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Biomedical subjects

T G Mitchell

Publications and source records attributed to T G Mitchell.

At least 55 records · Page 3Linked to original sources
PubMed1986

Binding of purified and radioiodinated capsular polysaccharides from Cryptococcus neoformans serotype A strains to capsule-free mutants.

Strains 6, 15, 98, 110, and 145 of Cryptococcus neoformans serotype A vary in capsule size, animal virulence, and susceptibility to in vitro phagocytosis. The isolated capsular polysaccharides (CPSs) differ in monosaccharide composition ratios and molecular size, as determined by gel filtration. The purpose of this investigation was to characterize the binding of CPSs to capsule-free mutants of C. neoformans and to examine CPSs from these strains for differences in their ability to bind, to determine whether such differences might explain the variation in the pathobiology of these strains. CPSs were partially periodate oxidized, tyraminated, iodinated with 125I, and used in binding studies with two capsule-free mutants of C. neoformans, strain 602 and Cap59. Binding was specific for yeast species and for polysaccharide and was saturable, which is consistent with a receptor-mediated mechanism of attachment. Binding occurred rapidly and was only slowly reversible. Binding was also independent of pH from pH 5.5 to 8, of cation concentrations, and of competition by sugars up to 1.0 M concentrations. Only a portion of CPS was capable of binding, and strains varied in the extent to which their CPS bound. CPS-15-IV (peak IV was the major polysaccharide peak on DEAE-cellulose chromatography of CPS from strain 15) had the highest proportion of binding (40%), followed by CPS from strains 98, 6, 145, 110, and 15-III (peak III was an earlier eluting fraction of CPS from strain 15). The CPSs differed similarly in their ability to competitively inhibit binding. Treatment of CPS, but not yeast cells, with proteinase XIV abolished binding without altering the CPS gross structure. Treatment of yeast cells with proteases, heat, or formaldehyde did not alter binding, and both strain 602 and Cap59 bound CPS similarly. Binding to encapsulated yeast cells was minimal.

Chemical Phenomena↗
PubMed1986

Immunoblot analyses of Candida albicans-associated antigens and antibodies in human sera.

We tested 10 patient sera for the presence of immunoglobulin G (IgG) antibodies to Candida albicans and for C. albicans antigens by immunoblot analysis (i.e., electrotransfer blot radioimmunoassay) (G. E. Smith and M. D. Summers, J. Virol. 39:125-137, 1981). We evaluated sera from two patients at risk for candidiasis, five patients with systemic candidiasis documented by culture, and two patients who had experienced transient candidemia. Both the specificity and the relative amount of IgG antibodies to C. albicans in each serum sample were readily visualized by this technique, as was the absence of antibody from serum of neonatal and immunocompromised patients. No antibody species appeared to be uniquely associated with candidiasis patients (i.e., each antibody species present in the candidiasis patient was also present in sera of normal individuals or "at-risk" patients). IgG from rabbits immunized with whole cells or with a cytoplasmic fraction of C. albicans was used to detect C. albicans antigens in patient sera. Although several antigens were detected in the sera from patients with candidiasis, the same antigens were also detected in sera from patients at risk and in normal human serum. No antigens were detected in human serum when preimmune rabbit sera were used. These results suggest that the antigens detected by the rabbit antisera were human serum proteins that cross-reacted with C. albicans antigens. These findings may have important implications in studies of both the pathobiology of C. albicans and the serodiagnosis of candidiasis.

Antibodies, Fungal↗
PubMed1984

Blastomyces dermatitidis chemotactic factor: kinetics of production and biological characterization evaluated by a modified neutrophil chemotaxis assay.

