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PubMed1994

Occurrences, immunoglobulin classes, and biological activities of antibodies in normal human serum that are reactive with Cryptococcus neoformans glucuronoxylomannan.

Serum obtained from normal human subjects contains antibodies reactive in an enzyme-linked immunosorbent assay with the glucuronoxylomannan (GXM) of Cryptococcus neoformans. The frequency of occurrence of class-specific antibodies among normal subjects was 28% for immunoglobulin G (IgG), 98% for IgM, and 3% for IgA. Anti-GXM antibodies with kappa light chains occurred in 98% of normal subjects, while the occurrence of lambda light chains was 28%. Each of five subjects with high levels of anti-GXM IgG antibodies had readily detectable antibodies of the IgG2 isotype; two of the five subjects had readily detectable IgG1 antibody. An examination of sera from human immunodeficiency virus-infected patients showed that human immunodeficiency virus infection was accompanied by a significant decrease in the occurrence of IgM antibodies and anti-GXM antibodies with kappa light chains; these decreases occurred early in infection when CD4 counts were still > or = 500 cells per microliter. A slight but not statistically significant decrease in the occurrence of anti-GXM IgG antibodies was seen only in patients with CD4 levels of < 200 cells per microliter. Sera from normal subjects with high levels of anti-GXM IgG antibodies were examined to identify any contribution of the antibodies to complement activation or to opsonization of the yeast cells. An analysis of the kinetics for activation and binding of C3 to the yeast cell showed no pattern of quantitative or qualitative differences between sera with high or low levels of anti-GXM IgG antibodies. Phagocytosis studies showed that the naturally occurring IgG antibodies did not contribute to opsonization of the yeast cells.

Antibodies, Fungal↗
PubMed1994

Susceptibility of melanized and nonmelanized Cryptococcus neoformans to nitrogen- and oxygen-derived oxidants.

Melanized Cryptococcus neoformans cells were less susceptible than nonmelanized cells to the fungicidal effects of nitrogen- and oxygen-derived oxidants. The results support the hypothesis that the phenoloxidase enzyme system contributes to virulence by protecting C. neoformans against nitrogen- and oxygen-derived oxidative antimicrobial molecules produced by immune effector cells.

Antifungal Agents↗
PubMed1994

Molecular and idiotypic analysis of antibodies to Cryptococcus neoformans glucuronoxylomannan.

Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) form the basis of two potential therapeutic intervention strategies, i.e., conjugate vaccines and passive antibody therapy. To better understand the molecular basis of the antibody response, the heavy- and light-chain immunoglobulin variable region (VH and VL, respectively) sequences of seven monoclonal antibodies (MAbs) to GXM were determined. Rabbit anti-idiotypic serum was made to the previously characterized murine MAb 2H1 and used to study MAb 2H1 idiotype expression in other GXM-binding MAbs and immune sera. MAb E1 originated from a C3H/HeJ mouse immunized with C. neoformans serotype A polysaccharide. MAbs 471, 1255, 339, 3C2, 386, and 302 originated from BALB/c mice immunized with polysaccharide of serotypes A, A, B, C, D, and D, respectively, conjugated to sheep erythrocytes. In the E1, VH uses V11 from the T15 gene family and JH3 and has a D segment of three amino acids, and the VL uses a VKSer-like gene family element and JK5. In MAbs 471 and 3C2, the VH uses VH7183-like gene family elements and JH2 and has D segments of seven amino acids, and the VL uses VK5.1 and JK1. In MAbs 1255 and 339, the VH uses VH10-like gene elements and JH4 and has six codon D segments, and the VL uses a VK21-like gene element and JK5. In MAbs 302 and 386, respectively, the VH uses VHGAM-like gene elements and JH2 and JH3 and has six and four codon D segments, and VL uses VK4/5-like gene elements and JK1.VH usage, MAb 2H1 idiotype expression, and fine specificity mapping define a minimum of three GXM epitopes which elicit protective antibodies. The results confirm that the antibody response is highly restricted, suggest a close relationship between molecular structure and serological properties, and provide insight into protein structural motifs important for GXM binding.

