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H R Buckley

Publications and source records attributed to H R Buckley.

At least 19 recordsLinked to original sources
PubMed1992

A role for complement receptor-like molecules in iron acquisition by Candida albicans.

Candida albicans, an opportunistic fungal pathogen of humans, is dependent upon iron for growth. Consequently, human serum inhibits C. albicans growth due to the presence of high affinity iron-binding proteins that sequester serum iron, making it unavailable for use by the organism. We report that in the inhibitory environment of human serum, the growth of C. albicans can be restored by the addition of exogenous hemoglobin or heme, but not by protoporphyrin IX, the heme precursor that does not contain iron. We further report that C. albicans can utilize cell surface proteins that are homologues of the mammalian complement receptors (CR) to rosette complement-coated red blood cells (RBC) and obtain RBC-derived iron for growth. The ability of Candida to acquire RBC-derived iron under these conditions is dependent upon Candida-RBC rosetting mediated by CR-like molecules. Unopsonized RBC do not support Candida growth in serum, and restoration of Candida growth in serum by complement-opsonized RBC is inhibited by monoclonal antibodies to the human CR type 3 (CR3). In addition, activation of the human alternative pathway of complement by Candida leads to "bystander" deposition of C3 fragments on the surface of autologous, unopsonized RBC, generating the ligands necessary for Candida-RBC rosetting. These results suggest that C. albicans has evolved a unique strategy for acquiring iron from the host, which exploits the host complement system, and which may contribute to the pathogenic potential of the organism.

Animals↗
PubMed1991

Detection of circulating candida enolase by immunoassay in patients with cancer and invasive candidiasis.

BACKGROUND: Invasive candidiasis is a major nosocomial infection that is difficult to diagnose. Few biochemically defined markers of invasive candidiasis are known. Initial findings suggested that the presence of candida enolase in the blood may be a novel marker for invasive candidiasis. METHODS: We tested 170 patients at high risk for invasive candidiasis for candida enolase antigenemia. All the patients had cancer and neutropenia. We detected antigen using a double-sandwich liposomal immunoassay for candida enolase in serially collected serum samples. Invasive candidiasis was proved by finding candida species in deep nonmucosal tissue, blood cultures, or both. Antigen testing was performed with the investigator blinded to tissue or culture diagnosis. RESULTS: Among 24 patients with proved invasive candidiasis, 149 serum samples were tested for enolase antigenemia; 80 were positive and 69 negative (sensitivity per sample, 54 percent). Multiple sampling improved the detection of antigenemia, which was found in 11 of 13 proved cases of deep tissue infection (85 percent) and in 7 of 11 proved cases of fungemia (64 percent). Specificity was 96 percent as measured against control groups including patients with mucosal colonization, bacteremia, and other deep mycoses. Antigenemia was detected in the absence of fungemia in 5 cases of deep tissue candidiasis, but was not detected in 6 cases of fungemia alone. CONCLUSIONS: Candida enolase antigenemia is a novel marker for invasive candidiasis. It may be a useful indicator of deep infection in patients with cancer and neutropenia and may complement the diagnostic usefulness of blood cultures.

Antigens, Fungal↗
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↗
PubMed1979

Characterization of antigens from Aspergillus fumigatus. IV. Evaluation of commercial and experimental preparations and fractions in the detection of antibody in aspergillosis.

Commercially available preparations of Aspergillus fumigatus, experimental extracts and fractions of A. fumigatus, and extracts of 5 other aspergilli were evaluated in a blind manner for their abilities to detect antibody in 66 serum specimens from patients with aspergilloma, allergic bronchopulmonary and invasive aspergillosis, and unconfirmed aspergillosis. Rocket immunoelectrophoresis was more sensitive than immunodiffusion in 2 dimensions. Although qualitative differences in antigen composition could be detected, commercial preparations compared favorably with the best experimental extracts in detecting positive specimens. Extracts from young, actively-growing myceliums were most effective and produced the largest numbers of precipitin bands. In contrast to all unfractionated preparations, several fractions of A. fumigatus were devoid of substances that react with C-reactive protein of serums, yet were as effective as the best preparations in detecting serums positive for Aspergillus.

Antibodies, Fungal↗
PubMed1978

Increasing the predictive value positive of the precipitin test for the diagnosis of deep-seated candidiasis.

Three hundred fifty human sera were tested by double immunodiffusion, crossed-line electrophoresis, and crossed immuno-affinoelectrophoresis with a concanavalin A intermediate gel for precipitating antibodies to antigens present in cytoplasmic extracts of Candida albicans. Sera from 48 of 287 hospitalized patients at risk of invasive candidiasis contained precipitating antibodies to Candida antigens. Of these 48 sera, 27 had precipitating antibodies only to cell-wall antigens present in the cytoplasmic extract, and 21 sera had precipitating antibodies to both cytoplasmic and cell-wall antigens. The latter sera came from patients who were 2.5 times as likely to have deep-seated candidiasis as those patients with precipitins exclusively to cell-wall antigens. Sera from seven of 22 patients with vaginal candidiasis and 10 of 41 patients with other fungal infections had precipitating antibodies to C. albicans cell-wall antigens; only two of these sera also contained precipitating antibodies to the cytoplasmic antigens. Crossed immunoaffinoelectrophoresis with concanavalin A reduced the number of false-positive results and increased the predictive value positive of the precipitin test for deep-seated candidiasis from 31% to 71%.

Antigens, Fungal↗
PubMed1977

Cell wall antigens in soluble cytoplasmic extracts of candida albicans as demonstrated by crossed immuno-affinoelectrophoresis with concanavalin A.

Crossed immuno-affinoelectrophoresis with Concanavalin A precipitated four antigens from soluble extracts of the yeast and mycelial phases of Candida albicans. Two of these antigens were also present in spheroplast preparations. One of the Concanavalin A precipitated antigens was destroyed by periodate and none were digested by pronase. Sequential treatment with pronase and periodate destroyed two of the remaining three antigens. The Concanavalin A precipitated antigens appear to be glycoproteins, two of which are associated with the cell wall of C. albicans.

Antigens, Fungal↗
PubMed1977

Characterization of antigens from type A and B yeast cells of Histoplasma capsulatum.

The antigenic composition of cytoplasmic extract and culture filtrate antigens of type A and B yeast cells of Histoplasma capsulatum grown in a synthetic medium was studied. These preparations from type A and B yeast cells contained varying amounts of protein, carbohydrates, and protein-carbohydrate complexes. The antigenic analysis of these preparations was performed by antigen-antiserum absorption with subsequent immunodiffusion and cross-immunoelectrophoresis with absorption in situ in an intermediate gel. All protein antigens observed in culture filtrate of either type A or B yeast cells were also present in the cytoplasmic extracts of the same type. The cytoplasmic extract of type A and B yeast cells each contained certain characteristic antigens that were not shared by the other type. Rocket immunoelectrophoresis with different molecular weight fractions of the antigenic preparations from both types indicated the polydisperse nature of Histoplasma yeast cell antigens.

Antigen-Antibody Reactions↗