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PubMed1999

New PCR primer pairs specific for Cryptococcus neoformans serotype A or B prepared on the basis of random amplified polymorphic DNA fingerprint pattern analyses.

Thirty-three strains of Cryptococcus neoformans were isolated from clinical specimens, including specimens from AIDS patients in Brazil, and were classified into two serotypes; we detected 31 and 2 strains of serotypes A and B, respectively. Random amplified polymorphic DNA (RAPD) fingerprint pattern analyses of these strains of serotypes A and B showed that the patterns were similar for strains of each serotype when three 10-mer primers were used as the RAPD primers. Comparative studies of the fingerprint patterns of the study isolates with those of the reference strains also showed that the RAPD patterns for strains of each serotype were related and that most of the fingerprint bands existed commonly for all strains of each serotype tested. The common RAPD bands (an approximately 700-bp band for serotype A and an approximately 450-bp band for serotype B) were extracted and the DNA sequences were determined. Using this information, we prepared two and one PCR primer pairs which were expected to be specific for C. neoformans serotypes A and B, respectively. Use of each PCR primer combination thus prepared for serotype A or B was 100% successful in identifying the respective C. neoformans serotypes, including the 33 clinical isolates tested in the present study. Among these combinations, one for serotype A was found to amplify DNA from C. neoformans serotype B as well as serotype A. Serotype B-specific PCR primer pairs amplified DNA from not only serotype B strains but also from serotype C strains. The usefulness of other serotype-specific PCR primers for clinical C. neoformans isolates is discussed.

AIDS-Related Opportunistic Infections↗
PubMed1997

Both CD4+ and CD8+ human lymphocytes are activated and proliferate in response to Cryptococcus neoformans.

The current studies were performed to determine the contribution of T-cell subsets to lymphocyte proliferation in response to Cryptococcus neoformans, the most common invasive mycosis in acquired immune deficiency syndrome. We demonstrate for the first time that both human CD4 and CD8 cells are activated in response to C. neoformans. Both CD4 and CD8 cells express interleukin-2 receptor alpha (IL-2R alpha) and transferrin receptor and proliferate in response to C, neoformans, however proliferation of CD8 cells was dependent upon CD4 cells. The requirement for CD4 cells was complex, since CD8 enriched cells failed to express mRNA for IL-2, suggesting that CD4-dependent IL-2 production was required for CD8-cell proliferation. However, IL-2 was not sufficient to restore CD8-cell proliferation. These studies provide experimental evidence in humans to support the clinical impression that CD4 cells are important in cryptococcosis, and suggest that the appropriate CD4-derived signals could allow CD8 cells to assist in host defence.

Adult↗
PubMed1993

Mobility of human neutrophils in response to Cryptococcus neoformans cells, culture filtrate antigen, and individual components of the antigen.

The mobility of human neutrophils (PMN) in response to encapsulated or nonencapsulated Cryptococcus neoformans cells or cryptococcal culture filtrate (CneF) and its components was studied by using a 48-well modified Boyden chamber. Encapsulated C. neoformans (isolate 184A) cells and CneF-184A stimulated directed migration of human PMN in the absence of serum (direct chemotactic activity) and activated a heat-labile component(s) in fresh human serum to become a chemoattractant(s) for human PMN (indirect chemotactic activity). At a 1:8 dilution (0.25 mg of carbohydrate per ml), CneF-184A displayed chemokinetic activity when assessed with a checkerboard assay. Nonencapsulated C. neoformans isolate 602 cells did not have direct chemotactic activity but did have indirect chemotactic activity. The capsule of C. neoformans is composed predominantly of glucuronoxylomannan (GXM). Purified GXM displayed both direct and indirect chemotactic activity. CneF-184A contains, in addition to GXM, a concanavalin A-binding mannoprotein (MP), whereas CneF-602 contains no GXM but does contain MP. CneF-184A showed direct chemotactic activity and CneF-602 did not. Both CneF-184A and CneF-602 displayed indirect chemotactic activity for human PMN. In addition, purified MP from CneF-184A, like CneF-602, showed only indirect chemotactic activity. These results indicate that GXM contributes to the direct chemotactic activity of PMN observed with the whole encapsulated yeast cells and the unfractionated CneF derived from the encapsulated cells. Both MP and GXM from encapsulated C. neoformans cells mediate indirect chemotactic activity on human PMN.

