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PubMed1981

Opsonization of encapsulated Cryptococcus neoformans by specific anticapsular antibody.

Antisera prepared in rabbits against either whole encapsulated cells of Cryptococcus neoformans or purified cryptococcal polysaccharide were opsonic for the encapsulated yeast. The opsonic activity was removed by absorption with whole cryptococci and was inhibited by free polysaccharide. As little as 0.13 microgram of cryptococcal polysaccharide produced a 50% inhibition of opsonization. Various degrees of neutralization by polysaccharides from the four cryptococcal serotypes suggested that the opsonins were type specific. Fractionation of antiserum on Bio-Gel A-5m (Bio-Rad Laboratories) and diethylaminoethyl cellulose showed that the opsonins were antibodies of the immunoglobulin G class. These opsonizing antibodies did not require heat-labile serum components for optimal phagocytosis of the yeast. Inhibition studies using 2-deoxy-D-glucose demonstrated that ingestion of encapsulated cryptococci opsonized with anticapsular antibody was a 2-deoxy-D-glucose-inhibitable process. This result differed from similar studies with non-encapsulated cryptococci which showed that ingestion of non-encapsulated cryptococci opsonized with normal serum was not inhibited by 2-deoxy-D-glucose.

Animals↗

PubMed1982

Immunization of mice by intracutaneous inoculation with viable virulent Cryptococcus neoformans: immunological and histopathological parameters.

Immune responses, including protection and delayed hypersensitivity, were evaluated in experimental murine cryptococcosis. Mice were immunized by the intracutaneous inoculation of viable virulent Cryptococcus neoformans yeasts. Response to the cutaneous infection was evaluated histologically and by cultural assays of the internal organs, as well as by intravenous challenge with the same strain. Protection was assessed by survival, histopathology, and quantitative organ culture. The intracutaneous inoculation of cryptococci resulted in a local inflammatory response that effectively limited dissemination of the organisms systemically and induced the development of delayed hypersensitivity demonstrable with a membrane extract of C. neoformans and with soluble cytoplasmic substances. A protective response was induced by the cutaneous inoculation of cryptococci as well, in that immunized animals survived longer, with about 25% of the challenged group ridding themselves completely of the cryptococci. Protection could be demonstrated by cultural analyses, but all animals, whether control or immunized, allowed considerable multiplication of the inoculum during the first 4 weeks after intravenous challenge. It would appear, therefore, that the protective mechanism(s) required additional antigenic stimulation before it could eventually function to eliminate all cryptococci from tissues. Histologically, there were no differences in pathology of the internal organs between immunized and unimmunized animals. Although the model described herein for the induction of immune responses in murine cryptococcosis has at least one drawback, viz., the presence of cryptococci in the skin lesion of many animals throughout the duration of the experiment, it does have the advantage that the immune responses were stimulated by a virulent strain and only minimal dissemination occurred. Therefore, lymphocytes could be removed from animals that were not contaminated with cryptococci for in vitro and in vivo transfer.

Animals↗

PubMed1982

In vitro interactions of immune lymphocytes and Cryptococcus neoformans.

CBA/J mice immunized subcutaneously with emulsions of heat-killed Cryptococcus neoformans in complete Freund adjuvant displayed delayed-type hypersensitivity to cryptococcal culture filtrate antigen and developed sensitized splenic lymphoid cells which inhibited the growth of C. neoformans in vitro. The in vitro assay of growth inhibition served to investigate further the kinetics of the effect of sensitized lymphoid cells on the pathogen. There was a close correlation between the delayed-type hypersensitivity response in mice and inhibition of growth of C. neoformans by lymphoid cells. Sensitized splenic lymphocytes capable of inhibiting the growth of the cryptococci were detected at day 6 after immunization and reached maximum levels by days 8 through 16. Inhibition of growth was highest with effector-to-target cell ratios of 300:1 or greater. Inhibition of growth of C. neoformans by sensitized lymphoid cells was detectable as early as 4 h after effector and target cells were mixed and increased gradually, reaching a maximum at 24 h, but dropped significantly by 48 h. By supplementing the reaction mixtures with fresh medium or additional sensitized effector cells during incubation, the inhibition of growth of C. neoformans could be maintained through 48 h. C. neoformans-sensitized effector lymphoid populations not only inhibited the growth of the pathogen in vitro but also restricted C. neoformans proliferation in various vital organs upon transfer to naive recipient animals, indicating that the in vitro growth inhibition assay may be a means of assessing the resistance of animals to C. neoformans. The effector cells from sensitized animals were nylon wool-nonadherent Thy-1+ and Ia+ lymphocytes.

