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T G Mitchell

Publications and source records attributed to T G Mitchell.

At least 19 recordsLinked to original sources
PubMed1995

Analysis of genes coding for small-subunit rRNA sequences in studying phylogenetics of dematiaceous fungal pathogens.

Because of their ability to display yeast-like growth forms in various environmental conditions, dematiaceous (melanized) hyphomycetes of the form-genera Exophiala, Rhinocladiella, and Wangiella have been informally termed "black yeasts." Cladistic analysis of 1,050 bp of the genes coding for small-subunit rRNA (SSU rDNA) supported a close relationship among species of these black yeasts with other dematiaceous hyphomycetes in the form-genera Fonsecaea, Phialophora, and Ramichloridium. The conventional categories of these fungi based on asexual states are not supported by phylogenetic analysis of SSU rDNA sequences. Isolates exhibiting annellidic modes of blastic conidiogenesis (e.g., Exophiala spp.) were not monophyletic and were placed as sister taxa to isolates that produce phialides or sympodulae. The results indicated very close relationships between isolates of Wangiella dermatitidis and Exophiala mansonii and between Rhinocladiella aquaspersa and Exophiala jeanselmei. This clade of dematiaceous hyphomycetes was a sister group to a clade comprising members of two orders of cleistothecial ascomycetes, Eurotiales and Onygenales. The etiological agents of chromoblastomycosis were found to be a closely related group (clade), while the agents of phaeohyphomycosis displayed a broader distribution on the SSU rDNA tree.

Ascomycota↗
PubMed1994

Unique oligonucleotide primers in PCR for identification of Cryptococcus neoformans.

On the basis of a comparison of rRNA sequences coding the internal transcribed spacer (ITS) and 5.8S regions of Filobasidiella neoformans, the teleomorph of Cryptococcus neoformans, and members of its most closely related taxa, unique oligonucleotides were designed for the specific amplification of DNA only from C. neoformans. Various combinations of these primers, designated CN4, CN5, and CN6, and the previously described fungal primers ITS1 and ITS2 were tested in PCR for their ability to amplify DNA from 37 strains of C. neoformans and 31 other isolates representing 18 species of yeasts. The combination of primers CN4 and CN5 amplified DNA from both varieties of C. neoformans but from none of the other species tested. Other pairs of primers (namely, CN5-CN6, CN4-ITS1, and CN6-ITS1) amplified DNA only from C. neoformans and from the saprophyte Filobasidiella depauperata, which is the only other member of the genus Filobasidiella. With appropriate controls, these specific oligonucleotides may be used as primers in PCR to identify C. neoformans.

Base Sequence↗
PubMed1992

Comparison of 5.8S ribosomal DNA sequences among the basidiomycetous yeast genera Cystofilobasidium, Filobasidium and Filobasidiella.

Nucleotide sequences obtained from regions of the ribosomal DNA repeat were compared by phylogenetic methods and combined with a statistical evaluation to clarify the relationships among the genera Cystofilobasidium, Filobasidium (F.) and Filobasidiella (Fl.), to assess the affinity of Filobasidiella neoformans and Filobasidiella depauperata, and to compare the varieties of Fl. neoformans. With appropriate primers, the nuclear 18S, 5.8S and internal transcribed spacer (ITS) regions of the ribosomal RNA genes (rDNA) of 10 strains were amplified with the polymerase chain reaction. The resulting DNA products were compared by digestion with endonucleases and analysis of restriction fragments. Single strands of the 5.8S rDNA and ITS regions were subsequently sequenced by the dideoxy method. Statistical support for the phylogeny inferred from parsimony analysis of aligned 5.8S rDNA sequences was determined by bootstrapping. There were no nucleotide substitutions in this region, nor in the ITS, among strains of Fl. neoformans that differ in variety and serotype. There was strong support for retaining Fl. depauperata and Fl. neoformans in the same genus, but nucleotide substitutions can be used to distinguish the two species. There was no support for combining the genera Filobasidium, Filobasidiella or Cystofilobasidium.

Base Sequence↗
PubMed1991

Killing of Cryptococcus neoformans strains by human neutrophils and monocytes.

The susceptibility of various strains of Cryptococcus neoformans to killing by human polymorphonuclear leukocytes (PMNs) and monocytes was investigated. Five previously characterized strains of C. neoformans serotype A, a capsule-free mutant, and six recent clinical isolates were compared. PMNs and monocytes were isolated from normal peripheral blood and allowed to adhere to the flat-bottom wells of microtiter plates. Yeast cells of C. neoformans were added in the presence of normal human serum, and the plates were incubated at 37 degrees C. After 4 h, killing was determined by comparing the quantitative plate counts of viable yeast cells in experimental wells with counts in control wells containing yeast cells in the absence of leukocytes. No appreciable growth of yeast cells occurred in the wells during the incubation period. Both PMNs and monocytes effectively killed yeast cells at effector-to-target ratios as low as 1:1, although monocytes failed to kill the capsule-free strain 602 at a 1:1 ratio. With 9 of 12 strains, PMNs killed C. neoformans more effectively than did monocytes. Significant interstrain variation in killing occurred for both monocytes and PMNs, and the recent, clinical isolates were more resistant to killing by monocytes and PMNs than were the previously characterized strains. The extent to which different strains were killed by monocytes and PMNs was not consistently related to the size of the capsule or the entire cell. Normal PMNs and monocytes are remarkably effective in killing strains of C. neoformans in the absence of specific antibody and appear to constitute a significant defense mechanism in the peripheral circulation.