Antigens, Fungal↗
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↗
PubMed1990

Cumulative positivity rates of multiple blood cultures for Mycobacterium avium-intracellulare and Cryptococcus neoformans in patients with the acquired immunodeficiency syndrome.

We examined the occurrence of low-grade Mycobacterium avium-intracellulare bacteremia and Cryptococcus neoformans fungemia in patients with the acquired immunodeficiency syndrome and the consistency of positive cultures obtained using a sensitive blood culture system (Isolator, E. I. Du Pont de Nemours, Wilmington, Del) for the recovery of these organisms. The blood culture records were reviewed, and the proportion of positive blood cultures yielding less than 1 colony-forming unit per milliliter of M avium-intracellulare or C neoformans was calculated. To determine consistency, a period of potentially detectable septicemia was defined as the period between 1 week before the first positive blood culture and the last positive blood culture, providing consecutive positive blood cultures were separated by less than 2 weeks. All positive and negative blood cultures obtained during the period of potentially detectable septicemia were considered in the data analysis. Overall, 40 (16.9%) of 236 cultures positive for M avium-intracellulare and 36 (57.1%) of 63 for C neoformans yielded less than 1 colony-forming unit per milliliter. Mycobacteremia was detected in 52 of 57 periods of potentially detectable septicemia in the first culture and in 56 of 57 in the first two (cumulative detection rates of 91.2% and 98.2%, respectively). Cryptococcemia was detected in 12 of 17 periods of potentially detectable septicemia in the first culture and in 15 of 17 in the first two (cumulative detection rates of 70.6% and 88.2%, respectively). Because of the sensitivity of the blood culture system and the consistency of M avium-intracellulare bacteremia and C neoformans fungemia in patients with the acquired immunodeficiency syndrome, it appears that two blood cultures are sufficient for the detection of most septic episodes caused by these organisms.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Activation of human peripheral blood mononuclear cells by interleukin-2 and granulocyte-macrophage colony-stimulating factor to inhibit Cryptococcus neoformans.

The abilities of selected cytokines to activate human peripheral blood mononuclear cells (PBMC) to inhibit and kill the opportunistic fungus Cryptococcus neoformans were studied. PBMC were cultured for 7 days in cell wells containing no cytokines, tumor necrosis factor (TNF), gamma interferon (IFN-gamma), 1,25-dihydroxycholecalciferol (vitamin D3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-2 (IL-2) and were then challenged for 24 h with a fixed number of CFU of C. neoformans. The number of CFU increased in wells containing no cytokines, TNF, IFN-gamma, or vitamin D3 and remained about the same in wells containing GM-CSF. In contrast, the number of CFU in wells containing IL-2-stimulated PBMC decreased, suggesting fungicidal activity. Optimal conditions for IL-2 stimulation included a minimum of 5 days of incubation of PBMC with IL-2, a concentration of 100 U of IL-2 per ml, and a high ratio of effectors to fungi. Separation of IL-2-stimulated PBMC based upon their adherence to plastic revealed that antifungal activity resided in the nonadherent fraction. These data demonstrate that IL-2 and GM-CSF are capable of stimulating PBMC-mediated antifungal activity and suggest that these cytokines may play physiological or pharmacological roles in host defenses against cryptococcosis.

Cells, Cultured↗
PubMedDate not supplied

Comparative study of antifungal activity of sertaconazole, terbinafine, and bifonazole against clinical isolates of Candida spp., Cryptococcus neoformans and dermatophytes.

