Biomedical subjects

S L Bragg

Publications and source records attributed to S L Bragg.

13 recordsLinked to original sources

PubMed1998

Epidemic leptospirosis associated with pulmonary hemorrhage-Nicaragua, 1995.

In October 1995, epidemic "hemorrhagic fever," without jaundice or renal manifestations, was reported in rural Nicaragua following heavy flooding; 2259 residents were evaluated for nonmalarial febrile illnesses (cumulative incidence, 6.1%) and 15 (0.7%) died with pulmonary hemorrhage. A case-control study found that case-patients were more likely than controls to have ever walked in creeks (matched odds ratio [MOR], 15.0; 95% confidence interval [CI], 1.7-132.3), have household rodents (MOR, 10.4; 95% CI, 1.1-97.1), or own dogs with titers >/=400 to Leptospira species (MOR, 23.4; 95% CI, 3.6-infinity). Twenty-six of 51 case-patients had serologic or postmortem evidence of acute leptospirosis. Leptospira species were isolated from case-patients and potential animal reservoirs. This leptospirosis epidemic likely resulted from exposure to flood waters contaminated by urine from infected animals, particularly dogs. Leptospirosis should be included in the differential diagnosis for nonmalarial febrile illness, particularly during periods of flooding or when pulmonary hemorrhage occurs.

Adolescent↗

PubMed1998

Comparison of two rapid latex agglutination tests for detection of cryptococcal capsular polysaccharide.

The Murex Cryptococcus Test was compared with the Cryptococcal Antigen Latex Agglutination System (CALAS) for detecting cryptococcal polysaccharide in 173 cerebrospinal fluid (CSF) specimens and 117 serum samples with 99% and 97% concordance, respectively. Eighteen CSF samples and 17 serum samples were positive in both assays, and 249 were negative. The sensitivity and specificity of the Murex relative to the CALAS were 90% and 100%, respectively, for CSF, and 81% and 100%, respectively, for serum. Six discrepancies were arbitrated by retesting, using a third analytic method, review of other laboratory and clinical data, or both. The reaction in 1 CSF specimen was considered false positive by the CALAS, and the reactions in 2 serum samples were false negatives by the Murex. For 3 patients with previous cryptococcal meningitis but no active disease, only the CALAS detected antigen, suggesting that the Murex has less analytic sensitivity in this context. Titer differences dictate that direct comparisons between the 2 tests are not feasible. There were no false-positive reactions in limited testing with either method using specimens from patients with concurrent noncryptococcal infections or in rheumatoid factor-positive serum samples. Infections caused by Cryptococcus neoformans serotypes A or AD were detected equally by both assays. Based on our study, we have elected to continue to use the CALAS for routine testing for cryptococcal antigen.

Adult↗

PubMed1994

Effects of histoplasmin M antigen chemical and enzymatic deglycosylation on cross-reactivity in the enzyme-linked immunoelectrotransfer blot method.

The enzyme-linked immunoelectrotransfer blot (EITB) method was evaluated as a suitable method for detecting antibodies against M antigen of Histoplasma capsulatum by use of both glycosylated and deglycosylated M protein of histoplasmin (HMIN). Sera from patients with histoplasmosis, paracoccidioidomycosis, blastomycosis, coccidioidomycosis, and aspergillosis were tested by the EITB with glycosylated M protein of HMIN. This assay demonstrated 100% sensitivity with histoplasmosis serum samples, all of which reacted with the 94-kDa glycoprotein (M antigen). Although the EITB is highly sensitive, it is not specific for histoplasmosis when glycosylated M protein is used as an antigen. A total of 81% of paracoccidioidomycosis, 25% of blastomycosis, 33% of coccidioidomycosis, 73% of aspergillosis, and 16% of tuberculosis serum samples cross-reacted with M protein of HMIN and yielded patterns indistinguishable from those obtained with histoplasmosis serum samples. The EITB reactions with both untreated M antigen and M antigen altered by periodate oxidation or by deglycosylation with endoglycosidases were compared. Cross-reactions with heterologous sera in the EITB could be attributed to periodate-sensitive carbohydrate epitopes, as reflected by the increase in the test specificity from 46.1 to 91.2% after periodate treatment of M protein. The EITB for the detection of antibodies to M antigen is a potential diagnostic test for histoplasmosis, provided that periodate-treated M protein is used as an antigen.

Antibody Specificity↗

PubMed1994

Immunochemical analysis of the H and M glycoproteins from Histoplasma capsulatum.

