Interferon gamma increases survival in murine experimental cryptococcosis

Se evaluo la efectividad del interferon-µ (IFN-µ) recombinante de rata en un modelo experimental de criptococosis desarollado en ratones Balb/C inoculados por via intraperitoneal con la cepa Rivas de Cryptococcus neoformas (C. neoformans). Se tuvieron en cuenta el tiempo de sobrevida de los animales, el aspecto macroscopico de los organos en la autopsia, la presencia de levaduras capsuladas en los tejidos y la siembra masiva de un homogenato de cerebro. El tratamiento con IFN-µ, en dosis diarias de 10.000 UI, no modifico estos parametros cuando la dosis infectante fue de 10 indice 7 levaduras y el tratamiento se retardo 5 dias post-infeccion (media de sobrevida de 21 vs. 23 dias en los grupos de control y tratados con IFN-µ, respectivamente)...

Facilitated isolation, purification, and analysis of glucuronoxylomannan of Cryptococcus neoformans.

Cryptococcus neoformans was cultured in a chemically defined medium. The culture was adjusted to 0.25% formaldehyde or autoclaved after 5 days of growth at 35 degrees C, and a cell-free supernatant was obtained by centrifugation. Solid calcium acetate was added to the supernatant to give a 5% solution, and the pH was adjusted to approximately 5 with glacial acetic acid. The polysaccharide (PS) was precipitated by the addition of 3 volumes of 95% ethanol. The PS was dissolved in 0.2 M NaCl, and insoluble calcium salts were solubilized by the addition of several drops of glacial acetic acid. The PS solution was treated by ultrasonic irradiation for 15 min. This concurrently decreased the molecular weight of the PS and reduced the viscosity of the solution. The ultrasonically irradiated PS was precipitated by differential complexation with hexadecyltrimethylammonium bromide at 23 degrees C, the complex was dissolved in 1 M NaCl, and the glucuronoxylomannan was precipitated by adding 3 volumes of ethanol. The glucuronoxylomannan was dissolved in 1 M NaCl and then ultrasonically irradiated for 2 h to reduce the molecular mass to a limiting value of approximately 100 kDa (GXMS). The purified GXMS was centrifuged, dialyzed, and finally recovered by lyophilization. GXMS was chromatographed on DEAE-cellulose at reasonable concentrations without the complication of high solution viscosity. The sugar composition and structure of GXMS were determined by gas-liquid chromatography, permethylation gas-liquid chromatography-mass spectrometry, and 13C nuclear magnetic resonance spectroscopy. The improved solution characteristics of GXMS were ideal for the determination of its chemical and serological properties.

Discriminant analysis of cellular fatty acids of Candida species, Torulopsis glabrata, and Cryptococcus neoformans determined by gas-liquid chromatography.

We used discriminant analysis of cellular fatty acid compositions determined by gas-liquid chromatography to differentiate yeastlike fungi (a total of 190 strains; including 37 Candida albicans strains, 21 Candida krusei strains, 13 Candida guilliermondii strains, 37 Candida tropicalis strains, 10 Candida pseudotropicalis strains, 24 Candida parapsilosis strains, 32 Torulopsis glabrata strains, and 16 Cryptococcus neoformans strains). Previous results with a standard strain of C. albicans indicated that reproducible fatty acid chromatograms can be obtained with cells grown in a medium of 2% Sabouraud glucose agar at 35 degrees C for between 48 and 72 h. These conditions were also maintained in cultures of the other organisms that we studied. The cellular fatty acid compositions of the organisms were determined quantitatively by gas-liquid chromatography and analyzed by discriminant analysis. The total correct identification expressed as relative peak percent was 95.8% (89.2% for C. albicans to 100% for C. krusei, C. guilliermondii, C. pseudotropicalis, T. glabrata, and C. neoformans). The total correct identification expressed as the common peak (palmitic acid) ratio was 94.7% (87.5% for C. parapsilosis to 100% for C. pseudotropicalis, T. glabrata, and C. neoformans). Both results suggest that cellular fatty acid compositions can be differentiated by this method.