The in vitro activity of sertaconazole was compared with that of terbinafine and bifonazole against 180 Candida spp., Cryptococcus neoformans yeasts and 53 dermatophytes. Minimum inhibitory concentrations (MICs) were determined by a microdilution method in Sabouraud's buffered liquid medium (pH 5.6). Sertaconazole (arithmetic mean MIC 1.24 mg/l) was statistically more active than bifonazole (MIC 6.54 mg/l) and terbinafine (MIC 12.61 mg/l) against yeasts strains, MIC values for sertaconazole being generally and specifically lower for each tested yeast species. MIC for C. parapsilosis (0.26 mg/l) demonstrated a higher activity of sertaconazole against this species, in contrast to C. tropicalis (MIC 1.49 mg/l). Against dermatophytes, MIC for terbinafine (0.05 mg/l) was lower than sertaconazole (MIC 0.41 mg/l) and bifonazole (MIC 1.04 mg/l). These results, obtained under the same experimental conditions, confirm the good antifungal activity of sertaconazole against both yeasts and dermatophytes with lower MICs obtained in the topical application. This in vitro activity correlates with the clinical efficacy of sertaconazole compared with other antifungal agents.

Antifungal Agents↗
PubMed1993

T-cell-dependent and T-cell-independent mechanisms of tolerance to glucuronoxylomannan of Cryptococcus neoformans serotype A.

Glucuronoxylomannan (GXM), a type 2 T-independent antigen, is the major component of the capsular polysaccharide (CnCAP) of Cryptococcus neoformans. Previous studies have described the tolerogenic effects of high doses of CnCAP on the specific humoral response. In this investigation, evidence for both high-dose and low-dose tolerance to GXM is presented. BALB/cBy female mice, primed with either 5 ng or 50 micrograms of GXM, then coimmunized 3 days later with immunogenic doses of both GXM and type 3 pneumococcal polysaccharide (SSS-III), showed an antigen-specific inhibition in their splenic plaque-forming cell (PFC) responses to GXM compared with control groups primed with normal saline. SSS-III PFCs remained unchanged between GXM-primed and normal saline-primed groups. Low-dose tolerance appeared to be T dependent, whereas high-dose tolerance appeared to be T independent. Low-dose tolerance to GXM could not be induced in athymic BALB/c nu/nu mice, whereas high-dose tolerance in the same mice could be induced. Furthermore, low-dose tolerance was adoptively transferred with B-cell-depleted splenocytes to naive BALB/c mice, while high-dose tolerance was not. Complement-mediated depletion of CD4+ but not CD8+ splenocytes from low-dose-primed mice abrogated the transfer of low-dose tolerance. These findings indicate T-dependent and T-independent mechanisms of antigen-specific B-cell tolerance to GXM in BALB/c mice at low and high antigen doses, respectively.

Animals↗
PubMed1982

The capsule of cryptococcus neoformans passively inhibits phagocytosis of the yeast by macrophages.

We examined the mechanism by which cryptococcal capsular polysaccharide inhibits phagocytosis of Cryptococcus neoformans by macrophages. O-Acetyl and carboxyl groups are major structural features of serotype D polysaccharide. Serotype D capsular polysaccharide or encapsulated whole cryptococci were de-O-acetylated by alkaline hydrolysis with 0.1 M NaOH. Carboxyl groups were reduced by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide followed by reduction with sodium borohydride. De-O-acetylated or carboxyl-reduced polysaccharides had phagocytosis-inhibiting properties that did not differ appreciably from the untreated polysaccharide; thus, neither the O-acetyl nor the carboxyl groups were essential phagocytosis-inhibiting determinants. Antiserum specific for these noninhibitory groups was obtained by adsorption of cryptococcal antiserum with de-O-acetylated or carboxyl-reduced whole cells to produce antiserum enriched respectively with antibody specific for the O-acetyl and carboxyl groups. These adsorbed antisera showed opsonic activity for the untreated yeast that was similar to the unadsorbed antiserum when these antisera were compared at identical levels of precipitating antibody. We also examined the ability of Fab' fragments of opsonic IgG to reduce the amount of anticapsular IgG needed to opsonize the yeast. No synergy was noted between Fab' fragments and undigested IgG in opsonization. These results are consistent with a passive mechanism for inhibition of phagocytosis. The capsule does not directly modulate phagocytic function, but instead, presents a surface that is not recognized by the phagocyte. This absence of recognition is corrected by opsonizing antibody specific for any surface determinant on the capsule.

