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Biomedical subjects

R A Calderone

Publications and source records attributed to R A Calderone.

At least 37 records · Page 2Linked to original sources
PubMed1988

Identification of C3d receptors on Candida albicans.

Pseudohyphal but not yeast forms of Candida albicans possess both iC3b and C3d receptors, as determined by rosetting with erythrocytes carrying iC3b (EAC3bi) or C3d (EAC3d). Rosetting with EAC3d was markedly reduced when pseudohyphae were heat killed or treated with trypsin or pronase but was not inhibited by several saccharides or aminosaccharides, including alpha-methyl-D-mannoside, or by pretreatment of pseudohyphae with concanavalin A. However, mannoproteins obtained by concanavalin A affinity chromatography of whole pseudohyphal extracts inhibited the attachment of EAC3d to C. albicans, whereas soluble (nonmannosylated) proteins were less active. Thus, although the C3d receptors appeared to be glycosylated, the oligosaccharide component of the receptor was apparently not involved in the recognition of C3d. To isolate these receptors, whole-cell extracts were separated by DEAE-Trisacryl chromatography. Fractions that inhibited rosetting were pooled and affinity purified by C3d-Thiol-Sepharose chromatography. The eluate from this affinity column inhibited attachment of C. albicans to EAC3d. Monoclonal antibodies to C. albicans were prepared, and three of these antibodies blocked rosetting. Western blotting (immunoblotting) with these antibodies indicated the presence of 62- and 70-kilodalton receptors for C3d in the extracts purified by C3d affinity chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Antibodies, Monoclonal↗
PubMed1986

Pathogenesis of vaginal candidiasis: studies with a mutant which has reduced ability to adhere in vitro.

A spontaneous, cerulenin-resistant mutant of Candida albicans (strain 4918-10) was found to adhere less readily to human vaginal mucosal cells in vitro than a wild type C. albicans (strain 4918). In a murine model of vaginal infection, strain 4918-10 was found to be less virulent than wild type C. albicans, i.e., the infection rate caused by 4918-10 was only 31% of that observed with wild type, 4918. A chitin-soluble extract (CSE) prepared from 4918 blocked attachment of yeast cells to human vaginal epithelial cells, while CSE from 4918-10 did not significantly reduce the attachment of yeasts to vaginal cells. Both 4918 and 4918-10 produced hyphae in vitro and in vivo, were negative for proteinase production and grew equally well at 28 degrees C and 37 degrees C. The data suggest that adherence to vaginal mucosa may be an important determinant in the pathogenesis of vaginal infection caused by C. albicans.

Adhesiveness↗
PubMed1986

Characterization of cerulenin-resistant mutants of Candida albicans.

Cerulenin, an inhibitor of fatty acid biosynthesis, has been used to study the role of the plasma membrane in germination of Candida albicans. To further elucidate this association, spontaneous, cerulenin-resistant mutants of C. albicans were isolated. Two of the mutants, 4918-2 and 4918-10, were compared biochemically with wild-type cells (4918). All strains grew equally well at 37 degrees C and synthesized fatty acids at comparable rates in the absence of the drug. In the presence of cerulenin, wild-type cells did not proceed through a logarithmic growth stage and exhibited a significantly impaired ability to incorporate [3H]acetate into newly synthesized lipid material. All strains were examined ultrastructurally. Alterations were observed in the membranous structures of cerulenin-treated wild-type cells. Such changes were not observed in cerulenin-treated mutant strains. Further examination of mutant strains revealed differences in cell wall protein and polysaccharide compositions when compared with those of wild-type cells. These apparent alterations in cell surface components may be correlated with the reduced abilities of mutant strains to adhere, in vitro, to mammalian cells.

Adhesiveness↗
PubMed1985

Yeast adhesion in the pathogenesis of endocarditis due to Candida albicans: studies with adherence-negative mutants.

Two spontaneous cerulenin-resistant mutants of Candida albicans, 4918-2 and 4918-10, were unable to adhere in vitro in fibrin-platelet clots. Because in vitro adherence correlates well with colonization of nonbacterial thrombotic endocarditis on traumatized valvular endocardium, 50% infectious dose studies were performed with a rabbit model of endocarditis. Wild-type C. albicans required 10(3.6) +/- 0.12 cfu in comparison with 10(5.73) +/- 0.31 and 10(7.3) +/- 0.21 cfu for mutants 4918-2 and 4918-10, respectively. The relative avirulence of mutant strains in producing endocarditis was not attributed to accelerated clearance of these strains from the bloodstream. In fact, clearance of wild-type and mutant strains was almost identical. In the same animals renal candidiasis was observed with all strains of C. albicans, although the number of cfu per gram of kidney was higher after infection with wild-type C. albicans. Thus, strains of C. albicans with reduced ability to adhere in vitro to a fibrin-platelet matrix are relatively avirulent in the rabbit endocarditis model.