Cell-wall glucans of Cryptococcus neoformans Cap 67.

Purified cell walls derived from Cryptococcus neofromans Cap 67, an acapsular mutant, consisted of 86% Glc and 7.3% GlcNAc. The integrity of the cell walls was disrupted in three successive extractions with 60% 4-methylmorpholine N-oxide (4-MMNO) at 120 degrees. Four 4-MMNO-soluble D-glucopyranans were isolated. Released within 0.5 h was water-insoluble Gi-1, followed by two water-soluble Gs fractions and water-insoluble Gi-2 over 17.5 h. A 4-MMNO-insoluble residue, containing 27% of GlcNAc, was also isolated. Gi-1 and Gi-2 were isolated as precipitates during dialysis of 4-MMNO extracts and were each reduced with NaBH4 to permit their investigation in alkaline solution. Gs-1 and Gs-2 were separated by ion-exchange chromatography of the water-soluble fractions. The structures of the D-glucopyranans were determined by 13C-n.m.r. spectroscopy and by g.l.c.-mass spectrometry of their per-O-methylated derivatives. Gi-1 was a (1----3)-alpha-D-glucopyranan (97%) with some (1----4)-D-glucosidic linkages (3%) and no chain-branching. Gs-1 and Gs-2 were (1----6)-beta-D-glucopyranans branched at O-3 (10-12%) with beta-D-Glcp-(1----3)-beta-D-Glcp side chains. Gs-2 may have approximately 2% more chain branching than Gs-1. Gi-2 was a D-glucopyranan with 80% of its structure like that of Gi-1, and 20% like that of Gs-1 and -2; the water-insolubility of Gi-2 suggests that these structures were covalently linked. Almost identical D-glucopyranans were obtained from aged cultures that had thickened walls (as observed by electron microscopy).

Antifungal activity in human cerebrospinal fluid and plasma after intravenous administration of Allium sativum.

Commercial Allium sativum (garlic) extract was given intravenously to two patients with cryptococcal meningitis and three patients with other types of meningitis. Plasma titers of anti-Cryptococcus neoformans activity rose twofold over preinfusion titers. Anti-C. neoformans activity was detected in four of five cerebrospinal fluid samples but not in pooled normal cerebrospinal fluid.

Reassessment of antigenic determinant of Saccharomyces cerevisiae serotype Ia.

We examined the immunochemical structure of the antigenic determinant of S. cerevisiae serotype Ia. The specific factor serum for S. cerevisiae serotype Ia was obtained either from factor 18 serum by adsorption with heat-killed cells of Candida glabrata, or from anti-S. cerevisiae Ia (M 6001) serum by adsorption with heat-killed cells of S. cerevisiae Ib (IFO 0751). We designated this adsorbed serum as factor 18a. Acetolysis of S. cerevisiae cell wall D-mannan gave five oligosaccharides. Signals of 1H-nuclear magnetic resonance spectra of mannooligosaccharides derived from S. cerevisiae mannan were assigned for their linkages by the aid of those of alpha-1,3'-linked mannooligosaccharides derived from glucuronoxylomannan of capsule of Cryptococcus neoformans serotype A-D. Agglutination-inhibition experiments revealed that the mannopentaose from S. cerevisiae mannan was the most effective inhibitor. Moreover, inhibitory activities of alpha-1,3'-linked mannotriose, mannotetraose, and mannopentaose which were derived from glucuronoxylomannan of C. neoformans were shown to be higher than those of mannotetraose with one terminal alpha-(1-3) linkage from homologous S. cerevisiae mannan. These results indicate that mannopentaose with terminal two alpha-(1-3) linkages is responsible for the specificity of S. cerevisiae Ia.

Type-specific polysaccharides of Cryptococcus neoformans. n.m.r.-spectral study of a glucuronomannan chemically derived from a Tremella mesenterica exopolysaccharide.