Animals↗
PubMed1995

Adherence to and damage of endothelial cells by Cryptococcus neoformans in vitro: role of the capsule.

Escape from the intravascular compartment is likely a critical step in the development of hematogenously disseminated cryptococcal infections, such as meningitis. The capsule of Cryptococcus neoformans is considered to be a virulence factor because of its antiphagocytic properties. To further investigate the role of the capsule in escape from the intravascular compartment, we used isogenic strain pairs, an acapsular mutant, and an encapsulated clinical isolate to determine the effects of the capsule of C. neoformans on adherence to, phagocytosis by, and damage of endothelial cells in vitro. Acapsular C. neoformans adhered significantly more to endothelial cells and caused greater endothelial cell injury than did encapsulated organisms. Coating of an acapsular strain with cryptococcal glucuronoxylomannan decreased both adherence to and damage of endothelial cells by 61.7% +/- 9.1% and 76.6% +/- 10.2%, respectively. Transmission electron microscopy demonstrated internalization of acapsular, but not encapsulated, organisms by endothelial cells. Internalization of an acapsular strain occurred through endothelial cell phagocytosis and was inhibited by cytochalasin D. Phagocytosis required a heat-labile serum factor, probably complement. These results suggest that acapsular or poorly encapsulated C. neoformans may be the form(s) that escapes from the vasculature during initiation of hematogenously disseminated disease.

Cell Adhesion↗
PubMed1995

Antibodies to Cryptococcus neoformans glucuronoxylomannan enhance antifungal activity of murine macrophages.

Monoclonal antibodies (MAbs) to the capsular polysaccharide of the pathogenic fungus Cryptococcus neoformans can prolong survival and decrease organ fungal burden in experimental murine cryptococcosis. To investigate the mechanism of antibody-mediated protection, the interaction of C. neoformans and murine macrophage-like J774.16 cells was studied in the presence and absence of MAbs differing in isotype. Immunoglobulin G2a (IgG2a) and IgG2b isotype switch variants were isolated from an IgM hybridoma to complete the IgG subclass set. IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA MAbs were studied for their ability to promote phagocytosis and reduce the number of CFU in C. neoformans and J774.16 cell cocultures. The MAbs in this set had similar if not identical fine specificities and were derived from a single B cell. All isotypes promoted phagocytosis; however, the IgG subclasses were more effective opsonins than IgM or IgA. All isotypes enhanced J774.16 anti-C. neoformans activity in vitro, as measured by a reduction in the number of CFU. The IgG1 MAbs were consistently more active in promoting opsonization and reducing the number of CFU. Addition of IgG1 MAb to C. neoformans and J774.16 cocultures resulted in rapid reduction in the number of CFU, which is consistent with fungal killing. Electron microscopy revealed that MAb-opsonized C. neoformans cells were internalized and appeared damaged. Administration of IgM, IgG1, IgG2a, and IgG2b isotype switch variant MAbs revealed that the IgG2a and IgG2b subclasses were the most and least effective isotypes, respectively, in prolonging survival in an intraperitoneal murine infection model. The results indicate that murine antibody subclasses differ in their ability to enhance macrophage anti-C. neoformans activity and suggest that antibody enhancement of macrophage function is a mechanism by which antibodies modify infection in vivo.

Animals↗
PubMed1995

Direct anticryptococcal activity of lymphocytes from Cryptococcus neoformans-immunized mice.

Assessment of the direct anticryptococcal activity of murine lymphocytes from both Cryptococcus neoformans-immunized and control mice was the focus of this investigation. We demonstrate that at a 2:1 effector cell-to-cryptococcal target cell ratio, effector cell populations comprised of alpha beta T-cell receptor-positive T lymphocytes (98 to 99% CD3+) from C. neoformans-immunized mice inhibited the growth of cryptococcal cells better than similar populations of lymphocytes from nonimmunized control mice. Almost immediately after mixing of cryptococci with the effector cells, C. neoformans-lymphocyte conjugates were observed. The percentage of conjugates increased over the first 30 min of incubation and then remained constant over the next 1.5 h. T-lymphocyte-enriched populations from C. neoformans-immunized mice formed significantly greater percentages of conjugates with cryptococci than control T lymphocytes at each time period that assessment was made. For growth inhibition to occur, direct contact between the effector and target cells was necessary, as evidenced by abrogation of cryptococcal growth inhibition when lymphocyte and cryptococcal cell populations were separated by a porous membrane during the growth inhibition assay. Vital staining of cryptococci after incubation with the T-cell-enriched populations showed that the T lymphocytes killed the cryptococcal cells.