Chemotactic activity for human polymorphonuclear neutrophils (PMNs) was detectable in culture filtrates (CFs) of Blastomyces dermatitidis and may have influenced the pathogenesis of blastomycosis. Production of this chemotaxin depended upon culture age and medium; peak levels were achieved after incubation for 17 days or more in minimal essential medium. This factor was also chemotactic for human monocytes. CF was temperature stable even after treatment at 100 degrees C for 60 min. The activity was stable under alkaline conditions but was destroyed below pH 7. Dialyzed, chemotactically active CF contained approximately 60 micrograms of carbohydrate per ml; total protein was estimated to be less than 0.8 micrograms/ml. Preincubation of PMNs with CF or N-formyl-L-methionyl-L-leucyl-L-phenylalanine deactivated their chemotactic response to each agent, whereas the chemotactic response to zymosan-activated serum was not affected. In addition, deactivation with N-formyl-L-methionyl-L-leucyl-L-phenylalanine reduced the response to CF. Pretreatment of CF with PMNs decreased chemotactic activity, which may reflect binding of the chemotaxin molecules to PMN receptors. A modified chemotaxis assay was developed in which commercial, disposable multiwell plates are used. This method was rapid, efficient, and inexpensive and permitted the assay of larger numbers of samples than was previously feasible with conventional chemotaxis methods.

Bacterial Proteins↗
PubMed1984

Effects of culture filtrates of Blastomyces dermatitidis on neutrophil locomotion.

A potent chemotactic activity for neutrophils is detectable in liquid culture filtrates of Blastomyces dermatitidis. The production of this activity is medium-dependent and culture age-dependent. The highest levels of cytotaxin were produced in filtrates of B. dermatitidis grown in tissue culture medium 199 for three or more weeks. This factor(s) stimulates directed as well as random migration. It functions directly and independently of serum. It is stable at -20 degrees C and 56 degrees C, and has a molecular weight greater than 10000 daltons. These properties define a new microbial chemotactic factor.

Blastomyces↗
PubMed1983

Urinary cobalt levels in patients with porous Austin-Moore prostheses.

Austin-Moore prostheses with a porous surface have been used recently to improve stabilization in total joint arthroplasty. However, these prostheses expose tissue directly to greater metallic surface area than previous nonporous designs. Cobalt levels were measured in the urine of patients who had received porous and nonporous prostheses, to assess any increased risk of metal toxicity posed by the porous design. Although a higher mean urinary cobalt concentration was found for patients with porous prostheses, the absolute levels were comparable with those of patients with nonporous prostheses. The prostheses with a porous surface appear to pose no significant added risk of metal toxicity as compared with the nonporous design.

Cobalt↗
PubMed1982

Comparison of an enzyme-linked immunoassay and a quantitative indirect fluorescent-antibody test with the conventional indirect fluorescent-antibody test for detecting antibodies to Toxoplasma gondii.

Two new methods for the detection of antibodies to Toxoplasma gondii, an enzyme-linked immunosorbent assay and a quantitative immunofluorescence assay, were evaluated and compared with the conventional indirect fluorescent-antibody slide test. Each of 100 human sera was assayed twice by the three procedures. Both the enzyme-linked immunosorbent assay and the quantitative immunofluorescence assay correlated well with serologically positive (indirect fluorescent-antibody titer greater than or equal to 1:32) and negative sera. The enzyme-linked immunosorbent assay was more specific, but less sensitive, than the quantitative immunofluorescence assay. However, the quantitative immunofluorescence assay was more reproducible and more rapid than the enzyme-linked immunosorbent assay.

Animals↗
PubMed1982

Human platelets labeled with In-111 8-hydroxyquinoline: kinetics, distribution, and estimates of radiation dose.

Platelets from nine normal male subjects were labeled with In-111 8-hydroxyquinoline (In-111 oxine) in the presence of plasma in either "closed" blood transfer packs or in "open" test tubes. The mean labeling efficiencies in these two systems were 27 and 53%, respectively. Mean survival time of In-111-labeled autologous platelets was 8.76 days, with a standard deviation of 1.05 according to the maximum-likelihood estimate of the gamma-function model. The initial recovery of In-111 platelets in the circulation was 57% with a standard deviation of 11%. The distribution of In-111 platelets in liver and spleen was quantitated by anterior, posterior, and transmission gamma-camera imaging. During the first 30 min, 38% of the injected dose accumulated in the spleen, 13% in the liver. No significant increase in In-111 radioactivity was observed in either of the two organs over a 3-9-day period. The bone marrow was an additional site of In-111 accumulation. The spleen was the critical organ with respect to radiation dose. The splenic dose was estimated to be 34 rad/mCi In-111 platelets, that of the liver 2.1 rad/mCi. With the injection of 100-150 microCi of In-111-labeled platelets in normal subjects, giving a splenic radiation of 5 rad, a complete 10-day survival study can be performed and uptake of In-111 in different organs can be measured quantitatively for at least 3-4 days.

