Biomedical subjects

R Calderone

Publications and source records attributed to R Calderone.

26 records · Page 2Linked to original sources

PubMed1994

Macrophage interactions with Candida.

The monocyte/macrophage, in comparison to the neutrophil, would appear to have a limited role in protection against C. albicans. This statement is based on the observations of several investigators who report that these cells have very little killing capacity unless they are activated by cytokines such as IFN-gamma and GM-CSF. The mechanisms of killing by these cells appear to include both oxidative and nonoxidative mechanisms, the latter perhaps being more important. The mechanisms of killing may be different for monocytes and macrophages. Granulocyte-macrophage colony-stimulating factor and its effect on monocytes has been studied using Candida as a target organism. Two explanations for the enhancement of monocyte killing by this cytokine have been proposed: GM-CSF augments both superoxide anion and the level of mannose receptors on treated monocytes. Both of these changes could be significant in the increased killing capacity of these cells.

Animals↗

PubMed1993

Analysis of mannoproteins from blastoconidia and hyphae of Candida albicans with a common epitope recognized by anti-complement receptor type 2 antibodies.

Mannoproteins of approximately 50 kDa from blastoconidia and 60 kDa from hyphae of Candida albicans reacted in Western blots (immunoblots) with either a polyclonal rabbit antiserum (CA-7) or a monoclonal antibody (CA-A) to the C. albicans C3d-binding protein (complement receptor type 2). The glycosylated nature of these proteins was demonstrated by their reactivity with concanavalin A and by selective labeling with the biotin-hydrazide reagent following periodate oxidation. Differences in the oligosaccharides of these proteins were observed in regard to their reactivity with lectin-peroxidase reagents and sensitivity to glycosidases such as N-glycanase or endoglycosidase F (but not endoglycosidase H). The 60-kDa mannoprotein reacted with wheat germ agglutinin, while the 50-kDa mannoprotein did not. Treatment of the 60-kDa mannoprotein with the glycosidases mentioned above resulted in its conversion into a species of 40 to 45 kDa. Enzyme treatment had no obvious effect on the electrophoretic mobility of the 50-kDa species from blastoconidia. Both the 50- and 60-kDa glycoproteins remained immunoreactive after treatment with the glycosidases. Reactivities of the two mannoproteins to neuraminidase also differed. Finally, the 50-kDa (blastoconidia) and the 60-kDa (hyphae) mannoproteins were purified by using ion-exchange chromatography and electroelution. The purified proteins differed in net charge, the 60-kDa species having a more acidic pI. Functional activity of the purified mannoproteins was demonstrated, as each inhibited the rosetting of antibody-sensitized sheep erythrocytes conjugated with iC3b or C3d by hyphae. Thus, an epitope(s) common to both a mycelial and blastoconidial mannoprotein is associated with a structurally different oligosaccharide for each growth form.

Animals↗

PubMed1992

Expression of the C3d-binding protein (CR2) from Candida albicans during experimental candidiasis as measured by lymphoblastogenesis.

The complement C3d-binding protein (CR2) of Candida albicans has been purified by immunoaffinity chromatography, and its specificity has been characterized by immunoblotting with monoclonal antibodies to the C. albicans CR2 and the mammalian CR2. Recent studies with immunoelectron microscopy indicated that the CR2 was expressed during a systemic infection in a murine model of candidiasis. As a continuation of these observations, the immunogenicity of the C. albicans CR2 was investigated in a lymphoblastogenesis assay. Lymph node cells as well as splenic lymphocytes from mice infected subcutaneously with viable blastoconidia of C. albicans reacted to the C. albicans CR2 to a significantly greater extent than did lymphocytes from uninfected mice (P less than 0.01). The maximum stimulation of splenic lymphocytes by the purified receptor occurred at a concentration of 0.54 micrograms of protein per ml after 72 h of incubation of lymphocytes and receptor. Also, splenocytes from infected or CR2-immunized mice exhibited significantly reduced responses to the T-cell-dependent mitogen phytohemagglutinin (P less than 0.01). These data indicate that lymphocytes from infected mice respond to the C. albicans CR2 in a lymphoproliferation assay to a greater extent than do lymphocytes from uninfected mice, indicating that the CR2 is expressed in vivo.

Animals↗

PubMed1990

Purification and characterization of the extracellular C3d-binding protein of Candida albicans.

A C3d-binding glycoprotein was purified from the culture filtrate of Candida albicans by preparative isoelectric focusing. The protein possessed a pI of 3.9 to 4.1 and could inhibit rosetting of EAC3d (sheep erythrocytes conjugated to C3d) by pseudohyphae of C. albicans. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol, the protein migrated as a doublet with apparent molecular masses of 55 and 60 kilodaltons (kDa) and as a 50-kDa band in nonreducing gels. These results were observed with Aurodye stain for proteins. Western immunoblot, and concanavalin A stain, which indicates that both bands contain carbohydrate as well as antigenic determinants. The treatment of purified glycoprotein with endoglycosidase F but not endoglycosidases H, N, and O resulted in a complete conversion of the doublet into a faster-migrating broad band with an apparent molecular mass of 45 kDa. When the amino acid analysis of the C3d-binding protein was compared with that of the CR2 from B lymphocytes, significant differences were observed. These data indicate that C. albicans secretes a C3d-binding protein during growth in vitro which appears to be different from the mammalian C3d receptor.

Amino Acids↗

PubMed1990

Isolation and characterization of cell surface mutants of Candida albicans.

