-Ag as detected by the ELISA, while 21.1% (23 of 109) showed IgM adsorption to the 110-kDa TP-Ag. The 120-kDa TP-Ag is a potentially valuable serodiagnostic reagent for detection of specific IgM by ELISA in patients with primary coccidioidomycosis.
Amino Acids↗
Isolation of tube precipitin antibody-reactive fractions of Coccidioides immitis.
Patients presenting with primary coccidioidal infection have been shown by earlier investigators to produce immunoglobulin M (IgM) precipitin antibodies to lysates of mycelial and spherule phases of Coccidioides immitis. This humoral response has been detected by tube precipitin (TP) and immunodiffusion (ID)-TP assays of patient sera, which are valuable aids in early diagnosis of coccidioidomycosis. Several reports of antigenic fractions which show reactivity with patient TP antibody have been published. However, confusion persists with respect to the nature of the specific serologically reactive macromolecule(s). In this study we isolated two TP antibody-reactive antigens (TP-Ags) from an alkali-soluble, water-soluble fraction of the inner conidial wall and a culture filtrate plus toluene lysate of the mycelial phase of C. immitis. The crude antigens were first separated by concanavalin A (ConA) chromatography. The TP-Ags were identified in ID-TP assays as 120- and 110-kilodalton (kDa) fractions which were electroeluted from reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of the ConA-bound conidial wall extract and ConA-bound culture filtrate plus lysate preparation, respectively. Following electroelution, the 120-kDa fraction was subjected to gel filtration chromatography which yielded a major 240-kDa and minor 120-kDa component. The apparent dimer may be a product of disulfide bond formation resulting from reassociation of the reduced, monomeric components (120 kDa). The latter was suggested by the presence of cysteine in the isolated fraction. The electroeluted 110-kDa fraction was subjected to ion-exchange chromatography. The DEAE-isolated, TP antibody-reactive fraction was identified as antigen 2 in the coccidioidin-anti-coccidioidin reference system. Homogeneity of the TP-Ags was demonstrated in silver-stained sodium dodecyl sulfate-polyacrylamide gels of the respective chromatographically isolated fractions. The two purified TP-Ags showed reactivity in the TP and ID-TP assays and were capable of binding patient IgM but comparatively little IgG antibody, as determined by an enzyme-linked immunosorbent assay. It appears that the diagnostic TP reaction between sera from patients with coccidioidomycosis and the ID reference antigens examined in this study is a composite of IgM binding to both a 120-kDa and a 110-kDa antigen.
Antibodies, Fungal↗
Composition, serologic reactivity, and immunolocalization of a 120-kilodalton tube precipitin antigen of Coccidioides immitis.
Diagnosis of coccidioidomycosis largely depends on serologic tests. In this investigation, the enzyme-linked immunosorbent assay (ELISA) was used to detect patient immunoglobulin M (IgM) precipitin antibody binding to a 120-kilodalton (kDa) fraction previously isolated from an alkali-soluble, water-soluble extract of the arthroconidial wall and mycelial culture filtrate plus toluene lysate of Coccidioides immitis. Results of the serologic response to this tube precipitin antigen (TP-Ag) in the ELISA correlated well with results of immunodiffusion assays of 30 serum samples from patients. Immunoelectron microscopic examinations of arthroconidia and spherules were performed with patient IgM precipitin antibodies isolated from sera eluted over a solid-phase immunosorbent column containing the purified 120-kDa TP-Ag. The antibody probe located the 120-kDa TP-Ag on the walls of in vitro-grown arthroconidia and spherules. Pronase digestion and heating (100 degrees C, 5 min) had no apparent effect on the activity of the 120-kDa TP-Ag, while periodate oxidation resulted in total loss of its immunodiffusion-TP activity. Analysis of the carbohydrate composition of the TP-Ag revealed xylose, 3-O-methylmannose (3-O-MM), mannose, galactose, and glucose. Competitive inhibition ELISAs were used to demonstrate that 3-O-MM is largely responsible for the reactivity of IgM precipitin antibodies with the 120-kDa TP-Ag. Synthetic 3-O-MM may be a useful probe for detection of anti-Coccidioides precipitin antibodies in the ELISA.
Amino Acids↗
Isolation of antigens with proteolytic activity from Coccidioides immitis.
Three antigens with proteolytic activity have been isolated from crude, water-soluble fractions of the saprobic phase of the fungal pathogen Coccidioides immitis. Two proteinases, identified in our immunoelectrophoresis reference system as Ag11 and AgCS, were isolated from the soluble conidial wall fraction (SCWF). Ag11 was previously shown to be a serine proteinase and was characterized in this study as a 60-kilodalton (kDa) fraction by gel filtration (GF). The purified proteinase demonstrated little or no reactivity with 21 serum samples from coccidioidomycosis patients in the enzyme-linked immunosorbent assay; this may be due to limited presentation of this antigen to the host during the course of coccidioidomycosis. AgCS was separated by GF chromatography into two fractions identified by molecular masses of 39 and 19 kDa. Most proteolytic activity was shown by substrate gel electrophoresis to be associated with the lower-molecular-mass fraction. AgCS was reactive with 18 of the 21 serum samples and shown to be the major component of a heat-stable antigen previously reported to be immunospecific for C. immitis. The third antigen with proteolytic activity was isolated from the 5-day mycelial culture filtrate and identified by GF as a 56-kDa fraction. Uniformly high levels of immunoreactivity between 18 of the 21 patient sera and the 56-kDa antigen were demonstrated. Antigens with proteolytic activity may play important roles in fungus-host interactions as well as morphogenesis of the pathogen.
Antigens, Fungal↗
Long-term follow-up of Perthes disease treated with spica casts.
Forty adults with a history of Legg-Calve-Perthes disease treated with spica casts were clinically and radiographically evaluated an average of 30 years post-treatment (range 14 to 40 years). Clinical results were 85% good, 5% fair, and 10% poor. Five percent showed significant degenerative joint disease on follow-up radiographs and 7% had undergone prosthetic replacement. Fifteen percent of the patients had a positive family history of Perthes disease. Factors associated with a better result included: diagnosis before age 9, shorter length of follow-up, minimal femoral head and neck involvement, and a congruous joint.
Age Factors↗