Adhesiveness↗
PubMed1985

Microbial adhesion to fibronectin in vitro correlates with production of endocarditis in rabbits.

Microbial adhesion to the constituents of nonbacterial thrombotic endocarditis (NBTE) is an important early event in the pathogenesis of infective endocarditis. Fibronectin is a ubiquitous mammalian glycoprotein with diverse functions which binds to certain bacteria but not to others. In this study, we determined that fibronectin is present on the surface of NBTE (after catheter-induced aortic valve trauma) but not on normal rabbit cardiac valvular endothelium. The adhesion of various bacteria and yeasts to human fibronectin in tissue culture wells was then measured. Microorganisms with a high isolation frequency from endocarditis cases (Staphylococcus aureus, Candida tropicalis, C. albicans, Streptococcus faecalis, S. sanguis) bound significantly better (P less than 0.01) to fibronectin in vitro than other organisms (Escherichia coli, C. krusei, Pseudomonas aeruginosa) rarely implicated in this disease. Microbial adhesion to fibronectin correlated closely with the propensity of each organism to produce endocarditis in rabbits (e.g., ID50) with preexistent NBTE. A similar distribution was noted after binding of soluble radiolabeled fibronectin to bacteria in suspension. The results suggest that fibronectin, expressed on the surface of NBTE, may mediate microbial adhesion of circulating organisms to initiate colonization during the early pathogenesis of infective endocarditis.

Animals↗
PubMed1984

In vitro binding of Candida albicans yeast cells to human fibronectin.

The binding of Candida albicans yeast cells to human fibronectin (Fn), a major glycoprotein of mammalian cells, was studied using an in vitro assay. Adherence was quantitated in microtiter dishes coated with Fn to which radiolabeled yeast cells were added. Under optimum conditions of the assay, i.e., 1 mM CaCl2 and 70 micrograms Fn protein, approximately 40% of the radiolabeled yeast cells adhered to the Fn. Adherence to Fn was greater at 30 degrees C than at 4 degrees C and was greater with viable yeast cells than with heat-killed cells. Candida albicans (two strains) and C. tropicalis adhered to Fn to a greater extent than C. pseudotropicalis, C. krusei, or Saccharomyces cerevisiae. Pretreatment of C. albicans with chymotrypsin, pronase, or papain, but not pepsin, decreased adherence to Fn. Blocking experiments using mannan, sugars, or amino sugars were carried out by preabsorbing the Fn with each of the above-mentioned compounds. Candida mannan blocked adherence of C. albicans to Fn. The mannan effect was dose dependent. However, adherence of C. albicans to Fn was not significantly reduced by mannose, glucose, or several other sugars. The role of FN as a receptor for the binding of C. albicans yeast cells to buccal and vaginal epithelial cells was investigated also using an in vitro assay. We determined, using indirect fluorescent antibody techniques, that both buccal and vaginal epithelial cells possessed Fn. In addition, yeast cells, when pretreated with Fn, showed reduced adherence with buccal and vaginal cells when compared with nontreated cells. These studies may indicate a role for Fn in the adherence of C. albicans to buccal and vaginal epithelial cells.

Candida↗
PubMed1984

Adherence of Candida albicans to buccal and vaginal epithelial cells: ultrastructural observations.

The adherence of Candida albicans to human buccal and vaginal epithelial cells was studied by transmission electron microscopy. Adherence to epithelial cells was confirmed by both a radiometric assay as well as direct microscopic examination of stained cell preparations. Ultramicroscopic preparations revealed that yeast cells were closely appressed to epithelial cell surfaces and were often partially enclosed within phagocyticlike invaginations of the epithelial cells. A murine model of vaginitis caused by C. albicans was also used to study adherence to epithelial cells and to follow the course of colonization. Ultramicroscopic preparations of murine vaginal tissue revealed that within 2 h postinfection, yeast cells could be seen adhering to epithelial cells. At 6 h postinfection, hyphae and yeast cells were not only found on the epithelial cell surface but also within the submucosal tissue. When observed on the epithelial cell surface, Candida cells were either attached to host cells, or when infected tissue was stained with ruthenium red, Candida cells were observed on the epithelial surface embedded within an electron-dense matrix. Fungal elements were abundant in the submucosa at 24 h postinfection and were still observed on the epithelial cell surface; all of this was accompanied by an inflammatory response.