Animals↗
PubMed1995

Effects of immunization with Cryptococcus neoformans cells or cryptococcal culture filtrate antigen on direct anticryptococcal activities of murine T lymphocytes.

Immunizing CBA/J mice with intact Cryptococcus neoformans cells or with a cryptococcal culture filtrate antigen (CneF) induces an anticryptococcal delayed-type hypersensitivity response. Recently, it has been shown that two phenotypically different T-cell populations are responsible for delayed-type hypersensitivity reactivity in mice immunized with intact cryptococcal cells, whereas only one of those populations is present in mice immunized with soluble cryptococcal antigens in complete Freund's adjuvant (CFA). The purpose of this study was to determine if differences occur with regard to direct anticryptococcal activity between T-lymphocyte-enriched populations from mice immunized with intact viable or dead cryptococcal cells and similar cell populations from mice immunized with the soluble cryptococcal culture filtrate antigen, CneF, emulsified in CFA. The percentage of lymphocytes which form conjugates with C. neoformans and the percentage of cryptococcal growth inhibition in vitro are greater with T-lymphocyte-enriched populations from mice sublethally infected with C. neoformans or from mice immunized with intact heat-killed cryptococcal cells in the presence or absence of CFA than with lymphocyte populations from mice immunized with CneF-CFA. Enhanced anticryptococcal activity of T lymphocytes could be induced by immunizing mice with heat-killed C. neoformans cells of serotype A, B, C, or D as well as by immunizing with a similar preparation of an acapsular C. neoformans mutant but not by immunizing with CFA emulsified with CneF prepared from any one of the C. neoformans isolates. These data indicate that the soluble cryptococcal culture filtrate antigens do not induce the same array of functional T lymphocytes as whole cryptococcal cells.

Animals↗
PubMed1995

Role of tumor necrosis factor and gamma interferon in acquired resistance to Cryptococcus neoformans in the central nervous system of mice.

Although naive C.B-17 and BALB/cBy mice die of meningoencephalitis within 5 weeks of intravenous infection with an opportunistic strain of Cryptococcus neoformans, immunized mice express an acquired, CD4+ T-cell-dependent immunity and survive an intravenous infection. Infusion of lymphocytes from immune mice into severe combined immunodeficiency (SCID) mice renders these mice more resistant to cryptococcal brain infection than uninfused controls. We have investigated the role of gamma interferon (IFN-gamma) and tumor necrosis factor (TNF) in acquired resistance to C. neoformans. Neutralization of either IFN-gamma or TNF impaired resistance of immune BALB/cBy or C.B-17 mice to cryptococci. At 10 days postinfection, there were approximately 10 times as many yeast cells in the brains of mice treated with either anticytokine antibody as in the brains of mice treated with control antibody. Simultaneous neutralization of IFN-gamma and TNF further exacerbated infection. Neutralization of IFN-gamma or TNF also impaired resistance in immune lymphocyte-infused SCID mice, resulting in significantly higher yeast burdens in brains of cytokine-neutralized mice than in brains of controls. Concurrent neutralization of IFN-gamma and TNF rendered SCID recipients of immune cells equivalent to uninfused SCID mice with respect both to brain yeast burdens at 10 days and to survival. Anti-TNF treatment alone also curtailed survival. Histological examination of the brains of cytokine-neutralized mice revealed deficiencies in ability to focus inflammatory cells at brain lesions. These data demonstrate that both IFN-gamma and TNF are important mediators of acquired resistance to cryptococcal meningoencephalitis.

Animals↗
PubMed1995

Determination of antigen binding specificities of Cryptococcus neoformans factor sera by enzyme-linked immunosorbent assay.