Adult↗
PubMed1980

Analysis of cytoplasmic antigens of the yeast and mycelial phases of Candida albicans by two-dimensional electrophoresis.

The extent of the macromolecular change accompanying yeast to mycelium morphogenesis of Candida albicans was analyzed by two-dimensional gel electrophoresis of the cytoplasmic proteins of the two growth forms after antibody cross-absorption experiments. Pure cultures of yeasts and true hyphae (i.e., without concomitant production of pseudohyphae) were grown in a synthetic low-sulfate medium (LSM). The two strains selected for this study were strain 4918, which produces pure mycelial (M) cultures in LSM at 37 degrees C (designated 4918-37M) and yeasts (Y) at 24 degrees C (4918-24Y), and strain 2252, which produces yeasts exclusively at both 24 and 37 degrees C in LSM (2252-24Y and 2252-37Y). The proteins of both strains were labeled at both temperatures with [35S]sulfate, and cytoplasmic fractions were prepared by mechanical disruption and ultracentrifugation. Rabbits were immunized with the 4918-24Y and 4918-37M cytoplasmic fractions to produce anti-yeast-phase and anti-mycelial-phase hyperimmune sera. Each radiolabeled cytoplasmic fraction was absorbed with anti-mycelial-phase immunoglobulin, anti-yeast immunoglobulin, and immunoglobulin from normal rabbit serum. Staphyloccal protein A was used to remove immune complexes. The labeled, nonabsorbed proteins were also analyzed by two-dimensional electrophoresis. Highly reproducible protein spot patterns were obtained which defined hundreds of proteins in each extract. The specificity of the immunoglobulin hundreds of proteins in each extract. The specificity of the immunoglobulin preparations was extremely broad, and as many as 168 cytoplasmic antigens were detected. Eighty-three antigens were recognized in the mycelial-phase extract only by the anti-mycelial-phase immunoglobulin. However, comparative analysis revealed that all of these proteins were present in at least one other extract. Therefore, none of them was unique to the mycelial morphology. Eleven antigens were detected in the 2252-37Y extract that were not present in the extracts from strain 4918, which indicates that proteins obtained from different strains may express similar antigenic determinants, but differ in their physiochemical properties.

Antigens↗
PubMed1980

Strain variation and morphogenesis of yeast- and mycelial-phase Candida albicans in low-sulfate, synthetic medium.

A low-sulfate synthetic medium was developed in which pure cultures of yeast- and mycelial-phase Candida albicans could be cultivated for investigations of the molecular biology of dimorphism. The medium contained ammonium ions, phosphate buffer, salts, glucose, and biotin. Morphogenesis was found to be dependent upon the strain of C. albicans. Of six strains tested in the low-sulfate medium at 37 degrees C, three formed mixed cultures of yeasts, true mycelium and pseudomycelium, two formed pure cultures of true mycelium, and one maintained yeast growth. All six strains produced pure cultures of yeasts at 24 degrees C. The buffering capacity of the medium maintained the pH at 6.9 even at high-density cell growth. The low concentration of sulfate and the absence of amino acids in the medium provided conditions in which to radiolabel cellular constituents with [35S]sulfate. For molecular investigations, the use of two strains is suggested, one forming yeasts and one forming true mycelium in low-sulfate medium at 37 degrees C, thus providing controls for both strain variation and for molecular changes induced by environmental change but unrelated to morphogenesis.

Amino Acids↗
PubMed1980

Morphogenesis of Candida albicans and cytoplasmic proteins associated with differences in morphology, strain, or temperature.