Animals↗
PubMed2006

The Cryptococcus neoformans catalase gene family and its role in antioxidant defense.

In the present study, we sought to elucidate the contribution of the Cryptococcus neoformans catalase gene family to antioxidant defense. We employed bioinformatics techniques to identify four members of the C. neoformans catalase gene family and created mutants lacking single or multiple catalase genes. Based on a phylogenetic analysis, CAT1 and CAT3 encode putative spore-specific catalases, CAT2 encodes a putative peroxisomal catalase, and CAT4 encodes a putative cytosolic catalase. Only Cat1 exhibited detectable biochemical activity in vitro, and Cat1 activity was constitutive in the yeast form of this organism. Although they were predicted to be important in spores, neither CAT1 nor CAT3 was essential for mating or spore viability. Consistent with previous studies of Saccharomyces cerevisiae, the single (cat1, cat2, cat3, and cat4) and quadruple (cat1 cat2 cat3 cat4) catalase mutant strains exhibited no oxidative-stress phenotypes under conditions in which either exogenous or endogenous levels of reactive oxygen species were elevated. In addition, there were no significant differences in the mean times to mortality between groups of mice infected with C. neoformans catalase mutant strains (the cat1 and cat1 cat2 cat3 cat4 mutants) and those infected with wild-type strain H99. We conclude from the results of this study that C. neoformans possesses a robust antioxidant system, composed of functionally overlapping and compensatory components that provide protection against endogenous and exogenous oxidative stresses.

Animals↗
PubMed2006

Cryptococcus neoformans capsular polysaccharide component galactoxylomannan induces apoptosis of human T-cells through activation of caspase-8.

The major virulence factor of Cryptococcus neoformans is its polysaccharide capsule composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM) and mannoproteins. A variety of immunomodulating activities have been described for GXM and mannoproteins but little is known about possible interactions of GalXM with the immune system. In the present article, we investigate the effect of purified soluble GalXM on human T lymphocytes. The results indicate that, GalXM (i) can affect selected immune responses; (ii) causes significant impairment of T cell proliferation and increases interferon-gamma and interleukin-10 production; and (iii) induces apoptosis of T lymphocytes through activation of caspase-8 that terminates with fragmentation of DNA. These results are the first to suggest a role for GalXM in C. neoformans virulence by demonstrating that it can target human T cells, and that it may impair the development of an effective specific T cell response.

Apoptosis↗
PubMed1993

The isolation of Cryptococcus neoformans from pigeon droppings and serotyping of naturally and clinically sourced isolates in China.

This is the first report on the isolation of Cryptococcus neoformans from pigeon droppings in China and their serotypes. C. neoformans colonies which produced brown colonies on caffeic acid-cornmeal agar were found in Twenty-five out of thirty-six samples of pigeon droppings. Fifty-one colonies randomly picked from the positive samples were identified as C. neoformans by a commercially available kit for carbon source assimilation test and Christensen's urea agar. Forty (78%) out of the 51 strains were serotyped as A and 11 (22%) as AD. At the same time, seventeen out of nineteen clinical isolates were serotyped as A and 2 as B. There are three findings in our results. One is that only C. neoformans var. neoformans strains could be isolated from pigeon droppings, although the variety gattii strains were found in the clinical isolates obtained in the same geographic site in China. The second is that serotype A strains were most frequently seen in natural and clinical materials in the southeast part of China, and serotype AD strains were isolated in pigeon droppings but not in clinical materials. The third is that the coexistence of serotype A and AD cells of C. neoformans strains in same samples of pigeon droppings were observed.

Animals↗
PubMed2004

Differential gene expression in auristatin PHE-treated Cryptococcus neoformans.

