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Biomedical subjects

H R Buckley

Publications and source records attributed to H R Buckley.

At least 37 records · Page 2Linked to original sources
PubMed1991

Identification and characterization of an immunodominant 58-kilodalton antigen of Aspergillus fumigatus recognized by sera of patients with invasive aspergillosis.

Sera from 38 patients with invasive aspergillosis were tested by Western immunoblotting for the presence of antibodies to antigens of Aspergillus fumigatus present in a mycelial extract of the organism. All of the sera contained antibodies to an antigen of molecular weight 58,000, which was which was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was the only antigen recognized in approximately 90% of the sera tested. The 58-kDa antigen is an abundant component of mycelial extracts composing approximately 50% of the Coomassie blue-stained protein. The antigen also contains carbohydrate, since it is stained by the carbohydrate stain periodic acid-Schiff and it binds to the lectin concanavalin A. It was purified by immunoaffinity chromatography employing a monoclonal antibody directed against an epitope on the 58-kDa antigen. Analysis of the purified antigen by gas-liquid chromatography revealed the presence of mannose, galactose, and glucose residues in a 2:1:2 ratio. The ratio of protein to carbohydrate is 1.16:1. The protein is slightly acidic, containing relatively high quantities of glutamic and aspartic acids, glycine, alanine, serine, and threonine. The 58-kDa antigen also contains phosphate groups as part of its structure. Serological activity was totally destroyed after treatment with sodium metaperiodate and was partially destroyed after treatment with pronase. The 58-kDa antigen was not able to hydrolyze protein.

Amino Acids↗
PubMed1991

Production and characterization of monoclonal antibodies to a 58-kilodalton antigen of Aspergillus fumigatus.

Eight monoclonal antibodies that recognize a serodiagnostically important 58-kDa antigen of Aspergillus fumigatus were produced and partially characterized. 2-7, 2-12, and 2-14 are of the immunoglobulin M class, and 2-2-1, 2-2-4, 2-2-6, 2-2-9, and 2-2-13 are all immunoglobulin G1(kappa) antibodies. Immunoblot analysis with A. fumigatus mycelial extract demonstrated that all of the monoclonal antibodies recognize a major 58-kDa antigen. The antigen was also detected by immunoblot analysis of 4- and 7-day culture filtrate preparations. 2-2-1, 2-2-4, and 2-2-6 cross-reacted with an antigen of approximately 55 kDa from an extract of Candida albicans. 2-7, 2-12, 2-14, and 2-2-4 formed a precipitin band with immunoaffinity-purified 58-kDa antigen by immunodiffusion. Results from indirect immunofluorescence assays with 2-7 and 2-2-9 showed fluorescent staining mainly on the surfaces of conidia and hyphae, indicating that the 58-kDa antigen may be cell wall associated. 2-2-9 and 2-2-13 and antibodies in patient and immune rabbit sera precipitated the [35S]methionine-labeled 58-kDa antigen. The 58-kDa antigen immunoprecipitated by each of the antibodies was enzymatically cleaved by Staphylococcus aureus V8 protease; one cleavage product, a 35-kDa fragment, was generated, indicating that the precipitated antigens share primary structure. Immunoblot analysis with an immunoaffinity-purified 58-kDa antigen showed that sera from patients with invasive aspergillosis reacted with the same antigen as that recognized by the monoclonal antibodies.

Animals↗
PubMed1990

Effect of Friend leukemia virus infection on susceptibility to Candida albicans.