Adhesiveness↗
PubMed1983

Inhibitory effect of cerulenin and sodium butyrate on germination of Candida albicans.

Candida albicans germination in liquid medium was inhibition by the antilipogenic agent cerulenin and the fatty acid sodium butyrate. Although these inhibitors prevented germ tube emergence at concentrations of 1 microgram/ml and 20 mM, respectively, neither significantly affected cell viability as judged by trypan blue staining or the rate of protein biosynthesis throughout the time course of the experiments. Cerulenin treatment resulted in inhibition of lipid biosynthesis, but lipid biosynthetic capabilities remained unaltered in sodium butyrate-supplemented cultures. Because each inhibitor blocks germination by different mechanisms, their utility in distinguishing events directly correlated to germination was examined. In this context, chitin synthase activity was inhibited by both compounds, confirming the importance of chitin biosynthesis in C. albicans germination.

Antifungal Agents↗
PubMed1983

Influence of preformed antibody on the pathogenesis of experimental Candida albicans endocarditis.

The influence of preformed antibody on the induction of experimental Candida albicans endocarditis was investigated by both in vitro and in vivo techniques. Preincubation of C. albicans with immune serum (raised in rabbits by intravenous injection of Formalin-killed yeast cells) decreased adhesion to the constituents of nonbacterial thrombotic endocarditis, e.g., fibrin plus platelets, in vitro. Two different methods, with radiolabeled or viable yeast cells, were confirmatory and demonstrated decreased adhesion of immune serum-treated C. albicans cells to 0 to 7.8% of control values (P less than 0.001). These results correlated with protection from the development of C. albicans endocarditis in the immunized rabbits. The mean (+/- standard deviation) infectious dose for 50% of the animals was 10(5.29) +/- 10(0.07) in 48 control animals versus 10(7.11) +/- 10(0.22) in 37 immunized rabbits (P less than 0.001). These studies suggest that humoral antibody may protect against C. albicans endocarditis, perhaps through inhibition of adhesion, a crucial early step in the pathogenesis of endocarditis.

Adhesiveness↗
PubMed1981

Role of surface mannan in the adherence of Candida albicans to fibrin-platelet clots formed in vitro.

An alkali-soluble extract from a cell wall preparation of Candida albicans was conjugated to sheep erythrocytes (SRBC) by using periodate oxidation or concanavalin A. Conjugated SRBC readily attached to a fibrin-platelet matrix, whereas nonconjugated SRBC did not. To determine the active component which promoted attachment of SRBC, the alkali-soluble fraction was treated with alpha-mannosidase, pronase, or glusulase or chemically degraded by acetolysis. The treated extract was then reconjugated with SRBC, and attachment was measured. When treated with alpha-mannosidase or degraded by acetolysis, the alkali-soluble extract failed to promote the adherence of SRBC to the fibrin-platelet matrix. Pronase- or glusulase-digested extract promoted attachment equally as well as untreated controls. In addition, when preabsorbed with antiserum to whole cells of C. albicans, the alkali extract abrogated the inhibition of adherence by antiserum, thus indicating its antigenicity. The extract consisted primarily of polysaccharide (72%) and contained a small amount of protein (less than 1%). Mannose and glucose (ratio, 3:1) were detected by gas-liquid chromatography. These data indicate that cell surface mannan may play an important role in the adherence of C. albicans to the fibrin-platelet matrices which form in vivo on the endocardium of heart valves.

Adhesiveness↗
PubMed1981

Platelet interactions with Candida albicans.

The interaction of human platelets and Candida albicans was studied. Platelet-rich plasma was obtained from freshly drawn blood or outdated platelet concentrates. From the platelet-rich plasma, a platelet extract was derived which stimulated germ tube formation by C. albicans when incubated with yeast cells at 37 degrees C. The active component(s) was heat stable, trypsin sensitive, and ribonuclease and deoxyribonuclease insensitive, and possessed cationic properties since it readily attached to carboxymethyl-Sephadex. The active component(s) seemed to bind to heparin also, since germ tube-promoting activity was eluted from a heparin-cyanogen bromide-activated Sepharose 4B column. In addition, platelet-derived growth factor (Collaborative Research, Inc.) stimulated germination when incubated with low amounts (0.4% final concentration) of bovine calf serum. The aggregation of platelets, prepared as platelet-rich plasma by C. albicans cell wall or alkali-extracted cell wall fractions, was also studied. Aggregation of platelets was observed when cell wall or cell wall fractions were incubated with platelet-poor plasma at 37 degrees C for 20 min and then added to platelet-rich plasma. The component of platelet-poor plasma which promoted aggregation of platelets by C. albicans cell wall or alkali-extracted fractions was inactivated at 56 degrees C (30 min) and by cobra venom factor, indicating a role for the alternate complement pathway in the aggregation response.