The competitive binding specificities of glucuronoxylomannan (GXM) and its derivatives to factor sera of Cryptococcus neoformans were studied by enzyme-linked immunosorbent assay. An effort was made to determine the epitope specificity of each factor serum. Despite the presence of antigenic factor 1 on all serotypes of C. neoformans, variations in inhibition ability were observed with different GXMs. The panspecific component of factor serum 1 (antibody 1) appeared to be due to the presence of more than one antibody component. The activity was dependent on the 6-O-acetyl substituent. GXMs of serotypes A and D inhibited factor serum 2 equally well, indicating a low titer for the antibody 7 component. Serotype B GXM was a poor inhibitor, and serotype C GXM did not inhibit factor serum 2. The activity of factor serum 2 was 6-O-acetyl dependent. GXMs from typical serotype A and serotype D isolates were excellent inhibitors of factor serum 3. GXMs from serotype B were poorly inhibitory and serotype C did not inhibit factor serum 3. The activity of factor serum 3 was 6-O-acetyl dependent. The activity of factor serum 4 was due predominantly to antibody component 6. The activity of factor 4 was directed mainly against serotype C, and it was independent of 6-O-acetyl substitution Factor serum 5 was specific for serotype B GXMs. The inhibitory effect was independent of 6-O-acetyl substitution, but the effect was diminished by reduction of the glucuronic acid. The GXMs with a typical serotype C structure inhibited antibody 6. O deacetylation of the GXMs did not affect their inhibitory activity. However, reduction of glucuronic acid reduced factor serum 6 binding. Factor serum 8 was specific to serotype D; native GXMs of serotype A were slightly inhibitory. O deacetylation of the serotype D GXMs abrogated the inhibitory effect. O deacetylation alone abrogates the activity of antibody components 1, 2, 3, and 8. Reduction of glucuronic acid reduces the inhibitory activity of the GXM to antibody components 4, 5, and 6. Partial GXM structures and methyl glycosides did not effectively inhibit the activity of any of the factor sera.

Animals↗
PubMed1995

Ingestion of acapsular Cryptococcus neoformans occurs via mannose and beta-glucan receptors, resulting in cytokine production and increased phagocytosis of the encapsulated form.

Cryptococcus neoformans is a pathogenic yeast and a major cause of opportunistic infection in AIDS patients. It is commonly found in an acapsular form in the environment, and infection is likely to occur by inhalation. The lung provides a suitable environment for capsule synthesis, and once encapsulated, C. neoformans becomes resistant to phagocytosis. A stable acapsular mutant of the organism is readily ingested by murine macrophages in vitro, indicating entry via constitutively competent receptors. We demonstrate in this report that this process is inhibitable by particles derived from Saccharomyces cerevisiae that are rich in mannan and beta-glucan, as well as more purified forms of these glycans. Furthermore, ingestion of the acapsular form of C. neoformans induces a range of proinflammatory cytokines, including tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor, which, as we have previously shown, enhance ingestion of serum-opsonized encapsulated C. neoformans in vitro. We demonstrate that ingestion of the acapsular form of the organism also enhances ingestion of the pathogenic encapsulated form. This is dependent on the production of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor by the macrophages, since addition of neutralizing antibodies to both cytokines inhibited the observed increase in ingestion. Together, these data demonstrate that ingestion of acapsular C. neoformans is mediated via mannose and beta-glucan receptors on the macrophage surface and that this process activates macrophages for enhanced phagocytosis of the encapsulated form via production of macrophage-derived cytokines.

Animals↗
PubMed1995

Effects of the two varieties of Cryptococcus neoformans cells and culture filtrate antigens on neutrophil locomotion.