Previous studies have demonstrated that Friend leukemia virus (FLV) induces a profound immunosuppression in susceptible mice. The studies described in this report indicate that mice infected with FLV have an increased susceptibility to subsequent infection with the opportunistic pathogen Candida albicans, as measured by increased numbers of C. albicans CFU in the kidneys of FLV-infected mice relative to uninfected controls. Experiments in which the NB-tropic and N-tropic strains of FLV were used suggest that virus replication or the resulting virus burden may be important in the observed increased susceptibility to C. albicans. Since neutrophils are believed to be important in the response of mice to systemic Candida infections, the effect of FLV infection on neutrophil candidacidal activity was investigated. The percentage of neutrophils present in unfractionated Proteose Peptone-elicited peritoneal exudates of mice infected with FLV for 14 days was significantly lower than in uninfected control mice or mice infected with FLV for 6 or 10 days. When neutrophils from FLV-infected and control mice were purified, adjusted to equal concentrations, and tested for in vitro candidacidal activity, neutrophils from mice infected with FLV for 14 days were deficient in their ability to kill C. albicans relative to normal controls and mice infected with FLV for 6 or 10 days. Addition of normal mouse serum increased killing in all groups but did not restore candidacidal activity of neutrophils from mice infected with FLV for 14 days to levels of control neutrophils or neutrophils from mice infected for 6 or 10 days with the virus. These results suggest a defect in neutrophil function, at the later stages of FLV infection, involving in vitro candidacidal activity. In addition, neutrophils from FLV-infected mice may be deficient in in vivo chemotactic activity. These defects in neutrophil function could account, at least in part, for the observed increased susceptibility of FLV-infected mice to C. albicans.

Animals↗
PubMed1987

Inhibition of germ tube development of Aspergillus fumigatus in cell-free transudate produced in subcutaneous chambers in rabbits.

The interaction of Aspergillus fumigatus conidia with host factors produced in rabbits was studied by means of subcutaneous, perforated plastic chambers. Transudate fluid from spore-free chambers, sampled 30 days after implantation, supported germ tube development and rapid hyphal growth of A. fumigatus in an assay in vitro. Inoculation of spores into chambers implanted 30 days previously produced a rapid infiltration of leukocytes, predominately neutrophils, into the chamber fluid. Cell-free supernatants, prepared from transudates collected 5-6 days after inoculation, inhibited germ tube development in vitro. This inhibition was also demonstrated using lysates derived from 3.6 X 10(6) leukocytes obtained from chambers 5 days after inoculation with 1 X 10(7) spores. However, lysates derived from greater than 10(7) leukocytes obtained from pre-inoculated chambers as well as peritoneal exudate cells did not inhibit germ tube development in vitro. The inhibitory activity of cell free supernatants was not altered by heating at 56 degrees C for 30 min but was destroyed by pronase treatment as well as boiling. These results provide evidence for a host defense mechanism against rapid hyphal extension mediated by the extracellular release of inhibitory factors by leukocytes.

Animals↗
PubMed1987

Purification of actin from Candida albicans and comparison with the Candida 48,000-Mr protein.

Actin was purified from Candida albicans cells by affinity chromatography by DNase-Sepharose and was recognized by immunoblotting with monoclonal antibody directed against chick muscle actin. The C. albicans 48-kilodalton protein recognized by sera from patients with invasive candidiasis was shown by DEAE chromatography and immunoblotting not to be identical with the purified C. albicans actin.

Actins↗
PubMed1985

Preparation and use of cytoplasmic antigens for the serodiagnosis of paracoccidioidomycosis.

A cytoplasmic antigen of Paracoccidioides brasiliensis (strain MTC) prepared from the yeast phase grown in the chemically defined medium of McVeigh and Morton is described. This antigen can be easily prepared, does not vary from lot to lot, and can be lyophilized without loss of activity or potency. In the immunodiffusion test, the cytoplasmic antigen demonstrated a sensitivity of 97% and a specificity of 100% when tested against 218 sera from 139 cases of paracoccidioidomycosis. When 177 sera from patients with fungal diseases other than paracoccidioidomycosis were tested by immunodiffusion, there were no false-positive reactions. In an enzyme-linked immunosorbent assay, the antigen was equally effective in identifying cases, giving a sensitivity of 100% and a specificity of 99% when a 1:640 titer was considered the threshold for clinical significance. Antigenic components in the cytoplasmic extract of P. brasiliensis were examined after fractionation by concanavalin A-Sepharose 4B column chromatography. The fraction of the cytoplasmic antigen that binds to the concanavalin A column is material identical to the specific b1 (antigen 1) precipitin band described by A. Restrepo and L. H. Moncada (Appl. Microbiol. 28:138-144, 1974).