Cryptococcus neoformans↗
PubMed1990

Human natural killer cells do not inhibit growth of Cryptococcus neoformans in the absence of antibody.

The interaction between human natural killer (NK) cells and yeast cells of Cryptococcus neoformans was investigated because experiments in mice indicated that NK cells inhibited the growth of C. neoformans. Strains of C. neoformans serotype A that differed in both resistance to alveolar macrophages and the size and composition of their capsules were evaluated. Human NK cells, which were isolated from normal peripheral blood, were activated by preincubation with interleukin-2 and alpha interferon to generate lymphokine-activated killer (LAK) cells. Yeast cells of C. neoformans were incubated with effector cells (NK or LAK cells); and inhibition of yeast cell growth was measured at 4, 8, and 24 h by comparing quantitative plate counts with controls consisting of yeasts in the absence of effector cells. The cytolytic activity of effector cells against target cells was confirmed by the release of radiolabel from 51Cr-labeled K-562 tumor cells. Neither NK nor LAK cells inhibited the growth of 13 strains of C. neoformans at effector to target cell ratios of as high as 500:1. Monocytes, which were isolated from the same populations of leukocytes as the NK cells, inhibited the growth of two strains of C. neoformans at effector to target cell ratios of 100:1 (92 and 46% inhibition), 50:1 (87 and 17%), and 1:1 (49 and 0%). NK cells could inhibit the growth of C. neoformans by an antibody-dependent cellular cytotoxicity mechanism in the presence of rabbit anticryptococcal antiserum at dilutions up to 1:4,000. Purified capsular polysaccharide of C. neoformans had no effect on the viability or tumoricidal activity of NK or LAK cells. These data suggest that human NK and LAK cells are not impaired by C. neoformans, and in the absence of antibody, which is rarely detectable in patients, they afford much less protection against C. neoformans than monocytes do.

Animals↗
PubMed1989

Phagocytosis and killing of Cryptococcus neoformans by rat alveolar macrophages in the absence of serum.

The in vitro interaction between yeast cells of Cryptococcus neoformans and Lewis rat alveolar macrophages (AM phi) was studied in the absence of serum. AM phi were harvested by lung lavage, and monolayers of adherent cells were established in wells of microtiter plates. Radiolabeled yeast cells were added to fresh AM phi monolayers, the plates were incubated at 37 degrees C under 5% CO2, nonadherent yeasts were removed, and phagocytosis (i.e., attachment or ingestion) was determined by measuring adherent radioactivity. AM phi were able to bind or ingest, and kill, encapsulated strains of C. neoformans in the absence of serum. Serum-free phagocytosis was suboptimal by comparison with phagocytosis in the presence of serum. The mechanism of serum-free phagocytosis involves a receptor on the AM phi with affinity for mannose-rich determinants present on the yeast cell walls and unrelated to the capsular polysaccharide. Opsonin-independent phagocytosis was only detected with nonencapsulated, small, and medium encapsulated strains of C. neoformans. Large encapsulated strains were not taken up without serum. Serum-free phagocytosis could be of critical importance in the alveolar spaces, where only marginal concentrations of serum opsonins are initially present.

Animals↗
PubMed1989

Strain variation in antiphagocytic activity of capsular polysaccharides from Cryptococcus neoformans serotype A.

Strains of Cryptococcus neoformans vary in resistance to phagocytosis in vitro. The binding of isolated capsular polysaccharide (CPS) to a capsule-free mutant of C. neoformans confers resistance to phagocytosis. The importance of capsule composition to differences among strains in susceptibility to phagocytosis was evaluated. CPSs from five strains of C. neoformans serotype A, designated 6, 15, 98, 110, and 145, which had previously been isolated and characterized as to molecular size, composition, and binding properties, were evaluated for relative antiphagocytic potencies. In the presence of 5% normal isologous serum, murine thioglycolate-elicited peritoneal macrophages phagocytized (i.e., attached to or engulfed) 80% of 51Cr-labeled cells of C. neoformans 602, a capsule-free mutant. Added CPS inhibited the uptake of these yeast cells. CPS from strain 110 was most potent, followed in decreasing order of inhibitory activity by CPSs from strains 6, 145, 98, and 15. The presence of 100 micrograms of strain 110 CPS per ml reduced uptake of cells of strain 602 from 80 to 50%. CPS had no effect on the uptake of 51Cr-labeled Saccharomyces cerevisiae. Cells of strain 602 that were preincubated with CPS and then washed were more resistant to phagocytosis than nonpretreated control cells, indicating the importance of bound, not free, CPS. Added CPS did not affect the uptake of wild-type, encapsulated cells of C. neoformans. Addition of endotoxin had no effect on phagocytosis. CPSs from strains of C. neoformans serotype A varied widely in their abilities to inhibit the uptake of capsule-free cells. The antiphagocytic activity of CPS did not correlate with the ability to bind to capsule-free mutant but was somewhat related to the capsule size of the strain from which the CPS was isolated.

