Effect of combined antibacterial and antifungal treatment in severe atopic dermatitis.
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Adult↗
Biomedical subjects
K C Hazen
Publications and source records attributed to K C Hazen.
Cell wall anchoring to cytoplasmic membrane of Candida albicans.
Cell wall ultrastructure of the opportunistic pathogenic yeast Candida albicans was investigated by stereoscopic freeze-etching technique. Three wall layers were distinguishable by this technique. No clear periplasmic space was evident. Bilayer membrane invaginations were extensive. The outermost regions of the membrane invaginations were lined with thin, spine-like fibrils, which extended into the cell wall. We suggest that the fibrils along the invaginations are involved in anchoring the cell wall to the membrane.
Candida albicans↗
New and emerging yeast pathogens.
The most common yeast species that act as agents of human disease are Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans. The incidence of infections by other yeasts has increased during the past decade. The most evident emerging pathogens are Malassezia furfur, Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Candida lusitaniae, and Candida krusei. Organisms once considered environmental contaminants or only industrially important, such as Candida utilis and Candida lipolytica, have now been implicated as agents of fungemia, onychomycosis, and systemic disease. The unusual yeasts primarily infect immunocompromised patients, newborns, and the elderly. The role of central venous catheter removal and antifungal therapy in patient management is controversial. The antibiograms of the unusual yeasts range from resistant to the most recent azoles and amphotericin B to highly susceptible to all antifungal agents. Current routine methods for yeast identification may be insufficient to identify the unusual yeasts within 2 days after isolation. The recognition of unusual yeasts as agents of sometimes life-threatening infection and their unpredictable antifungal susceptibilities increase the burden on the clinical mycology laboratory to pursue complete species identification and MIC determinations. Given the current and evolving medical practices for management of seriously ill patients, further evaluations of the clinically important data about these yeasts are needed.
Carrier State↗
Expression of surface hydrophobic proteins by Candida albicans in vivo.
Candida albicans modulates cell surface hydrophobicity during growth and morphogenesis in vitro. To determine if surface hydrophobicity is expressed during pathogenesis, we generated a polyclonal antiserum against yeast hydrophobic proteins. The antiserum was then used for indirect immunofluorescence analysis of tissues from mice colonized and chronically infected with C. albicans. Results demonstrated that yeast hydrophobic proteins are exposed on fungal cells present in host tissues. The polyclonal antiserum distinguished between hydrophobic and hydrophilic cell surfaces in vitro and gave similar staining patterns and intensities for C. albicans cells in vivo. Of the yeast forms present within tissue lesions, approximately half exhibited moderate to intense immunofluorescence with the antiserum. Immunoblot analysis indicated that antigens recognized by the antiserum are predominantly low-molecular-mass hydrophobic proteins that are expressed by different C. albicans isolates and are expressed regardless of growth temperature. Taken together, the immunofluorescence and immunoblot analyses of antigens indicate that C. albicans displays surface hydrophobic proteins during pathogenesis and these proteins are available for hydrophobic interactions with host tissues. The effect of hydrophobic protein exposure on the virulence of C. albicans is discussed.
Candida albicans↗
Cell surface hydrophobicity and medically important fungi.
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Cell Membrane↗
Chronic sinusitis. Relationship of computed tomographic findings to allergy, asthma, and eosinophilia.
OBJECTIVE: To develop a technique for evaluating the severity of chronic sinus disease and to examine the correlation with allergy, asthma, and eosinophilia. DESIGN: A survey of 104 patients undergoing surgery for chronic sinusitis. SETTING: A university hospital ear, nose, and throat clinic. PATIENTS: A referral population of adult patients being scheduled for endoscopic sinus surgery was eligible; 104 completed questionnaires and agreed to participate. MAIN OUTCOME MEASURES: Computed tomographic scans were reviewed and scored for extent of disease. Serum samples were assayed for total IgE and specific IgE antibodies to common inhalant allergens. Peripheral blood samples were analyzed for total eosinophil count. Surgical biopsy specimens were examined for eosinophilia and cultured for bacteria and fungi. RESULTS: Extensive disease was present in 39% of subjects and correlated well with asthma, specific IgE antibodies, and eosinophilia, but not with elevated total IgE. Among patients with peripheral eosinophilia, 87% had extensive disease. All cultures grew aerobic bacteria; anaerobes and fungi were uncommon. CONCLUSIONS: We present a system for quantitation of disease extent using computed tomographic scans of patients with chronic sinusitis. The well-accepted associations of chronic sinusitis with asthma and allergy appear to be restricted to the group with extensive disease. The presence of peripheral eosinophilia in patients with sinusitis indicates a high likelihood of extensive disease.