A glucuronomannan (GM) was derived by removal, through Smith degradation, of xylose from the native (3-O-acetylglucurono)xylomannan exopolysaccharide isolated from Tremella mesenterica. 13C-N.m.r. chemical shifts measured at various pD values were compared for p-nitrophenyl beta-D-glucopyranosiduronic acid (1) and two GMs (2 and 3) differing in GlcA content (Man:GlcA; 2, 10:1; and 3, 5:1). Also measured and compared were pKa values for 1 and 2. One-dimensional and two-dimensional (COSY and HETCOR) n.m.r. data allowed unambiguous assignments of pD-sensitive chemical shifts due to 2-O-beta-D-GlcpA substituents attached to a (1----3)-linked alpha-D-Manp backbone. The pKa and n.m.r. data indicated that the CO2H groups in either GM are independent of each other, and are similar in behavior to those of p-nitrophenyl beta-D-glucopyranosiduronic acid molecules. The n.m.r. data confirmed the previous, chemically deduced, structural role of GlcpA in the native polysaccharide from T. mesenterica, and indicated that significant pD-induced changes occur in the stabilities of the glycosidic orientations in the GM. Previous 13C-n.m.r. assignments for 2-O-beta-D-GlcpA in polysaccharides derived from Cryptococcus neoformans serotype A-variant were confirmed, except for the signal due to the anomeric carbon atom. This signal is now known to be pD-sensitive. In acidic solutions, it is coincident with the signal (104.5 p.p.m.) due to the anomeric carbon atoms of the unsubstituted alpha-D-Manp backbone residues. In basic solutions, the 2-O-beta-D-GlcpA anomeric carbon resonance is shifted upfield by approximately 0.2 p.p.m., and is observed as a separate signal.

Structure determination of Cryptococcus neoformans serotype A-variant glucuronoxylomannan by 13C-n.m.r. spectroscopy.

A series of polysaccharides was derived by physical and chemical methods from an antigenic, O-acetyl-containing, glucuronoxylomannan (GXM), isolated from the growth medium of Cryptococcus neoformans (CDC B2550) serotype A-variant having composition ratios of Man:Xyl:GlcA:OAc = 10:4:3:6. 13C-N.m.r. spectra of derivatives provided new structural evidence for GXM. Treatment of GXM with Li in ethylenediamine gave a xylomannan (XM, with Man:Xyl = 5:2). Smith degradation of XM gave a mannan (M). Ultrasonic treatment of GXM gave GXM-sonicated (GXMS). Treatment of GXM with 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide.HCl and then with NaBH4 gave reduced GXMS (RGXMS), or with aq. trifluoroacetic acid gave partially acid-hydrolyzed GXMS. Periodate oxidation of GXM and NaBH4 reduction of the product gave a polyalcohol-mannan (PM). Treatment of GXMS, RGXMS, and PM with NH4OH at pH 11 gave the respective O-deacetylated analogs. Comparison among the 13C-n.m.r. spectra of GXM, the various derivatives, and reference monosaccharides allowed the following conclusions: M is (1----3)-alpha-D-mannopyranan; XM consists of the M backbone with 91% of the Xyl on nonadjacent Man residues as 2-O-beta-D-Xylp substituents and with 9% as 4-O-D-Xylp substituents on other Man residues. GXM consists of the XM structure, but with non-D-xylosylated Man residues substituted with 2-O-beta-D-GlcpA substituents and with 6-O-acetyl groups distributed approximately equally on Man residues that have other substituents and those that have none. The molecular mechanics program MM2 was used to estimate the relative energies of anomeric orientations of the typical glycosidic linkage in M. The results suggest that 6'-OH----O-2 H-bonding is significant in the minimal-energy orientation of M, with phi = -36 degrees and psi = 51 degrees, and that two other glycosidic orientations may be important in the 2-O- or 6-O-substituted derivatives of M.