Mutant strains of Candida albicans were obtained by selecting for cells that escaped agglutination by a polyclonal antiserum raised against standard C. albicans serotype A isolate B311. Mutants were obtained from strains B311 and B792 and from four strains isolated from patients with acquired immunodeficiency syndrome. All 15 tested mutants retained characteristic sugar assimilation patterns. All but one of the mutants retained the ability to form germ tubes and chlamydospores. Two mutants from an acquired immunodeficiency syndrome-derived isolate were deficient in binding complement ligands iC3b and C3d, whereas another mutant was deficient in binding ligand iC3b but not C3d. The hyphae of these three mutants lacked antigens when examined by Western immunoblotting with monoclonal antibody Ca-A, which detects several glycoproteins, including C3d-binding proteins. One of the complement-binding-deficient mutants was tested for its ability to colonize the gastrointestinal tract of rabbits but did not differ from the wild-type parent in site or degree of colonization. The proton magnetic resonance spectra of bulk mannan carbohydrate extracted from tested mutants showed the loss of a signal characteristic of the mannosyl alpha-PO4 linkage; each mutant also had a distinct pattern of other changes.

Agglutinins↗

PubMed1988

Candida albicans C3d receptor, isolated by using a monoclonal antibody.

Pseudohyphae of Candida albicans possess a receptor for C3d, a fragment of the complement component C3. This receptor was partially purified by using a monoclonal antibody (CA-A) that previously had been shown to inhibit the binding of C3d to C. albicans pseudohyphae. Purified immunoglobulin G from ascites fluid (CA-A) was coupled to a cyanogen bromide-activated Sepharose column, and an affinity-purified fraction (A2) from C. albicans pseudohyphae was obtained. This fraction inhibited rosetting of the EAC3d receptor by pseudohyphae and appeared to contain glycoprotein, since receptor activity could be removed when A2 was incubated with lectins specific for mannose and glucose. A2 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two polypeptides of approximately 60 and 70 kilodaltons (kDa) were consistently identified in reducing gels. The 60-kDa protein was identified as a glycoprotein by concanavalin A binding. A2 was further analyzed by high-pressure liquid chromatography (HPLC). Of three fractions obtained by HPLC, one containing the 60-kDa protein was found to have receptor activity. When analyzed by HPLC, this protein was found to contain mannose and glucose in approximately equal amounts. Both immunofluorescence and electron microscopy of pseudohyphae treated with CA-A identified A2 as a surface moiety. Thus, the C3d receptor of C. albicans, isolated with CA-A, is a glycoprotein of approximately 60 kDa.

Antibodies, Monoclonal↗

PubMed1987

Characterization with crossed immunoelectrophoresis of some antigens differentiating a virulent Candida albicans from its derived, avirulent strain.

Antigenic differences between a wild-type virulent Candida albicans 4918 (wt) and its spontaneous avirulent mutant (m-10) were found with crossed immunoelectrophoresis. Yeast cell extracts as well as soluble protein and mannoprotein fractions obtained by affinity chromatography on concanavalin A (Con A) were analyzed. Sera from patients with candidiasis and antisera from rabbits infected with live wt cells and boosted with wt extracts or rabbits immunized with purified wt cell wall preparation were used as counter reactants. Qualitative differences in serum precipitins formed by patients with suspected or culture-proven candidiasis to polysaccharide antigens of wt and m-10 origin were observed. In comparison, except for a spike-formed precipitate detected only with the wt extract, the serum from infected rabbits precipitated the wt and m-10 cell wall polysaccharide antigens about equally. The same type of precipitate was also found with the Con A wt mannoprotein fractions but was again lacking with the m-10 mannoproteins. This precipitate, with extremely slow electromobility in the first dimension, may be related to some special immunodeterminant of the wt mannan molecule. No substantial differences in the precipitation patterns of the Con A wt and m-10 proteins were found when analyzed with patients' sera or rabbit anti-cell sera. However, using these protein fractions with anti-cell wall sera revealed a larger number of precipitates for the wt as opposed to the m-10 strain. The observed antigenic differences between the virulent- and the avirulent-derived strains seem to be mainly associated with cell wall determinants (components) and might be related to the greater adherence and infectivity of the wild strain.

Animals↗

PubMedDate not supplied

Characterization of the phosphoribosylpyrophosphate synthetase gene from Candida albicans.

Phosphoribosylpyrophosphate (PRPP) synthetase catalyzes the transfer of phosphate from ATP to D-ribose-5-phosphate during the synthesis of purine and pyrimidine nucleotides and tryptophan and histidine biosynthesis in a variety of organisms. We cloned and sequenced the PRPP synthetase gene (PRS1) of Candida albicans because, in Saccharomyces cerevisiae, a deficiency in PRPP synthetase activity interacts with a mutation in ELM4-1 (elongated morphology) to cause constitutive pseudohyphal growth in nitrogen-rich media. In order to study the role of the C. albicans PRS1 in growth and morphogenesis, we used gene disruption to isolate PRS1 mutants; however, while heterozygous PRS1 clones were readily obtained, homozygous, null strains were not recovered indicating that PRS1 is probably essential for growth of the organism. Heterozygotes in PRS1 produced approximately 35% less PRPP synthetase (P = 0.0004) and exhibited a similar reduction in transcript levels. Confirmation of a heterozygous, single disruption in PRS1 was obtained by I-SceI digestion of chromosomal-sized DNA and Southern blot hybridizations. While no role in morphogenesis is elucidated by this work, the data strongly suggests that PRS1 is an essential gene in C. albicans and supports earlier results that indicated the presence of a single PRS gene in C. albicans.

Candida albicans↗