Adult↗
Potential use of BacT/Alert automated blood culture system for antifungal susceptibility testing.
A simple, rapid susceptibility test is needed to determine the possible resistance of yeasts to commonly used antifungal agents. The BacT/Alert automated blood culture system was evaluated as one technology for development of such a test. Yeast nitrogen base was used as the growth medium, and amphotericin B was the test antifungal agent. Isolates of various Candida species, Torulopsis glabrata, Saccharomyces cerevisiae, and Cryptococcus neoformans were evaluated. The results suggest that detection of amphotericin B resistance of yeast isolates within 12 to 14 h after inoculation of test medium is possible.
Amphotericin B↗
Hydrophobic cell wall protein glycosylation by the pathogenic fungus Candida albicans.
Cell surface hydrophobicity influences adhesion and virulence of the opportunistic fungal pathogen Candida albicans. Previous studies have shown that cell surface hydrophobicity is due to specific proteins that are exposed on hydrophobic cells but are masked by long fibrils on hydrophilic cells. This observation suggests that hydrophobic cell wall proteins may contain little or no mannosylation. In the present study, the glycosylation levels of three hydrophobic cell wall proteins (molecular mass range between 36 and 40 kDa) derived from yeast cells were examined. One hydrophilic protein (90 kDa) was also tested. Various endoglycosidases (endoglycosidase F-N-glycosidase F, O-glycosidase, beta-mannosidase, N-glycosidase F), an exoglycosidase (alpha-mannosidase), and trifluoromethane sulfonic acid were used to deglycosylate the proteins. All four proteins were reactive to the lectin concanavalin A, demonstrating that they were mannoproteins. However, gel electrophoresis of the control and treated proteins revealed that mannosyl groups of hydrophobic proteins were less than 2 kDa in size, while the mannosyl group of the hydrophilic protein had a molecular mass of approximately 20 kDa. These results suggest that unlike many hydrophilic proteins, hydrophobic proteins may have low levels of glycosylation. Changes in glycosylation may determine exposure of hydrophobic protein regions at the cell surface.
Candida albicans↗
Surface hydrophobic and hydrophilic protein alterations in Candida albicans.
Cell surface hydrophobicity influences pathogenesis of Candida albicans. Previous studies suggested that stationary-phase hydrophilic and hydrophobic cells, obtained by growth at 37 and 23 degrees C, respectively, may have similar hydrophobic proteins. However, whether hydrophilic and hydrophobic surface proteins differ during the growth cycle at 37 degrees C is unknown. Freeze-fracture analysis revealed surface fibrillar layer differences between hydrophobic late-lag and hydrophilic stationary-phase yeast cells grown at 37 degrees C. Hydrophilic protein differences were also observed between these populations. However, similar hydrophobic proteins were detected among the late-lag and stationary phase cells grown at 37 degrees C and hydrophobic stationary-phase cells grown at 23 degrees C. These results suggest that hydrophobic proteins remain constant but hydrophilic proteins vary during growth. Thus, conversion from surface hydrophilicity to hydrophobicity by C. albicans may only require alterations in the hydrophilic fibrillar protein components.
Candida albicans↗
Hydrophobic surface protein masking by the opportunistic fungal pathogen Candida albicans.