Cryptococcus neoformans var. gattii (serotype B and C) isolates have a relative predilection for immunocompetent hosts, and C. neoformans var. neoformans (serotype A and D) isolates have a relative predilection for immunocompromised hosts, suggesting that normal host resistance to the former may be relatively inefficient compared with that to the latter variety. In order to assess the possibility that normal cellular host defense is inadequate in protecting against C. neoformans var. gattii, we compared the two varieties of C. neoformans cells and their culture filtrate antigens (CneF) with respect to effects on neutrophil (polymorphonuclear leukocyte [PMN]) locomotion. In a 48-well modified Boyden chamber, the cells and CneF of C. neoformans var. neoformans (serotype A and D) isolates stimulated chemotaxis and chemokinesis of human PMN and activated a complement component(s) in pooled human serum to become a chemoattractant(s) for human PMN. In contrast, the cells and CneF of C. neoformans var. gattii (serotype B and C) isolates did not stimulate chemotaxis or chemokinesis in human PMN but rather inhibited chemokinesis and chemotactic responses of PMN to pooled human serum and formylmethionyl leucyl phenylalanine. Neither of the CneF from the C. neoformans var. gattii isolates was cytotoxic to PMN. Furthermore, with the mouse model, we found that CneF from C. neoformans var. neoformans caused migration of PMN into gelatin sponges implanted in naive and immunized mice, whereas CneF from C. neoformans var. gattii inhibited PMN migration into sponges. Our results, combined with findings of others showing reduced PMN infiltration in lungs of mice infected with C. neoformans var. gattii compared with PMN infiltration in lungs of mice infected with C. neoformans var. neoformans, indicate that the relative inadequacy of normal host resistance mechanisms to prevent infection with C. neoformans var. gattii results, in part, from inhibition of PMN migration to the site of the organism.

Animals↗
PubMed1995

Analysis of human monoclonal antibodies elicited by vaccination with a Cryptococcus neoformans glucuronoxylomannan capsular polysaccharide vaccine.

The Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) has been conjugated to tetanus toxoid (GXM-TT) as an investigational vaccine. GXM-TT elicits antibodies that are protective in C. neoformans-infected mice. In an effort to characterize the fine specificity and molecular structure of human GXM-TT-elicited antibodies, we generated two GXM monoclonal antibodies (MAbs) from peripheral blood lymphocytes of a volunteer GXM-TT recipient and studied serum GXM antibody idiotype expression in 10 additional vaccinees. The MAbs, 2E9 and 3B6, are the immunoglobulin M(lambda) isotype and bind capsular polysaccharides of C. neoformans serotypes other than the serotype A that was used for immunization. Neither antibody competes with murine GXM MAbs for antigen binding, suggesting that the human MAbs recognize a different epitope. The B-cell superantigen staphylococcal protein A binds both MAbs, and human immunodeficiency virus gp120 binds 2E9. MAb nucleic acid sequence analysis revealed that both antibodies use an identical V lambda 1a-J lambda genetic element with different, somatically mutated, members of the VH3 gene family and different DH and JH gene elements. The gene elements used by both MAbs occur in fetal B-lymphocyte repertoires, autoantibodies, and other polysaccharide antibodies. Post-GXM-TT vaccination GXM antibodies from 10 additional vaccinees expressed a shared idiotype defined by rabbit antiserum raised against MAb 2E9. Our data suggest that the human GXM antibody response is restricted and raise questions regarding the importance of specific variable-region elements and superantigens in the generation of human antibody responses to encapsulated pathogens.

Amino Acid Sequence↗
PubMed1995

Cryptococcus neoformans melanin and virulence: mechanism of action.

Black melanin-like pigments are produced by several neurotropic fungi, including Cryptococcus neoformans. Pigment production is associated with virulence. In media containing phenolic substrates such as L-dopa, C. neoformans cells become black as a result of pigment accumulation. Pigmented and nonpigmented C. neoformans cells were studied with transmission electron microscopy and electron spin resonance (ESR) spectroscopy. Transmission electron microscopy showed electron-dense cell walls, and ESR spectroscopy revealed a stable free-radical population in pigmented cells. The ESR signals of pigmented cells were increased by light, alkaline pH, and Zn2+ and decreased by acid pH, indicating that the black pigment was a type of melanin. A mutant deficient in melanin synthesis (mel) generated by UV radiation lacked ESR-detectable radicals, was less virulent for mice, was more susceptible to killing by nitrogen- and oxygen-derived radicals, and had 100-foldless phenoloxidase activity than the parent strain. The interaction of melanized C. neoformans, nonmelanized C. neoformans, and the hypomelanotic mel mutant with J774.16 murine macrophage-like cells was studied. Melanized cells were more resistant to antibody-mediated phagocytosis and the antifungal effects of murine macrophages than nonmelanized cells. Small increases in the intensity of the ESR signals of melanized cells in solutions containing chemically generated oxygen- and nitrogen-derived radicals indicated electron transfer to or from melanin. Melanin appears to contribute to virulence by protecting fungal cells against attack by immune effector cells.