Antibodies, Fungal↗
PubMed1984

Growth characteristics of the yeast phase of Paracoccidioides brasiliensis in a chemically defined medium.

The growth of four clinical strains of Paracoccidioides brasiliensis was investigated using the chemically defined medium of McVeigh and Morton. Emphasis was placed upon controlling conditions of inoculum preparation, age of inoculum used, and the homogeneity of samples used for analysis. The medium was evaluated for its ability to support growth of the yeast phase of P. brasiliensis at 37 degrees C. Cultures were followed for 240 h and growth patterns were determined by measuring optical density, dry weight, nucleic acids and protein. The medium is excellent for growing the yeast phase of P. brasiliensis. The exponential phase lasted an average of 135 h and the stationary phase 72 h; a decline began after 207 h. This defined medium supports abundant growth of the yeast phase of P. brasiliensis and may thus prove useful in the preparation of yeast-phase antigens.

Culture Media↗
PubMed1984

Identification and molecular weight characterization of antigens from Candida albicans that are recognized by human sera.

Antigenic components in the cytoplasmic extract of Candida albicans were examined after fractionation by concanavalin A-Sepharose and DEAE-Sephacel ion-exchange chromatography. Fractions from the DEAE column were tested by fused rocket immunoelectrophoresis for their reactivity with antibodies in the sera of 20 patients with disseminated candidiasis. Three groups of fractions (regions A, B, and C) from the DEAE column were defined by their reactivity with these sera. Immunoblot analysis with 20 human sera identified 18 antigenic components in regions A, B, and C. Region A contained nine antigens, region B contained four antigens, and region C contained five antigens. Region A contained an antigen with an apparent molecular weight of 48,000 that was recognized by 7 of 10 sera from patients with disseminated candidiasis. Immunoprecipitation experiments with labeled proteins from region A and 51 human sera also demonstrated the presence of a major antigen whose apparent molecular weight is 48,000 to 52,000. The 48- to 52-kilodalton protein is an abundant protein in region A and is the most frequently recognized protein by antibodies in the sera of patients with disseminated candidiasis. Patients with disseminated candidiasis had significantly higher levels of antibody (immunoglobulin G) (P less than 0.001) directed against the 48- to 52-kilodalton protein than did patients with noninvasive forms of candidiasis, patients with other fungal infections, or normal, healthy persons.

Antibody Formation↗
PubMed1984

Production and characterization of three monoclonal antibodies to Candida albicans proteins.

Three monoclonal antibodies, designated A2C7, C2C7, and F19, were produced which recognize proteins from Candida albicans. All are of the immunoglobulin G1 heavy chain and kappa light chain class. A2C7 and C2C7 immunoprecipitated three proteins contained in a partially purified fraction (region A) of a mycelial cytoplasmic extract of C. albicans. The apparent molecular weights of these proteins are 120,000 (120K) to 135K, 44K to 52K, to 38K. Monoclonal antibody F19 was reactive with proteins of 42K, 43K, and 50K in immunoblotting experiments. F19 was also able to form a precipitin band in agarose gel with protein(s) contained in region A. Limited proteolytic digestion of the three proteins immunoprecipitated by A2C7 and C2C7 demonstrated that both monoclonal antibodies recognized the same three Candida proteins and that there exists a significant degree of relatedness in primary structure among the three proteins. Proteins with apparent molecular weights of 120K to 135K, 44K to 52K, and 35K to 38K that were immunoprecipitated by sera from two patients with invasive candidiasis and by the serum from a rabbit immunized against a 48K (44K to 52K) Candida protein were also analyzed by limited proteolysis. Patterns of peptide fragments generated by enzymatic digestion of these proteins showed that the proteins recognized by the monoclonal antibodies are the same proteins recognized by antibodies in the sera of patients during an invasive Candida infection and by antibodies in the serum of the immune rabbit.

Animals↗
PubMed1984

Critical role of germ tube formation in the pathogenesis of candidal vaginitis.