Ultrastructural and biochemical analyses of hydrophobic and hydrophilic yeast cell surface proteins of Candida albicans were performed. Hydrophobic and hydrophilic yeast cells were obtained by growth at 23 and 37 degrees C, respectively. In addition, hydrophilic yeast cells were converted to surface hydrophobicity by treatment with tunicamycin and dithiothreitol. When freeze-etched cells were examined, the temperature-induced hydrophilic cells had long (0.198 micron), compact, evenly distributed fibrils while temperature-induced hydrophobic cells had short (0.085 micron), blunt fibrils. Hydrophobic microsphere attachment to the hydrophobic cells occurred at the basement of and within the short fibril layer. Dithiothreitol-induced hydrophobic cells had the long fibrils removed; tunicamycin-induced hydrophobic cells retained some of the long fibrils, but the fibrils were less compact and more aggregated than the untreated controls. These results suggest that the long fibrils prevent hydrophobic microsphere attachment to the hydrophobic area of the cell surface. This was confirmed by assessing the hydrophobic avidity of hydrophobic yeast cell populations differing in fibril density and arrangement. 125I-labelled surface proteins from hydrophobic and hydrophilic cells were compared after separation by hydrophobic interaction chromatography-high-performance liquid chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The yeast cell populations had hydrophilic proteins of similar molecular masses (greater than 200 kDa), but the hydrophilic cells possessed at least two additional proteins (ca. 63 and 69 to 71 kDa). Hydrophobic surface proteins appeared to be similar. However, the amount of total radiolabelled hydrophobic proteins was approximately 10-fold higher for the hydrophobic cells than for the hydrophilic cells. This result agrees with the ultrastructural observations which showed that yeast cell surface hydrophobic proteins are masked by hydrophilic high-molecular-mass surface fibrils. Taken together, the data indicate that yeast cell hydrophobicity is not determined by differences in surface hydrophobic proteins but by the presence of hydrophilic, surface fibrils.
Candida albicans↗
Differential adherence of hydrophobic and hydrophilic Candida albicans yeast cells to mouse tissues.
Using an ex vivo binding assay, we previously demonstrated that yeast cells grown at 37 degrees C display binding specificity in mouse spleen, lymph node, and kidney tissues. In spleen and lymph node tissues, binding was predominantly in regions rich in macrophages. Here, we tested the possibility that hydrophobic and hydrophilic cells bind differentially to host tissues. Hydrophobic and hydrophilic yeast cells of four Candida albicans strains were incubated for 15 min at 4 degrees C with cryostat sections of organs that had been rapidly frozen after removal from BALB/cByJ mice. Unattached cells were removed by washing, and the sections were examined. Hydrophobic cells bound diffusely and abundantly to all tissues, while hydrophilic cell attachment was restricted to specific sites. For example, hydrophobic cells bound to the white and red pulp and the marginal zones in spleens, whereas hydrophilic cells attached primarily to the marginal zones. Hydrophobic yeast cells attached throughout lymph node tissue including paracortical areas, but hydrophilic cell attachment occurred primarily at the subcapsular and trabecular sinuses, EDTA inhibited the adherence of hydrophilic cells but not hydrophobic cells to mouse tissues. Hydrophobic C. albicans strains displaying similar levels of hydrophobicity differed quantitatively in their levels of attachment to kidney and spleen tissues, confirming our earlier observation that surface hydrophobicity is not the sole determinant in adherence to host cells. Other studies have shown that hydrophobic and hydrophilic cells display different virulence characteristics related to their surface properties and that hydrophobic cells are more virulent than hydrophilic cells. Taken together, the present results suggest that the enhanced virulence of hydrophobic cells over hydrophilic cells may be due, in part, to the potential of hydrophobic cells to bind throughout various organs following clearance from the bloodstream.
Animals↗
Improved assay for surface hydrophobic avidity of Candida albicans cells.
A simple method that distinguishes among hydrophobic avidity levels of highly hydrophobic isolates of the pathogenic fungus Candida albicans is described. This method involves mixing polystyrene microspheres at different concentrations with a constant concentration of yeast cells and plotting the data in accordance with the Langmuir isotherm equation. A 10-fold difference between the C. albicans isolates with the lowest and highest avidity (KH) values was found. This method may also demonstrate that surface hydrophobic sites with different avidities are present within a yeast cell population.
Candida albicans↗
Partial biochemical characterization of cell surface hydrophobicity and hydrophilicity of Candida albicans.