Animals↗
PubMed1995

Variable efficacy of passive antibody administration against diverse Cryptococcus neoformans strains.

The efficacy of monoclonal antibody (MAb 2H1) against diverse strains of Cryptococcus neoformans was studied in a murine model of intravenous infection. For six of eight strains, administration of MAb prior to infection prolonged survival of mice. For two strains, 371 and SB4A, administration of MAb prior to infection did not prolong survival in multiple experiments with inocula ranging from 10(2) to 10(6) yeast cells per mouse. Mice infected with strains 371 and SB4A had fewer CFU than non-MAb-treated controls, but the CFU reduction was not sufficient to affect survival. Serum glucuronoxylomannan (GXM) levels varied for the different C. neoformans strains. For mice that did not receive MAb 2H1, there was a positive correlation between lung fungal burden and serum GXM levels. MAb 2H1-treated mice had significantly reduced serum GXM levels. The results indicate that the efficacy of MAb 2H1 administration in prolonging survival and/or reducing organ CFU varies with the C. neoformans strain.

Animals↗
PubMed1995

Binding of host collectins to the pathogenic yeast Cryptococcus neoformans: human surfactant protein D acts as an agglutinin for acapsular yeast cells.

Cryptococcus neoformans is an opportunistic pathogen in AIDS patients causing disseminated disease and lethal meningitis after inhalation of acapsular or sparsely encapsulated yeast cells. In this study we have investigated whether a recently described family of primitive opsonins, termed collectins, contribute to innate resistance against C. neoformans. The pulmonary surfactant proteins SP-A and SP-D as well as the serum collectins mannose-binding protein and CL-43 bound in a calcium-dependent manner to acapsular C. neoformans in vitro. Binding was concentration dependent and abolished by competition with defined mono- and oligosaccharides. In contrast, no binding of the collectins was observed with the encapsulated form of the yeast. Furthermore, binding of purified collectin SP-D, but not SP-A, mannose-binding protein, or CL-43, led to a concentration-dependent agglutination of acapsular C. neoformans. These data indicate that collectins recognize carbohydrate structures in the cell wall of an initial infectious form of C. neoformans and may play a role in early antifungal defenses in the lung.

Agglutinins↗
PubMed1995

Tissue localization of Cryptococcus neoformans glucuronoxylomannan in the presence and absence of specific antibody.

During infection, Cryptococcus neoformans capsular glucuronoxylomannan (GXM) is released into tissues, where it may be associated with a variety of deleterious immunological effects. Relatively little is known about the organ distribution and cellular localization of GXM antigen. Intravenous administration of GXM to rats resulted in persistent serum levels which declined with a half-life of 14.3 h in the first 74 h and 3 h thereafter, coincident with the appearance of serum antibodies to GXM. GXM was sequestered primarily in spleen tissue, with localization to marginal zone and follicular cells. Administration of the murine immunoglobulin G1 monoclonal antibody (MAb) 2H1 resulted in > 99% reduction in serum GXM level within 3 h. MAb 2H1 administration resulted in liver GXM deposition, with cellular localization primarily to Kupffer cells. GXM was also found in the spleens of MAb 2H1-treated rats, with localization to the marginal zones and follicles. Endotracheal administration of GXM resulted in low serum levels, with lung tissue having the highest GXM organ levels, localized primarily to alveolar macrophages. The results indicate that (i) intravenous administration to rats produced persistent serum GXM levels with a half-life similar to that found in mice and rabbits; (ii) endotracheal administration of GXM resulted in low serum levels; (iii) in the absence of specific antibody, GXM organ deposition occurs primarily in the spleen and is localized primarily to marginal zone macrophages; (iv) in the presence of specific immunoglobulin G1 antibody, GXM organ deposition occurs primarily in the liver and is localized primarily to Kupffer cells; and (vi) reticuloendothelial cells sequester GXM in the presence and absence of specific antibody.