A variant strain of Candida albicans incapable of hyphal production at 37 degrees C was used to study the role of germ tube formation in the pathogenesis of experimental vaginal candidiasis in rats. No difference was observed in the in vitro adherence at 25 degrees C of blastoconidia of the variant strain to vaginal epithelial cells when compared with the parent wild-type, germ tube-producing strain and multiple clinical isolates of C. albicans. However, after exposure to conditions favoring germ tube production, the adherence of the variant strain to epithelial cells was significantly less than that of germinated strains (P less than 0.01). In vivo animal studies revealed that the variant strain was less likely to result in vaginal colonization and infection than the wild-type strain and the other clinical isolates. Furthermore, infection, when established, was milder, often transient, and with significantly lower titers of cultured vaginal microorganisms obtained by lavage. Electron microscopic studies confirmed the failure of the variant strain to produce hyphae in vivo. The capacity of C. albicans to produce hyphae appears to be an important but nonessential virulence factor in the pathogenesis of candidal vaginitis.

Adhesiveness↗
PubMed1984

Purification and partial characterization of a Nocardia brasiliensis extracellular protease.

Nocardia brasiliensis possess proteolytic activities that can be readily detected in a variety of media. In a modified formulation of a growth medium originally used for Streptomyces aureofaciens, N. brasiliensis was found to secrete proteolytic enzymes, one of which was capable of hydrolyzing casein. This enzyme was purified to homogeneity from cell-free culture filtrates of N. brasiliensis. The purification procedure included ion-exchange chromatography on carboxymethyl-Sepharose, gel filtration on Sephadex G-100, and affinity chromatography, using a hemoglobin-Sepharose resin. The molecular weight of the N. brasiliensis protease was found to be 25,000 by gel filtration and 35,000 by sodium dodecyl sulfate-discontinuous gel electrophoresis. The enzyme is inhibited by o-phenanthroline and 8-hydroxyquinoline-5-sulfonic acid but is not affected by EDTA. Average values for its kinetic parameters were 0.288 mumol of hemoglobin solubilized per min per mg of enzyme for Vmax and 0.76 mM for Km, using hemoglobin as the substrate.

Chromatography, Gel↗
PubMed1983

Effects of culture density on the kinetics of germ tube formation in Candida albicans.

The relationship between culture density or phase of growth at 24.5 degrees C and the ability of Candida albicans to form germ tubes when shifted to 37 degrees C was investigated. Evidence is presented demonstrating germ tube production from liquid synthetic medium cultures at all phases of growth. Previous studies reported that only cells from stationary phase cultures were competent to form germ tubes. Comparisons between exponential and stationary phase cultures indicate more rapid and more synchronous germ tube production from cells growing in the exponential phase.

Candida albicans↗
PubMed1983

Differential protein synthesis in Candida albicans during blastospore formation at 24.5 degrees C and during germ tube formation at 37 degrees C.

To identify proteins synthesized during blastospore to germ tube transformation in Candida albicans, membrane and cytoplasmic protein fractions labelled with 14C were analysed by SDS-PAGE and autoradiography. Four cytoplasmic proteins were detected in pulse-labelled lysates prepared from cells forming blastospores and germ tubes at 37 degrees C, but not in pulse-labelled lysates prepared from cells forming blastospores at 24.5 degrees C. Three proteins detectable in 24.5 degrees C lysates were diminished in 37 degrees C lysates.

Autoradiography↗
PubMed1982

Isolation of a variant of Candida albicans.

During the course of Candida albicans antigen production, a variant of this organism was encountered which did not produce hyphae at 37 degrees C. Presented here are some of the characteristics of this variant. It produces hyphae at 25 degrees C on cornmeal agar and synthetic medium plus N-acetylglucosamine and Tween 80. At 37 degrees C, it does not produce hyphae on these media, although C. albicans normally does produce hyphae under these circumstances. In liquid synthetic medium, this variant does not produce hyphae at 37 degrees C. The variant strain was analyzed for DNA, RNA, protein content, and particle size. After 50 to 70 h in balanced exponential-phase growth, particle size distribution was narrow, and there were no differences in the DNA, RNA, or protein content per particle in the two strains. When balanced exponential-phase cultures were brought into stationary phase, both strains contained the same amount of DNA per cell.

Candida albicans↗