Hydrophobic yeast cells of Candida albicans are more virulent than hydrophilic yeast cells in mice. Results of experiments performed in vitro suggest that surface hydrophobicity contributes to virulence in multiple ways. Before definitive studies in vivo concerning the contribution of fungal surface hydrophobicity to pathogenesis can be performed, biochemical, physiological, and immunochemical characterization of the macromolecules responsible for surface hydrophobicity must be accomplished. This report describes our initial progress toward this goal. When hydrophobic and hydrophilic yeast cells of C. albicans were exposed to various enzymes, only proteases caused any change in surface hydrophobicity. Hydrophobic cell surfaces were sensitive to trypsin, chymotrypsin, pronase E, and pepsin. This indicates that surface hydrophobicity is due to protein. Papain, however, had no significant effect. The hydrophobicity of hydrophilic cells was altered only by papain. The proteins responsible for surface hydrophobicity could be removed by exposure to lyticase, a beta 1-3 glucanase, for 30 to 60 min. When 60-min lyticase digests of hydrophobic and hydrophilic cell walls were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 12.5% resolving gel, each protein population contained a single unique protein that was not evident in the other protein population. However, when the cell wall surface proteins of hydrophobic and hydrophilic cells were first labeled with 125I and then removed by lyticase and analyzed by SDS-PAGE, at least four low-molecular-mass (less than 65 kilodaltons) proteins associated with hydrophobic cells were either absent or much less abundant in the hydrophilic cell digests. This result was seen for both C. albicans strains that we tested. When late-exponential-phase hydrophilic cells were treated with tunicamycin, high levels of surface hydrophobicity were obtained by stationary phase. These results indicate that the surface hydrophobicity of C. albicans reflects changes in external surface protein exposure and that protein mannosylation may influence exposure of hydrophobic surface proteins.
Bacterial Outer Membrane Proteins↗
Characteristics of Candida albicans adherence to mouse tissues.
An ex vivo binding assay originally described for determining lymphocyte homing receptors was adapted for studying Candida albicans-host cell interactions in unfixed tissue sections. BALB/cByJ mice were sacrificed, and various organs were removed, rapidly frozen on dry ice, and sectioned. C. albicans yeast cells were suspended to 1.5 x 10(8) cells per ml in Dulbecco modified Eagle medium supplemented with 5% newborn calf serum, and 100 microliters of the suspension was added to tissue sections for 15 min with rotation at 4 degrees C or at 22 to 24 degrees C. The sections were then fixed in glutaraldehyde, washed, and examined. Stationary-phase yeast cells adhered better than log-phase cells, and adherence characteristics were similar at 4 degrees C and 22 to 24 degrees C. Yeast cells from nine strains of C. albicans showed similar tissue specificity. Adherence to lymph node tissue was confined to subcapsular spaces and trabecular sinuses. In the spleen, yeast cells bound to the marginal zones. In both tissues, an association of yeast cells with tissue macrophages was suggested by results with macrophage-specific monoclonal antibodies and fluorescent or immunoperoxidase staining techniques. C. albicans adhered to convoluted tubules, glomeruli, and the tunica media of arterioles in the kidney. During experimentally induced fungemia in mice, C. albicans yeast cells associated with the same tissue sites as in the ex vivo assay, except that binding to renal arterioles was not seen in the in vivo test. A strain of Saccharomyces cerevisiae showed some adherence patterns in common with C. albicans, which indicates that tissue adherence is not sufficient for virulence. Mechanisms of attachment were not determined, but strains of C. albicans varied quantitatively in their ability to attach, and binding was inhibited by chelators of divalent cations.
Adhesiveness↗
Isolation of hydrophobic and hydrophilic variants of Candida albicans.