The antifungal pentapeptide auristatin PHE was recently shown to interfere with microtubule dynamics and nuclear and cellular division in the opportunistic pathogen Cryptococcus neoformans. To gain a broader understanding of the cellular response of C. neoformans to auristatin PHE, mRNA differential display (DD) and reverse transcriptase PCR (RT-PCR) were applied. Examination of approximately 60% of the cell transcriptome from cells treated with 1.5 times the MIC (7.89 micro M) of auristatin PHE for 90 min revealed 29 transcript expression differences between control and drug-treated populations. Differential expression of seven of the transcripts was confirmed by RT-PCR, as was drug-dependent modulation of an additional seven transcripts by RT-PCR only. Among genes found to be differentially expressed were those encoding proteins involved in transport, cell cycle regulation, signal transduction, cell stress, DNA repair, nucleotide metabolism, and capsule production. For example, RHO1 and an open reading frame (ORF) encoding a protein with 91% similarity to the Schizophyllum commune 14-3-3 protein, both involved in cell cycle regulation, were down-regulated, as was the gene encoding the multidrug efflux pump Afr1p. An ORF encoding a protein with 57% identity to the heat shock protein HSP104 in Pleurotus sajor-caju was up-regulated. Also, three transcripts of unknown function were responsive to auristatin PHE, which may eventually contribute to the elucidation of the function of their gene products. Further study of these differentially expressed genes and expression of their corresponding proteins are warranted to evaluate how they may be involved in the mechanism of action of auristatin PHE. This information may also contribute to an explanation of the selectivity of auristatin PHE for C. neoformans. This is the first report of drug action using DD in C. neoformans.

Antifungal Agents↗
PubMed1997

Mannoproteins of Cryptococcus neoformans induce proliferative response in human peripheral blood mononuclear cells (PBMC) and enhance HIV-1 replication.

To investigate the possible role of Cryptococcus neoformans var. neoformans in HIV disease progression, and to identify the responsible cryptococcal components, an in vitro cell culture model was set up to study the C. neoformans-induced enhancement of HIV replication in HIV-1-infected PBMC. Similar to whole C. neoformans, cell-wall membrane fraction and mannoproteins induced proliferation of PBMC and enhancement of lymphotropic HIV replication in HIV-infected PBMC, while galactoxylomannan did not. MoAbs capable of interfering with MHC class II-mediated antigen presentation prevented the induction of cell proliferation by whole C. neoformans or cryptococcal mannoproteins. MoAb binding to adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) also inhibited C. neoformans-induced cell proliferation. In addition, anti-MHC class II MoAb inhibited the enhancement of HIV replication by C. neoformans. The results suggest that: (i) C. neoformans may accelerate HIV disease progression by stimulation of HIV replication through MHC class II-mediated antigen presentation; and (ii) cryptococcal mannoprotein may be one of the responsible components. The ability to enhance HIV replication in PBMC in vitro is not unique for C. neoformans. However, this is the first report to study in detail a yeast-induced enhancement of HIV replication in PBMC.

Antibodies, Monoclonal↗
PubMed1989

Persistent Cryptococcus neoformans infection of the prostate after successful treatment of meningitis. California Collaborative Treatment Group.

STUDY OBJECTIVE: To assess the frequency of persistent Cryptococcus neoformans infection in patients with the acquired immunodeficiency syndrome (AIDS) after receiving apparently adequate treatment for meningitis. DESIGN: Blood, urine, and cerebrospinal fluid were cultured at the conclusion of primary therapy to assess the adequacy of treatment. SETTING: Outpatient clinics at three medical centers. PATIENTS: Patients had C. neoformans grown in culture from cerebrospinal fluid. Primary therapy consisted of either 2.0 g of amphotericin B alone; 6 weeks of combination therapy with flucytosine; or, if flucytosine was poorly tolerated, an adjusted minimum total amphotericin B dose. To meet criteria for adequate treatment of meningitis all patients had two sequential cerebrospinal fluid samples which were culture negative. MEASUREMENTS AND MAIN RESULTS: Nine of forty-one patients grew C. neoformans from urine after completion of primary treatment, but none had urinary symptoms. Fungi were visualized in expressed prostatic secretions in 4 of these patients. One patient refused further treatment and developed cryptococcemia within 5 weeks. Three patients received additional amphotericin B; all had persistent funguria without systemic relapse. Six patients received fluconazole; 4 became urine culture negative, and 2 had systemic relapse. CONCLUSION: The persistence of urinary C. neoformans after adequate therapy for meningitis suggests that the urinary tract (probably the prostate) is a sequestered reservoir of infection from which systemic relapse may occur.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Involvement of endogenously synthesized interleukin (IL)-18 in the protective effects of IL-12 against pulmonary infection with Cryptococcus neoformans in mice.