The H and M antigens of Histoplasma capsulatum are glycoproteins, and both possess epitopes found on the C antigen, a cross-reactive galactomannan shared by the major genera of systemic dimorphic fungi. We modified the H and M glycoproteins by chemical and enzymatic digestion to determine the relative contributions of the carbohydrate and protein moieties to the immunological reactivities and the apparent molecular weights of these antigens. Endoglycosidases with known action patterns were used to determine the nature of the glycopeptide bonds in the H and M antigens. The effects of these treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lectin binding, and enzyme-linked immunoelectrotransfer blots probed with polyclonal and monoclonal antibodies (MAbs). Oxidation with 100 mM periodate destroyed the common fungal epitope recognized by MAb CA1-CB4 and nearly all of the concanavalin A-binding sites on both the H and M antigens; it also caused the molecular mass of the M antigen to shift from 94 to 88 kDa. Treatment of samples with O-glycanase had little, if any, effect on the H and M glycoproteins. On the other hand, treatments with endo-beta-N-acetylglucosaminidase H, and particularly peptide N-glycoproteins F (PNGase F), produced pronounced shifts in the M(r) but did not completely eliminate concanavalin A- or MAb CA1-CB4-binding sites. PNGase F treatment caused the molecular mass of the H antigen to shift from 116 to 94 kDa and that of the M antigen to shift from 94 to 74 kDa. The susceptibilities of the H and M glycoproteins to endo-N-acetyl-beta-D-glucosaminidases suggest that their glycosidic moieties are N linked.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody Specificity↗

PubMed1993

Purification and characterization of the extracellular aspartyl proteinase of Candida albicans: removal of extraneous proteins and cell wall mannoprotein and evidence for lack of glycosylation.

Aspartyl proteinase (AP) is an extracellular enzyme of Candida albicans implicated as a pathogenic factor. Previous reports on the purification and characterization of AP suggested that a single DEAE-Sephadex chromatographic step was sufficient for the removal of extraneous proteins and that the final product was glycosylated. We purified AP using a chromatographic series consisting of DEAE-Sephadex A25, Sephadex G75 and rechromatography on DEAE-Sephadex A25. Use of DEAE-Sephadex alone did not remove extraneous proteins and removed little contaminating mannoprotein (MP). The addition of a Sephadex G75 column to the purification scheme removed the majority of contaminating MP and proteins. The final DEAE-Sephadex A25 chromatographic step resulted in (a) removal of detectable extraneous proteins, (b) removal of immunologically detectable MP by dot blot and Western blot enzyme immunoassay, (c) loss of periodic acid-silver stain positivity, and (d) a high AP yield (1295 U l-1) and specific activity (1749 U mg-1). We conclude that a single DEAE-Sephadex A25 purification step is insufficient to remove extraneous proteins and MP, which could interfere with the production of AP-specific antibodies and the dissection of moieties responsible for immune reactivity. Reports of periodic acid-Schiff or anthrone positivity of AP preparations may reflect the presence of extraneous MP, which can be removed by the chromatographic series we describe.

Aspartic Acid Endopeptidases↗

PubMed1993

Heterogeneity of the purified extracellular aspartyl proteinase from Candida albicans: characterization with monoclonal antibodies and N-terminal amino acid sequence analysis.

Three dominant proteins (41, 48, and 49 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in purified preparations of the extracellular aspartyl proteinase (AP) of Candida albicans. All three proteins bound to the specific carboxyl proteinase ligand, pepstatin A, and were associated with maximum AP activity. The N-terminal amino acid sequence for the 48- and 49-kDa proteins matched that reported by others for AP, whereas the sequence for the 41-kDa protein was unique and was not homologous to any known protein. Time course studies demonstrated the simultaneous presence of all three proteins, supporting evidence that the 41- and 48-kDa proteins were not breakdown products of AP. Previous studies did not detect carbohydrate in SDS-polyacrylamide gels of purified AP preparations stained with periodic acid and silver, making glycosylation an unlikely explanation for the observed differences in the molecular masses of the proteins. Some monoclonal antibodies directed against the 49-kDa protein reacted with the 41- and 48-kDa proteins, indicating cross-reactive epitopes. Other monoclonal antibodies, however, reacted only with the 49-kDa protein. We conclude that three pepstatin A-binding proteins occur in purified AP preparations: two have the same amino acid N terminus as that reported for AP, whereas the third has a unique sequence. All three proteins should be considered when undertaking studies to determine the role of AP in candidal pathogenesis or when preparing specific antibodies for antigen capture assays.