We have previously demonstrated that most isolates of C. albicans are hydrophobic when grown at room temperature (RT, ca. 22-24 degrees C) and hydrophilic when grown at 37 degrees C. Variants of our standard strain LGH1095 were isolated that are hydrophobic at 37 degrees C and hydrophilic at RT. After repeated phase partitioning with cyclohexane-water cell populations that were 6-16% hydrophobic at RT and 66-80% hydrophobic at 37 degrees C were obtained. Subsequent limiting dilution experiments provided clones which were more hydrophobic at RT or hydrophilic at 37 degrees C. These were then recloned until the resultant populations were consistently under 5% cell surface hydrophobicity (CSH) at RT or over 95% at 37 degrees C. Treatment with several detergents as well as sugars did not decrease the CSH of these cells. Lipase and several proteases also had no effect. When treated with trypsin at a concentration twice that used to lower CSH of normal cells to less than 5%, the hydrophobic variant only decreased in CSH by 50%. Both variants were capable of germinating, although at different levels depending on prior growth temperature. Sensitivity to the germination inhibitor morphogenic autoregulatory substance (MARS) was similar to that of the parent strain.
Candida albicans↗
Participation of yeast cell surface hydrophobicity in adherence of Candida albicans to human epithelial cells.
Recent studies have revealed that hydrophobic cells of the opportunistic pathogenic fungus Candida albicans are more virulent than hydrophilic cells. One critical step in the pathogenic process is adherence to host tissues. Adherence of C. albicans to epithelial tissues is mediated primarily by specific adhesin-receptor interactions, but whether cell surface hydrophobicity (CSH) of the yeast cells may also contribute has not been definitively demonstrated. Nineteen isolates of C. albicans were grown in Sabouraud dextrose broth at either 23 or 37 degrees C and tested for CSH by a polystyrene microsphere assay and for the ability to adhere to HeLa cells, a human cervical epithelioid carcinoma cell line. For 13 isolates, growth at 23 degrees C resulted in significantly higher levels of CSH than did growth at 37 degrees C. Three isolates were hydrophobic and two were hydrophilic regardless of growth temperature. One isolate was more hydrophobic after growth at 37 degrees C. Of the isolates that were more hydrophobic after growth at 23 degrees C, 86.5% (11 of 13) were also more adherent to HeLa cells. Growth temperature did not appear to determine adherence ability, as all isolates that did not differ in CSH after growth at either temperature also did not differ in ability to adhere. No correlation (r = 0.44) was obtained between CSH and adherence when the isolates grown at 23 degrees C were evaluated as a group. Higher correlation (r = 0.65) was obtained when the isolates were grown at 37 degrees C. Interestingly, a significantly positive correlation between CSH and adherence was obtained when individual isolates were analyzed. To accomplish this analysis, the isolates were allowed to vary in CSH over time in tissue culture medium without serum, and the corresponding adherence values determined. Only isolates that varied in CSH by greater than 10% were used. Correlation statistical analysis in which the coefficient of determination (r2) was calculated indicated that poor correlation between CSH and adherence for the isolates evaluated as a group was likely due to the fact that CSH had little effect on adherence once a moderately high level of CSH was attained. These results indicate that CSH is involved in adherence but is not the predominant mechanism and that the effect of CSH on adherence is isolate dependent.
Adhesiveness↗
Cryoprotection of antibody by organic solutes and organic solute/divalent cation mixtures.
Antibodies are globular glycoproteins that protect animals from microbial and toxic insult. These proteins have proven to have substantial commercial and research value but are variably susceptible to freeze-thaw damage, thus limiting their usefulness. Several carbohydrates and divalent cations were examined alone and in combination to determine whether they could protect antibody from freeze-thaw damage. The amino acid proline was also tested. Two antibodies, derived from different sources and specific for different antigens, were tested by a direct enzyme-linked immunosorbent assay (ELISA). Confirmation of antibody freeze-lability was obtained by rocket electrophoresis and radial immunodiffusion tests. Neither carbohydrate nor divalent cation alone fully protected antibody activity from freeze-thaw damage. However, several combinations protected antibody activity completely when compared to their effect on antibody maintained at room temperature. In the case of affinity-purified antibody, full protection of antibody activity relative to an untreated control was obtained. In several instances, cryoprotection of antibody by solute-divalent cation combinations was synergistic and not an additive effect of each component. Alkaline phosphatase, an enzyme typically linked to antibody for an ELISA, was not freeze-thaw labile. These results indicate that antibody function can be fully protected from damage due to freeze-thaw treatment by organic solutes in combination with divalent cations.
Alkaline Phosphatase↗