Animals↗

PubMed1982

Phenoloxidase activity and virulence in isogenic strains of Cryptococcus neoformans.

A naturally occurring Mel- variant of Cryptococcus neoformans was isolated from the wild type. The effect of phenoloxidase activity on virulence was analyzed on genetically constructed Mel+ and Mel- isolates. The traits Mel+ and virulence in mice, as measured by cumulative mortality and replication potential in brain tissue, cosegregated among the progeny of a Mel+ X Mel- cross. Revertants (MelR) isolated during the course of the cumulative mortality experiment were used to compare virulence in isogenic sets of Mel- and MelR. In two separate sets of such isolates, Mel+ phenotype and virulence coreverted. Measurement of substrate uptake and phenoloxidase activity showed that loss of detectable phenoloxidase was the basis for the Mel- phenotype and that enzyme activity reappeared in the MelR isolates. An intermediate phenotype, Melbg, was also described. Cosegregation and coreversion of the melanin phenotype and virulence suggest that phenoloxidase is a virulence factor in C. neoformans.

Catechol Oxidase↗

PubMed1982

Chemotaxis of human neutrophils and monocytes induced by Cryptococcus neoformans.

Chemotaxis of human neutrophils and monocytes was stimulated by sera activated with greater than or equal to 1.25 x 10(6) Cryptococcus neoformans. Leukocytes from five renal transplant recipients had depressed chemotactic responses to C. neoformans-activated sera when compared with normal subjects (P less than 0.05). Concentrations of cryptococcal capsular polysaccharide less than 1 mg/ml failed to generate chemotactic factors from sera.

Chemotactic Factors↗

PubMed1983

Dissociation of a hydrophobic surface from phagocytosis of encapsulated and non-encapsulated cryptococcus neoformans.

Cryptococcus neoformans is surrounded by a capsular polysaccharide that inhibits phagocytosis of the yeast by macrophages. This capsular polysaccharide also confers several physicochemical properties to the cell surface, including a negative surface charge and a hydrophilic surface. The present study was designed to determine whether a hydrophobic surface was necessary or sufficient for phagocytosis of C. neoformans cells. The hydrophobic nature of the cell surface was measured by hydrophobic interaction chromatography on octyl-Sepharose. Liability to phagocytosis was determined by use of mouse peritoneal macrophages. The surface properties of C. neoformans cells were modified by (i) preincubation of cryptococcal cells with nonimmune serum or immune anticapsular serum, (ii) chemical modification of the carboxyl and O-acetyl groups in the capsular polysaccharide, and (iii) use of various serotypes of C. neoformans with different degrees of O-acetyl and xylosyl substitution. The results showed that it was possible to experimentally vary the surface hydrophobic-hydrophilic characteristics of the cell surface; however, the antiphagocytic character of the capsule remained unchanged. The results further suggest that a hydrophobic surface was neither necessary nor sufficient for phagocytosis of C. neoformans cells by macrophages.

Chemical Phenomena↗

PubMed1984

Localization on encapsulated Cryptococcus neoformans of serum components opsonic for phagocytosis by macrophages and neutrophils.