Amino Acid Sequence↗

PubMed1993

Multilocus enzyme typing of Cryptococcus neoformans.

Multilocus enzyme electrophoresis was adapted for subtyping Cryptococcus neoformans. The two cryptococcal varieties were clearly distinguishable. Isolates of the C. neoformans var. neoformans were sorted according to serotype and were sorted into four to five subtypes within each serotype. Nearly no two isolates of the C. neoformans var. gattii displayed the same enzyme electrophoretic type. This method may be a useful adjunct to current methods for classification and epidemiologic studies of cryptococci.

Cryptococcus neoformans↗

PubMed1993

Evaluation of cation exchange chromatography for the isolation of M glycoprotein from histoplasmin.

Cation exchange chromatography was evaluated to purify the M antigen from histoplasmin (HMIN). Two H and M antigen-containing fractions, soluble (S) and precipitate (PP), resulted from the initial 0.025 M, pH 3.5 citrate buffer dialysis step. The PP fraction contained 62% of the M antigen activity and was resolubilized. Both fractions were chromatographed on CM Sepharose CL-6B. Polysaccharide C antigen was abundant in the S fraction and most of it did not bind to CM Sepharose. M antigen-enriched fractions were eluted with 0.5 M NaCl. Re-chromatography of the relevant S fraction (S-II) and PP fraction (PP-II) by linear gradient fast protein liquid chromatography (FPLC) removed protein and C impurities. M antigen purified by FPLC from the PP-II fraction was depleted of other antigens when Western blots were probed with anti-M, anti-H and anti-C monoclonal antibodies (Mabs). M antigen was identified as a 94 kDa glycoprotein containing a specific-protein epitope and an epitope that reacted with a Mab against the polysaccharide C antigen. M antigen can be purified from HMIN by tandem cation exchange chromatography of the precipitable fraction on an open CM Sepharose CL-6B column followed by linear gradient FPLC.

Blotting, Western↗

PubMed1986

Monoclonal antibodies against the M-protein and carbohydrate antigens of histoplasmin characterized by the enzyme-linked immunoelectrotransfer blot method.

Monoclonal antibodies (MAbs) of two different specificities were produced by immunizing mice with the semipurified M antigen of histoplasmin. One type, from clone CB4, was an immunoglobulin M that precipitated a polysaccharide present in histoplasmin and also formed immunoprecipitates with a cross-reactive polysaccharide present in extracts of Blastomyces dermatitidis and Coccidioides immitis. The second type of MAb, from clone EC2, was an immunoglobulin G that reacted in the enzyme-linked immunoelectrotransfer blot (EITB) assay with a doublet of proteins with an apparent molecular size of 70 to 75 kilodaltons. This molecule is proposed as the authentic M protein antigen that is recognized by M antibodies in sera from mice and rabbits immunized with Histoplasma capsulatum and from persons with histoplasmosis. The M factor also occurs in an abundant disulfide-bridged dimer which has a molecular size of 150 kilodaltons and is nonimmunoreactive under the conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Animals↗

PubMed1986

Monoclonal antibodies against Candida tropicalis mannan: antigen detection by enzyme immunoassay and immunofluorescence.

Three strains of mice were immunized with Candida tropicalis cell walls, and antibodies against mannan were detected by indirect enzyme immunoassay (EIA) in 3 of 9 BALB/c mice, 4 of 11 C57BL/6 mice, and 4 of 8 CFW mice. Responding mice produced immunoglobulin M (IgM), but IgG was not detected in their sera. Fusion of the high-responder BALB/c mouse with a plasmacytoma cell line resulted in 41 clones secreting antimannan monoclonal antibodies (MAbs). Four clones selected for propagation included one IgM and one IgG MAb that reacted with mannans of Candida albicans serotypes A and B and of C. tropicalis and two IgM MAbs specific for an epitope only in the mannans of C. albicans serotype A and C. tropicalis. One of the IgM MAbs, CB6, was an effective substitute for rabbit antibodies in the double-antibody sandwich EIA to detect antigenemia produced in rabbits infected with C. albicans A or C. tropicalis. It could function either as the peroxidase-conjugated indicator antibody or as the capture antibody. Two MAbs, CB6 (C. tropicalis and C. albicans A specific) and AC3 (C. tropicalis and C. albicans A and B specific), functioned in place of polyclonal antisera in the serotyping of C. albicans by immunofluorescence. There was 95.8% agreement in the results of serotyping using MAbs as reagents compared with rabbit antisera. Competitive inhibition in EIA between CB6 and monospecific antisera against C. albicans factors 1, 4, and 6 indicated that CB6 binds to an epitope which is probably factor 6. Serologic similarity between factor 4 and the binding site of MAb AC3 was also determined.