The extent of change in cytoplasmic proteins which accompanies yeast-to-mycelium morphogenesis of Candida albicans was analyzed by two-dimensional gel electrophoresis. Pure cultures of yeasts and true hyphae (i.e., without concomitant production of pseudohyphae) were grown in a synthetic low-sulfate medium. The two strains selected for this study were strain 4918, which produces pure mycelial cultures in low-sulfate medium at 37 degrees C and yeast cells at 24 degrees C, and strain 2252, which produces yeast cells exclusively at both 24 and 37 degrees C in low-sulfate medium. The proteins of both strains were labeled at both temperatures with [35S]sulfate, cytoplasmic fractions were prepared by mechanical disruption and ultracentrifugation, and the labeled proteins were analyzed by two-dimensional electrophoresis. Highly reproducible protein spot patterns were obtained which defined hundreds of proteins in each extract. Ten protein spots were identified on the two-dimensional gels of the 4918 mycelial-phase extract which were not present in the 4918 yeast-phase extract. These proteins appeared to be modifications of preexisting yeast-phase proteins rather than proteins synthesized de novo in the mycelial cells because 5 were absorbed by rabbit anti-yeast-phase immunoglobulin and each of the 10 was also present in extracts of strain 2252 grown at 24 and 37 degrees C, indicating that they were neither unique to filamentous cells nor sufficient for induction or maintenance of the mycelial morphology. Thirty-three proteins were identified in the 4918 yeast-phase extract which were not present in the 4918 mycelial-phase extract. Pulse-chase experiments revealed the synthesis of new proteins during yeast-to-mycelial conversion, but none of these was unique to mycelial cells. No differences in the major cytoplasmic proteins of any of the yeast- or mycelial-phase extracts were identified. This finding suggests that the major structural proteins of the cytoplasm are not extensively modified and argues instead that proteins unique to either phase may serve a regulatory function.

Autoradiography↗
PubMed1975

Evaluation of a cytocentrifuge method for measuring neutrophil granulocyte chemotaxis.

An in vitro method for the assessment of neutrophil granulocyte chemotaxis employing a cytocentrifuge has been evaluated. The cytocentrifuge is used to directly sediment leukocytes on a Millipore filter, and thus (1) eliminates errors due to abnormal or unequal settling of the cells and (2) eliminates the need for serum, plasma, or albumin on the cell side of the chemotactic chamber to obtain optimal chemotaxis. In addition, other advantages of the method are that it requires smaller volumes of blood, less handling of the cells, and less time is required for visual counting of the neutrophils. The method has been utilized to assess chemotactic activity of neutrophils from 80 control subjects and over 100 patients with various diseases and to study the effects of various pharmacologic agents on neutrophil chemotactic responsiveness.

Centrifugation↗
PubMed1972

In vitro phagocytosis and intracellular fate of variously encapsulated strains of Cryptococcus neoformans.

Five isolates of Cryptococcus neoformans type A with stable capsular thicknesses were used. Three of the isolates had capsules of medium size, one had a minimal capsule, and the other, a large capsule. Peritoneal exudate cells from Lewis rats were cultured on cover slips in Leighton tubes containing medium 199 and 20% fresh, isologous normal rat serum. Yeast cells were added to the Leighton tube cultures, and, 2 hr later, the extracellular yeasts were rinsed out. Cover slips were removed from some tubes for Wright staining and measurement of both phagocytosis and loss of macrophages. The remaining tubes were reincubated and sampled at 24 or 48 hr. To determine fate of yeast cells after ingestion, washed cover slips were inverted onto agar slide cultures, and specific macrophages were observed in situ for subsequent multiplication of their intracellular yeasts. More than half of the macrophages survived 24 to 48 hr of exposure to different strains of C. neoformans, with small, medium, or large capsules. Phagocytic activity was dependent upon a heat-labile factor in normal rat serum. The number of yeast ingested by macrophages was inversely proportional to the capsular size. Although most of the ingested yeasts were resistant to intracellular killing, the agar culture technique clearly demonstrated that many were unable to multiply, presumably dead. Three of the isolates were more susceptible than the other two, and the fate of these yeasts after engulfment was not correlated with their capsular size.

Animals↗