Previous studies have shown that the cryptococcal capsule inhibits phagocytosis of Cryptococcus neoformans by macrophages and neutrophils. In this study, the binding sites of potential serum opsonins in immune and nonimmune sera were determined by immunoelectron microscopy, and the results were compared with the results of phagocytosis of the yeasts by mouse peritoneal macrophages and human neutrophils. Immunoglobulin G (IgG) from normal human serum showed low-density binding at the capsular surface and at sites throughout the capsule. Complement component C3 from normal serum bound heavily at the capsular surface. IgG from rabbit capsular antiserum showed relatively dense deposition at the capsular surface and at sites throughout the capsule. Cells opsonized with heat-inactivated human serum were engulfed poorly by both macrophages and neutrophils, indicating that the low-density deposition of IgG produced by normal serum was not adequate for opsonization. Yeasts opsonized with normal human serum were engulfed in large numbers by neutrophils and to a lesser extent by macrophages, indicating that neutrophils in particular were able to effectively utilize the opsonically active C3 which normal human serum deposited at the capsular surface. Yeasts opsonized with rabbit anticapsular serum were engulfed by both macrophages and neutrophils, indicating that the high density of surface IgG produced by capsular antiserum is an effective opsonin for both cells. These results suggest that the complement-neutrophil system is a possible defense mechanism in the nonimmune host.

Animals↗

PubMed1985

Effects of first-order Cryptococcus-specific T-suppressor cells on induction of cells responsible for delayed-type hypersensitivity.

Cell-mediated immunity is an important aspect of host resistance against Cryptococcus neoformans. Using a CBA/J murine model, we demonstrated that injection of cryptococcal antigen (CneF) at dosages sufficient to stimulate the antigenemia observed in cryptococcosis patients induces specific T-cell-mediated suppression of the cryptococcal delayed-type hypersensitivity response. The purpose of this study was to establish whether Lyt 1+, first-order T-suppressor (Ts1) cells block the induction of T cells responsible for delayed-type hypersensitivity (TDH cells) or whether they function by inducing Lyt 2+, efferent suppressor (Ts2) cells. In one set of experiments, suppression was observed when Ts1 cells were adoptively transferred to recipient animals the day before, the day of, or the day after immunization; however, when Ts1 cells were transferred after TDH cells were present, no suppression occurred. In other experiments, putative TDH cells from lymph nodes (LN) or spleens were adoptively transferred from mice after immunization or after a suppressive dose of CneF or adoptive transfer of Ts1 cells and immunization. Delayed-type hypersensitivity could not be transferred with LN or spleen cells from mice receiving the suppressive dose of CneF or the Ts1 cells, even when the LN or spleen cells were treated with anti-Lyt 2.1 antibody and complement to remove any Ts2 cells. Delayed-type hypersensitivity was readily transferred with LN or spleen cells from immunized mice whether the cells were or were not treated with anti-Lyt 2 and complement. Furthermore, the cells in the tolerized LN cell pools responsible for suppression of TDH cell induction were Lyt 1+ 2-, I-J+ cells, which is the phenotype of the Ts1 cells. Taken together, these data indicate that Ts1 cells inhibit the induction of TDH cells. This finding, coupled with the previous demonstration that Ts1 cells or a Ts1 cell-derived soluble factor (TsF1) induces Ts2 cells, establishes that the cryptococcal Ts1 cells are bifunctional in the suppressive pathway.

Animals↗

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↗

PubMed1986

Encapsulation and melanin formation as indicators of virulence in Cryptococcus neoformans.