Animals↗

PubMed1986

Monoclonal antibodies against isoelectrically focused Nocardia asteroides proteins characterized by the enzyme-linked immunoelectro-transfer blot method.

Sera from rabbits immunized with culture filtrates and homogenates of Nocardia asteroides B1042 gave at least eight precipitin bands by immunoelectrophoresis. At least 20 proteins with isoelectric points (pls) in the pH 4 to 5.4 range were observed in isoelectric focusing patterns. The enzyme-linked immunoelectro-transfer blot (EITB) assay showed that several of the isofocused proteins reacted with rabbit antisera and with sera from nocardiosis and tuberculosis patients. Antibodies against three proteins with pls of 4, 4.43, and 4.68 (antigenic factors 1,6,8) were present in nocardiosis patients' sera. The proteins were excised from isofocused gels, and IgG monoclonal antibodies (MAbs) were produced by the hybridoma method. MAbs against factors 1 and 6 did not crossreact with cytoplasmic antigens of Mycobacterium chelonae, M. intracellulare serotypes 4B and 8A, M. fortuitum, M. gordonae, or M. kansasii in the EITB method. Factor 8 (MAb) crossreacted with antigens of M. intracellulare and M. fortuitum.

Animals↗

PubMed1985

Relative hydrophobicities of Actinomyces viscosus and Actinomyces naeslundii strains and their adsorption to saliva-treated hydroxyapatite.

The present study examined 42 strains of Actinomyces spp. to determine whether adsorption to saliva-treated hydroxyapatite (SHA) of the selected strains of this prominent group of dental-plaque bacteria correlated with hydrophobicity. The relative hydrophobicity of the strains was determined by their adsorption to hydrophobic gels (i.e., phenyl-Sepharose) and their aggregation in ammonium sulfate. Within serogroups the relative hydrophobicity for the strains was similar. The relative adsorption of strains to SHA was also similar within the respective serogroups. Strains which were relatively hydrophobic, as judged by their binding to the hydrophobic gel and aggregation in low concentrations of ammonium sulfate, adsorbed well to SHA. Strains which adsorbed poorly to SHA were relatively hydrophilic since they did not bind well to the hydrophobic gel and were only aggregated in relatively high concentrations of ammonium sulfate. Tween 80, a nonionic detergent known to inhibit hydrophobic interactions, blocked binding of cells to the hydrophobic gel, suggesting that hydrophobic interactions had been inhibited. However, Tween 80 exhibited no influence on the adsorption of cells to SHA. Thus, although there was a strong statistical correlation between the relative hydrophobicity of a strain and its adsorption to SHA, the data were consistent with the view that other interactions, such as ionic bonds and interactions between complimentary macromolecules, are involved in adsorption of the Actinomyces strains to SHA.

Actinomyces↗

PubMed1981

Serotyping Cryptococcus neoformans by immunofluorescence.

Four serotypes of Cryptococcus neoformans designated A, B, C, and D are currently recognized. Although an agglutination test is most often used to serotype C. neoformans in cultures, this test is not appropriate for typing the fungus in fixed tissues. A study to prepare fluorescent-antibody reagents for typing C. neoformans in cultures and to determine whether they can be used to type this fungus in fixed tissues was carried out. Antisera to one strain belonging to each of the four serotypes were prepared in rabbits by intravenous injection of whole Formalin-killed cryptococci. Each antiserum was labeled with fluorescein isothiocyanate and then adsorbed with cells of each of the heterologous serotypes. The adsorbed conjugates were then tested against six serotype A isolates and five isolates of each of the other three serotypes. Labeled serotype A or D antiserum adsorbed with either B or C cells stained the A and D, but not the B or C, isolates. Labeled serotype B antiserum adsorbed with A cells stained the B and C, but not the A or D, isolates. Labeled A antiserum absorbed with D cells differentiated A from D; labeled C antiserum absorbed with B cells differentiated C from B. Of the 21 test isolates, 17 could be serotyped in paraffin sections of tissues of experimentally infected mice.

Animals↗