Animals↗
PubMed1995

Mechanisms of inhibition of Cryptococcus neoformans by human lymphocytes.

Recently, our laboratory and others have demonstrated that human peripheral blood T and NK lymphocytes directly inhibit the growth of Cryptococcus neoformans. In this study, we further define the conditions under which lymphocyte-mediated fungistasis against C. neoformans occurs and examine whether mechanisms implicated in lymphocyte-mediated activities against other target cells are also involved in anticryptococcal activity. The addition of whole or broken heat-killed C. neoformans modestly inhibited lymphocyte-mediated fungistasis, whereas other particulates had no effect. The hydroxyl radical scavenger catechin, but not diethyl urea or propyl gallate, profoundly inhibited fungistasis. Salicylic acid inhibited fungistasis in a dose-dependent fashion. However, two other cyclooxygenase inhibitors, piroxicam and indomethacin, had no effect, suggesting that the mechanism of inhibition by salicylic acid was cyclooxygenase independent. Reagent prostaglandin E2, at concentrations shown by others to inhibit NK cell-mediated bactericidal and tumorlytic activities, had no effect on lymphocyte-mediated fungistasis. The addition of selected monoclonal antibodies or ligands reactive with receptors on human lymphocytes had no significant effect on lymphocyte-mediated fungistasis. Acapsular, small-capsuled, and large-capsuled C. neoformans organisms were inhibited by lymphocytes to an approximately equal extent. These data demonstrate that lymphocyte-mediated activity against C. neoformans proceeds regardless of the presence of capsule and by mechanisms at least in part dissimilar from those seen with other target cells.

Antibodies, Monoclonal↗
PubMed1996

Proteins in the cell wall and membrane of Cryptococcus neoformans stimulate lymphocytes from both adults and fetal cord blood to proliferate.

Cryptococcus neoformans is an encapsulated yeast that infects patients who have defective cell-mediated immunity, including AIDS, but rarely infects individuals who have intact cell-mediated immunity. Studies of the immune response to C. neoformans have been hampered by a paucity of defined T-lymphocyte antigens, and hence, the understanding of the T-cell response is incomplete. The goal of this study was to separate C. neoformans into its component parts, determine whether those components stimulate lymphocyte proliferation, perform preliminary characterization of the proteins, and establish the potential mechanism of lymphocyte proliferation. The lymphocyte response to fungal culture medium, whole organisms, disrupted organisms, and the yeast intracellular fraction or cell wall and membrane was studied by determining thymidine incorporation and by determining the number of lymphocytes at various times after stimulation. The cell wall and membrane of C. neoformans stimulated lymphocyte proliferation, while the intracellular fraction and culture filtrate did not. The optimal response occurred on day 7 of incubation, with 4 x 10(5) peripheral blood mononuclear cells per well and with 13 microg of cryptococcal protein per ml. The number of lymphocytes increased with time in culture, indicating that thymidine incorporation was accompanied by proliferation. Proteinase K treatment of the cell wall and membrane abrogated lymphocyte proliferation, indicating that the molecule was a protein. [35S]methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography were performed to analyze the proteins contained in the cell wall and membrane, intracellular fraction, and culture filtrate. At least 18 discrete bands were resolved from the cell wall and membrane. Since a large percentage of healthy adults responded to the cryptococcal cell wall and membrane, a mitogenic effect was investigated by testing proliferation of fetal cord blood lymphocytes. The percentage of fetal samples that proliferated in response to the cell wall and membrane was similar to the percentage of fetal samples that proliferated in response to Staphylococcus enterotoxin B, a microbial mitogen. Thus, a protein in the cell wall and membrane of C. neoformans is a potent stimulant of lymphocyte proliferation and has mitogenic properties, which may have important implications for cell-mediated immunity to C. neoformans.

Adult↗