Acapsular (Cap-) mutants of Cryptococcus neoformans var. neoformans that produce melanin (Mel+) on diphenol media at 30 degrees C but not at 37 degrees C were found to be avirulent for mice. Compared with wild-type isolates, the mutants had a lower rate of L-3,4-dihydroxyphenylalanine uptake at 37 degrees C and showed an insignificant level of phenoloxidase activity at both temperatures. To study the relationship of Cap and Mel phenotypes to virulence in mice, we crossed one of the mutants (Cap- Mel-) with a wild type (Cap+ Mel+) to obtain four classes of progeny (Cap+ Mel+, Cap+ Mel-, Cap- Mel+, and Cap- Mel-). The progeny with the Cap+ Mel+ phenotype and the wild-type parent (Cap+ Mel+) were inoculated into mice (10(6) cells per mouse) and, within 40 days, produced fatal infection in 90 to 100% of the animals. None of the other three phenotypes produced fatal infection within the same period. While progeny with the Cap+ Mel- phenotype did produce fatal infection after 40 days, 70 to 90% of the mice survived at least until day 70. However, in the isolates recovered from the brain tissue of a mouse that died on day 68, nearly 40% of the CFU had reverted to the Cap+ Mel+ type. The virulence of one of these revertant Cap+ Mel+ isolates was compared with that of a Cap+ Mel- isolate recovered from the same tissue. One hundred percent of the mice inoculated with the revertant died within 35 days, while no fatal infection was produced in the mice inoculated with the Cap+ Mel- isolate within the same period. The isolates with the Cap- Mel+ or Cap- Mel- phenotype not only failed to produce fatal infection but failed to revert to the Cap+ Mel+ type in the mouse brain during the experimental period. These results indicate that both the Cap+ phenotype and the Mel+ phenotype are important indicators of virulence in C. neoformans.

Animals↗

PubMed1986

Correlation of natural killer cell activity and clearance of Cryptococcus neoformans from mice after adoptive transfer of splenic nylon wool-nonadherent cells.

Previous reports demonstrate that natural killer (NK) cells inhibit the growth of Cryptococcus neoformans in vitro, but conclusive evidence supporting the effectiveness of NK cells in host resistance to cryptococci is not available. The objective of these studies was to assess the ability of NK cells to clear C. neoformans from the lungs, livers, and spleens of infected mice. CBA/J mice were depleted of NK cells, as well as other natural effector cells, by an intraperitoneal injection of cyclophosphamide (Cy), 240 mg/kg of body weight. One day later, 7.5 X 10(7) nylon wool-nonadherent (NWN) spleen cells, either untreated or treated with anti-asialo GM1 and complement to remove NK cells, were adoptively transferred to Cy-pretreated mice. On day 2 after Cy treatment, the mice were injected intravenously with 2 X 10(4) cryptococci. At 4 and 6 days after Cy treatment, tissues were assayed for NK reactivity, using a 4-h 51Cr-release assay, and for in vivo clearance of cryptococci as reflected by mean log10 CFU per organ. We observed that Cy treatment depleted NK activity against YAC-1 targets and reduced in vivo clearance of C. neoformans from the tissues of infected mice. Additionally, Cy treatment depleted the total lung and spleen cellularity and the total number of peripheral blood lymphocytes when compared with those in normal untreated control mice. Also, spleen weights were significantly decreased in comparison with those of untreated animals 4 days after Cy treatment. Adoptive transfer of untreated NWN spleen cells into Cy-depressed mice restored the NK cell activity which correlated with enhanced clearance of cryptococci from lungs, livers, and spleens. In contrast, treatment of NWN spleen cells with anti-asialo GM1 and complement before adoptive transfer abrogated the ability of these cells to restore NK activity or reduce the numbers of cryptococci present in tissues of infected mice. Taken together, these data indicate that NK cells are the cells effective in diminishing the numbers of cryptococci in tissues of infected mice. Consequently, NK cells may play a role in first-line host resistance against C. neoformans.

Animals↗

PubMed1986

Antibody-dependent natural killer cell-mediated growth inhibition of Cryptococcus neoformans.