Animals↗
PubMed1989

Phagocytosis of Cryptococcus neoformans by rat alveolar macrophages.

A radiolabeling method was developed to investigate the interaction in vitro between Cryptococcus neoformans and Lewis rat alveolar macrophages (AM phi). AM phi were harvested by lung lavage, monolayers of adherent cells were established in wells of microtiter plates and [51Cr]-labeled yeast cells were added to the monolayers. After removal of extracellular yeasts, the adherent radioactivity associated with the AM phi was directly proportional both to the number of yeasts added and to the number of yeasts per AM phi as determined by microscopic examination of Giemsa-stained monolayers. Phagocytosis (attachment and/or ingestion) of radiolabeled C. neoformans by AM phi was a sensitive, quantitative and reproducible assay for the evaluation of the AM phi-C. neoformans interaction. AM phi were able to phagocytose encapsulated strains of C. neoformans. The extent of phagocytosis was inversely related to the capsule size. Normal rat serum (NRS) was an excellent source of opsonins for the ingestion. Inactivation of serum complement or depletion of C3 by affinity chromatography removed most of the opsonic activity of NRS. Specific antibodies against C. neoformans did not increase phagocytosis.

Agglutination Tests↗
PubMed1989

Killing of Cryptococcus neoformans by rat alveolar macrophages.

The addition of [51Cr]-labeled yeast cells of Cryptococcus neoformans to monolayers of Lewis rat alveolar macrophages (AM phi) provided a sensitive and reproducible in vitro assay of phagocytosis. AM phi and yeast cells were incubated in 10% (v:v) normal rat serum for 1 h, non-AM phi associated yeast cells were removed and the AM phi-associated radioactivity (phagocytosis) determined. Replicate wells were replenished with fresh medium and reincubated. At different times, yeast-AM phi monolayers were treated with a non-cryptococcocidal mixture of DNAse and sodium deoxycholate to release the yeast cells from the AM phi. The fate of the yeast cells was critically evaluated by [51Cr]-release and viable plate counts. Killing was detected by plate counts within an hour following phagocytosis and did not increase significantly during the next 5 h. Strains of C. neoformans with small, medium, or large capsules varied in their susceptibility to killing from 10% to 95% but susceptibility to killing was not directly related to capsule size and the extent of phagocytosis. Release of 51Cr did not correlate with viability as determined by culture. The 51Cr was associated with two pools in the yeast cells; one, representing 15-20% of the radiolabel, was easily released and was probably bound to low molecular weight compounds in the cytoplasm. The majority of label was tightly bound to the particulate alkali-soluble cell wall fraction.

Animals↗
PubMed1986

Strain variation in composition and molecular size of the capsular polysaccharide of Cryptococcus neoformans serotype A.

The capsule of Cryptococcus neoformans is an important virulence factor. In this investigation capsular polysaccharides (CPSs) were isolated by ethanol precipitation from culture filtrates of C. neoformans serotype A strains 6, 15, 98, 110, and 145. Capsule sizes on India ink examination ranged from barely perceptible (strain 15) to greater than the diameter of the yeast cell (strain 6); the others were intermediate in size. On ion-exchange chromatography on DEAE-cellulose each CPS eluted at 0.2 M NaCl; CPS of strain 15 had two major peaks, designated III and IV. On gel-permeation chromatography CPSs of strains 6, 98, 110, and 145 eluted at the void volume of Sepharose CL-2B in the presence or 0.1 M EDTA, while the CPS of strain 15 eluted in two peaks. Sephacryl S-1000 resolved CPSs of all five strains in the following order, from largest to smallest molecular size: 145 greater than 110 greater than 98 greater than 6 much greater than 15. All five CPSs contained mannose, xylose, and glucuronic acid, while the carboxyl-reduced CPS of strain 110 also contained a large percentage of an inositol-like compound. The CPS of strain 110 contained approximately 30% uronic acid by weight, while the others had 15 to 20%. The composition of peak IV from the CPS of strain 15 resembled those of the other strains; peak III of strain 15 contained a substantial amount of galactose. Each CPS contained less than 0.2% protein by weight. The significant differences in molecular size and sugar composition among CPSs of these strains of C. neoformans serotype A may partially explain strain differences in virulence and biological properties of the organism.

Amino Acids↗
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↗