Candida albicans↗
PubMed1980

Adherence of Candida albicans to a fibrin-platelet matrix formed in vitro.

The adherence of Candida albicans to a fibrin-platelet matrix formed in vitro was studied. Platelet-rich plasma obtained from rabbits was incubated with thrombin and CaCl2 to form a clot in tissue culture dishes. Such clots were then infected with 3 x 10(7) C. albicans cells per 0.3 ml prelabeled with [U14C]-glucose, and the percent adherence was measured after 30 min of incubation by counting the radioactivity in saline washes of the clot as well as a streptokinase-streptodornase digest of the corresponding clot. Heat- and formaldehyde-killed cells did not adhere as well as viable cells. Pretreatment of C. albicans with trypsin, chymotrypsin, and pronase reduced adherence to the clots. Normal rabbit serum and anti-Candida antiserum also inhibited adherence 40 and 100%, respectively, Diethylaminoethyl-purified anti-Candida gamma globulin (1:8) completely inhibited adherence, whereas purified normal serum gamma globulin did not. Several Candida spp. and Saccharomyces cerevisiae showed differences in their ability to adhere to clots. C. albicans and C. stellatoidea presented the highest adherence, whereas C. krusei, C. guilliermondii, and S. cerevisiae adhered less readily. Other species were intermediate in their ability to adhere.

Blood Platelets↗
PubMed1979

Regulation and solubilization of Candida albicans chitin synthetase.

A cytoplasmic component which inhibited the activation of chitin synthetase was studied in the dimorphic fungus Candida albicans. The inhibitor was found to be heat stable and trypsin sensitive and was only effective when incubated with a vacuolar protease, an activator of chitin synthetase, before the activation of chitin synthetase. In addition, the particulate chitin synthetase from the yeast form of C. albicans was solubilized by a sodium cholate-digitonin extraction and subsequently was purified approximately 30-fold by Sepharose column chromatography and Amicon XM 100 filtration. Activity of the soluble enzyme was increased by the addition of trypsin or phosphatidyl serine. The molecular weight of the enzyme was estimated to be 400,000.

Candida albicans↗
PubMed1978

Candida albicans endocarditis: ultrastructural studies of vegetation formation.

Candida albicans endocarditis was established in rabbits after transaortic catherization. Within 30 to 90 min after infection, C. albicans was observed by scanning electron microscopy on the valve surface. The organisms were predominantly associated with host deposits of erythrocytes, phagocytes, platelets, and fibrinous-appearing material, which collectively appeared on the valve surface in response to trauma. Within 48 h after infection, vegetations composed of these same host components were observed on the heart valves, although Candida cells were not demonstrable on the valve or vegetation surface. When the vegetations were examined by transmission electron microscopy, Candida blastospores were only observed within phagocytic cells (predominantly monocytes) enmeshed within the vegetation matrix. Many of the intracellular organisms were undergoing degradation, as evidenced by a reduction in electron density of the cell wall. Other fungi had highly electron-dense cell walls and germ tubes. Phagocytic cells containing germinating Candida were highly vacuolated and were observed at various stages of cell lysis. After 7 days of infection, the vegetation contained a dense meshwork of fibrin and Candida pseudohyphae with 10(8) to 10(9) colony-forming units/g of vegetation. The mature vegetation was devoid of phagocytic cells and continued to grow until the death of the animal.

Animals↗
PubMed1978

Inhibition of specific amino acid uptake in Candida albicans by lysosomal extracts from rabbit alveolar macrophages.

Lysosomal-rich fractions, obtained from normal rabbit alveolar macrophages, were extracted and tested for their effects on Candida albicans. The uptake and incorporation of various compounds (amino acids, uridine, 2-deoxy-D-glucose, and Rb+) by C. albicans were measured in the presence and absence of extract. These studies demonstrated that the extract had a specific effect on the uptake of certain amino acids by C. albicans. Of the amino acids tested, the uptake of methionine valine, lysine, phenylalanine, and leucine was drastically reduced in the presence of extract, whereas proline and glutamic acid uptake was unaffected. Those amino acids whose uptake was inhibited have been shown to be transported in other yeasts by a general amino acid permease. The existence of a general amino acid permease in C. albicans is compatible with our data. Additionally, the extract had no effect on the uptake of uridine, 2-deoxy-D-glucose, and Rb+. Leakage of 86Rb by C. albicans was detected in the presence of the extract. Viability of Candida, as measured by colony-forming ability, decreased after a 16-h incubation of C. albicans with the extract.

Amino Acids↗