Previous data from this laboratory indicate that normal murine nylon wool nonadherent splenic cells with characteristics of natural killer (NK) cells effectively inhibit in vitro growth of Cryptococcus neoformans, a yeastlike pathogen. Since NK cells have been shown to be involved in antibody-dependent, cell-mediated cytotoxicity against immunoglobulin G (IgG)-coated tumor cells and xenogenic erythrocytes, we were interested in assessing the effects of the IgG fraction of rabbit anticryptococcal serum on NK cell-mediated inhibition of C. neoformans growth. Early in the study it became apparent that the conventional method of determining the numbers of CFU that was used previously for assessment of viable cryptococci at the end of the growth inhibition assay was not reliable for these studies, owing to minor clumping of the organisms in the presence of anticryptococcal antibody. Therefore, the BACTEC radiometric system was evaluated and determined to be a reliable replacement for the CFU count method. Using the BACTEC methodology, we showed that the anticryptococcal antibody significantly augmented the in vitro ability of NK cells to inhibit the growth of C. neoformans compared with normal rabbit serum or tissue culture medium. Furthermore, the antibody alone did not have an adverse effect on the organism, confirming that reduced growth indices obtained from test wells containing antibody, NK cells, and cryptococci were due to the effects of the NK cells. Maximum anticryptococcal activity of the NK cells was observed in the presence of 16 micrograms of IgG per ml; however, significant augmentation of anticryptococcal activity was seen with antibody concentrations as low as 3 micrograms/ml. Using different populations of murine splenic cells which had varying degrees of NK cell activity, we were able to show that NK cell activities, as determined by 51Cr release from YAC-1 targets, directly correlated with antibody-dependent, cell-mediated growth inhibition against cryptococci, suggesting that NK cells were effector cells in the antibody-dependent assays. Furthermore, in every case, the antibody-dependent activity of NK cells against C. neoformans was higher than the spontaneous activity of NK cells against the organism, emphasizing that NK cell activity against cryptococci can be augmented by specific antibody. When NK cell numbers were enriched by Percoll fractionation of nylon wool nonadherent splenic cells, antibody-dependent and spontaneous growth inhibitory activities of the effector cells were concomitantly augmented, confirming that NK cells were the effector cells in antibody-dependent growth inhibition of cryptococci.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Immunoadsorption of Cryptococcus-specific suppressor T-cell factors.

In the murine cryptococcal suppressor cell circuit, two different T-cell suppressor factors, TsF1 and TsF2, have been identified which specifically suppress the delayed-type hypersensitivity (DTH) response to cryptococcal culture filtrate antigen (CneF). TsF1 is produced by a first-order T suppressor (Ts1) cell population and suppresses the afferent limb of the DTH response, whereas TsF2 is produced by a second-order T suppressor (Ts2) cell population and suppresses the efferent limb of the cryptococcal DTH response. The objective of this study was to ascertain whether TsF1 or TsF2 could bind to cryptococcal antigen. To assess this, adsorption of TsF1 and TsF2 was performed with heat-killed Cryptococcus neoformans cells and by solid-phase immunoadsorption (SPIA) on columns containing cryptococcal antigens, i.e., CneF covalently bound to Sepharose 4B. The suppressive effect of TsF1 was removed by adsorption with intact heat-killed cryptococci and by SPIA on CneF-Sepharose 4B. The binding of cryptococcal TsF1 to the cryptococcal SPIA column was shown to be specific since Sepharose 4B columns either coupled with Saccharomyces cerevisiae mannan or blocked with glycine did not adsorb the suppressor activity. In contrast, the suppressive component of TsF2 did not bind to heat-killed cryptococci, CneF-Sepharose 4B, S. cerevisiae mannan-Sepharose 4B, or glycine-Sepharose 4B columns. These results, together with the finding that cryptococcal antigen, anticryptococcal antibody, and C1q-binding immune complexes were not demonstrated in either TsF1 or TsF2, establish that TsF1 and TsF2 can be differentiated on the basis of their affinity for cryptococcal antigen.

Animals↗

PubMed1986

Activation of the complement system by Cryptococcus neoformans leads to binding of iC3b to the yeast.

The complement system plays a key role in resistance to cryptococcosis. In the present study, we examined several factors that influence the binding of C3 cleavage fragments to Cryptococcus neoformans. Binding of C3 was determined by using normal human serum supplemented with 125I-labeled C3. Incubation of encapsulated cryptococci in 20% serum led to the binding of approximately 3.2 X 10(6) molecules of C3 to each cell. The binding of C3 was markedly inhibited by heating the serum at 56 degrees C for 30 min or by chelation of the serum with EDTA. Chelation of the serum with EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] reduced binding of C3 by 37%. These results indicated that activation of C3 cleavage fragments and their binding to C. neoformans was primarily dependent upon the alternative pathway. Bound C3 could be removed by incubation with 1.0 M hydroxylamine (pH 10) but not by incubation with 3.5 M NaSCN or with phosphate-buffered saline containing 0.1% sodium dodecyl sulfate. These results suggested that C3 fragments were bound to C. neoformans by ester bonds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of C3 fragments eluted from the yeast showed the presence of protein bands consistent with the presence of iC3b. C3b was not detected on the yeast after incubation with serum for time intervals as short as 2.5 min, indicating a rapid conversion of cell-bound C3b to iC3b. These results indicate that iC3b is the ligand which most likely interacts with the phagocyte C3 receptors involved in the phagocytosis of C. neoformans.