We previously demonstrated that interleukin (IL)-12 protected mice against fatal pulmonary infection with a highly virulent strain of Cryptococcus neoformans, which correlated well with the production of interferon (IFN)-gamma as well as IL-18 in the primary infected site. In the present study, we examined the role of endogenously synthesized IL-18 in IL-12-induced host resistance to this pathogen. There was little or no production of IFN-gamma and IL-18 both at mRNA and protein levels in lungs of mice infected with C. neoformans, while treatment with IL-12 induced a marked production of these cytokines. Caspase-1 mRNA was expressed in infected mice even without IL-12 treatment. Administration of neutralizing anti-IFN-gamma monoclonal antibody (mAb) clearly inhibited production of IFN-gamma and IL-18 induced by IL-12, while control IgG did not show such an effect. However, administration of IFN-gamma did not induce the production of both cytokines in infected mice, although tumor necrosis factor (TNF)-alpha and IFN-gamma-inducible protein (IP)-10 were synthesized by the same treatment. Finally, neutralizing anti-IL-18 antibody (Ab) significantly interfered with the production of IFN-gamma and elimination of the microorganism from the lung induced by IL-12 treatment. Furthermore, both IFN-gamma synthesis and host protection caused by IL-12 were profoundly diminished in IL-18 gene-disrupted mice. Considered collectively, our results indicated that host protection against C. neoformans induced by IL-12 involved endogenously synthesized IL-18 and that the production of IL-18 was mediated at least in part by endogenous IFN-gamma.

Animals↗
PubMed2001

Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts.

Actin cytoskeleton and microtubules were studied in a human fungal pathogen, the basidiomycetous yeast Cryptococcus neoformans (haploid phase of Filobasidiella neoformans), during its asexual reproduction by budding using fluorescence and electron microscopy. Staining with rhodamine-conjugated phalloidin revealed an F-actin cytoskeleton consisting of cortical patches, cables and cytokinetic ring. F-actin patches accumulated at the regions of cell wall growth, i. e. in sterigma, bud and septum. In mother cells evenly distributed F-actin patches were joined to F-actin cables, which were directed to the growing sterigma and bud. Some F-actin cables were associated with the cell nucleus. The F-actin cytokinetic ring was located in the bud neck, where the septum originated. Antitubulin TAT1 antibody revealed a microtubular cytoskeleton consisting of cytoplasmic and spindle microtubules. In interphase cells cytoplasmic microtubules pointed to the growing sterigma and bud. As the nucleus was translocated to the bud for mitosis, the cytoplasmic microtubules disassembled and were replaced by a short intranuclear spindle. Astral microtubules then emanated from the spindle poles. Elongation of the mitotic spindle from bud to mother cell preceded nuclear division, followed by cytokinesis (septum formation in the bud neck). Electron microscopy of ultrathin sections of chemically fixed and freeze-substituted cells revealed filamentous bundles directed to the cell cortex. The bundles corresponded in width to the actin microfilament cables. At the bud neck numerous ribosomes accumulated before septum synthesis. We conclude: (i) the topology of F-actin patches, cables and rings in C. neoformans resembles ascomycetous budding yeast Saccharomyces, while the arrangement of interphase and mitotic microtubules resembles ascomycetous fission yeast Schizosaccharomyces. The organization of the cytoskeleton of the mitotic nucleus, however, is characteristic of basidiomycetous yeasts. (ii) A specific feature of C. neoformans was the formation of a cylindrical sterigma, characterized by invasion of F-actin cables and microtubules, followed by accumulation of F-actin patches around its terminal region resulting in development of an isodiametrical bud.

Actin Cytoskeleton↗