Complement Activation↗

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↗

PubMed1987

Production, characterization, and antibody specificity of a mouse monoclonal antibody reactive with Cryptococcus neoformans capsular polysaccharide.

Two monoclonal immunoglobulin G1 antibodies reacting with Cryptococcus neoformans capsular polysaccharide (CNPS) were produced in mice by using a carefully defined procedure for immunization with unmodified CNPS purified from C. neoformans serotype A. Since the antibodies were found to have the same pattern of specificity, only one of them (E1) is described. This anti-CNPS monoclonal antibody reacted with the glucuronoxylomannan component of CNPS but not with the constituent monosaccharides or with the mannose alpha(1----3)-linked oligosaccharide structures present on CNPS. E1 appeared to be specific for C. neoformans serotype A by agglutination of whole cells; it was specific for soluble CNPS A by gel immunoprecipitation. However, indirect immunofluorescence and competitive-binding enzyme-linked immunosorbent assay experiments showed low levels of cross-reactivity with serotypes B and D but not with serotype C. Concentrations 10,000 times higher for serotypes B and D cells than for serotype A cells were required for a 50% inhibition of E1 anti-CNPS A activity as measured by enzyme-linked immunosorbent assay. Among the other yeasts tested, a cross-reaction was only detected with Trichosporon beigelii. The four serotypes of C. neoformans could be distinguished based on intensities and patterns of fluorescence in an indirect immunofluorescence assay using the monoclonal anti-CNPS A antibody. Monoclonal anti-CNPS A antibodies could be useful for fundamental studies on the glucuronoxylomannan structure, as well as for clinical applications such as serotyping and possibly the serological diagnosis of cryptococcosis.

Animals↗

PubMed1987

Urease inhibition by EDTA in the two varieties of Cryptococcus neoformans.

Cryptococcus neoformans var. neoformans (74 isolates) and C. neoformans var. gattii (44 isolates) were used to test urease activity after growth on both yeast extract-glucose-peptone agar (YEPG) and on YEPG supplemented with 100 microM EDTA. Every isolate grown on YEPG agar for 48 h at 30 degrees C produced a positive reaction within 1 h in a modified rapid urease assay at 37 degrees C. However, isolates grown on YEPG with 100 microM EDTA showed a distinct pattern which corresponded to their varietal status. All but 1 of 74 C. neoformans var. neoformans isolates (98.7%) produced a positive reaction within 1 to 4 h, while none of 44 C. neoformans var. gattii isolates produced a positive reaction within the same period. The urease inhibition results and the canavanine-glycine-bromthymol blue agar test results showed 100% correlation among isolates of C. neoformans var. gattii and 98.7% correlation among isolates of C. neoformans var. neoformans. Two representative isolates of C. neoformans var. gattii (serotypes B and C) were further tested for urease during a prolonged incubation period in urea broth. These isolates failed to show a positive reaction even after 11 h of incubation. The uptake of EDTA was negligible in the two varieties. Extracts of cells grown on YEPA agar showed a high level of urease activity in both varieties. Extracts of cells grown on the agar with 100 microM EDTA showed a marked reduction (86%) of urease activity in one isolate of C. neoformans var. gattii but showed only a 30% reduction in one isolate of C. neoformans var. neoformans. Based on these results, the differential effect of EDTA on the two varieties of C. neoformans appeared to be due to greater inhibition of urease synthesis in C. neoformans var. gattii.

Cryptococcus↗