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.

Ultrastructure of acapsular mutant Cryptococcus neoformans cap 67 and monosaccharide composition of cell extracts.

Acapsular mutant Cryptococcus neoformans cap 67 was grown in Pine's citrate broth medium for 3 days and the cells then transferred to a nitrogen-free medium for 6 days. The cells were subjected to a four stage extraction with buffered Triton-X100, cold dilute alkaline borohydride, hot dilute acetic acid, and a second alkaline extraction. Galactoxylomannan antigens were recovered from the culture supernates of both 3 day-old and 9 day-old yeast cells. The alkaline extracts contained water-soluble galactoxylomannan and a water-insoluble glucan. Dilute acid treatment released a minor amount of carbohydrate from the cells. The second alkaline extraction yielded increased amounts of glucan and galactoxylomannan from the 9 day-old cells. Soluble non-dialyzable cell extracts were antigenically identical in immunodiffusion with the culture supernate antigens. After the extraction sequence, all of the galactose, xylose, and mannose were removed from the cells. The walls retained their shape after extraction but their layers were loosened. Cells resuspended in nitrogen-free medium for six days developed thickened walls with alternating electron-dense and electron-lucent layers. The major constituent of the thickened 9 day-old cell walls was glucose, only 5% glucosamine was detected.

Lipid composition of Cryptococcus neoformans.

The lipid composition of Cryptococcus neoformans grown in Sabouraud's dextrose broth (shake culture) was analysed. The organism contained extremely low amounts of lipid (0.96% dry weight basis) of which 86.1% were nonpolaris lipids, 3.4% phospholipids and the rest were glycolipids and pigments. Alkoxylipids (41%), tryglicerides (18%), diglycerides (7.4%), free fatty acids (5.4%), sterols (4.7%), sterol ester (3.9%) and monoglycerides (2.2%) were found in the nonpolar lipid fraction of C. neoformans. The phospholipid composition (expressed as relative abundance) was: phosphatidylinositol (11.5%), lysophosphatidyl ethanolamine (10.9%), cardiolipin (10.1%), a glycophospholipid (9.5%), lysophosphatidyl choline (4.7%), phosphatidic acid (4.1%), phosphatidyl choline (28.1%), phosphatidyl ethanolamine (14.5%) and an unidentified lipid (6.5%). Phosphatidyl serine, sphingolipids and cerebrosides, generally found in yeast-like fungi, were absent. Probable reasons for the abnormally low lipid content are discussed.

Virulence, capsule size and lipid composition interrelation of Cryptococcus neoformans.

Virulence and lipid composition were studied in three isolates of Cryptococcus neoformans. Virulence was evaluated by injecting mice intraperitoneally with 10(7) cells and recording organ involvement and spontaneous death over a 25 day period. Though the least virulent strain contained the least amount of total lipid and phospholipids, none of the lipids showed any quantitative relation to virulence. There was no major difference in the phospholipid composition among the three strains. Fungal cells with bigger capsules had a lower lipid content. The role of lipid in the defence mechanism of pathogenic fungi during the host invasion process is discussed.

Capsular polysaccharides of Cryptococcus neoformans.

Polysaccharides of Cryptococcus neoformans are considered to have a role in the virulence of this encapsulated fungus. The structure has been determined for the most abundant polysaccharide, a glucuronoxylomannan of varying xylose and ester content. The structural complexity of the capsular material increases from serotype D to A to B to C, but even for the simplest capsule (type D), the immunodeterminants seem to occur only on the side chains. The interactions of some of these groups with antibodies is discussed.

Genetic and phenotypic characterization of capsule mutants of Cryptococcus neoformans.

Stable mutants with reduced capacity to produce capsules were isolated from suspensions of Cryptococcus neoformans after treatment of the wild type with a mutagen. The mutants could be assigned one of two phenotypes, hypocapsular or acapsular. Hypocapsular mutants were immunochemically and physicochemically indistinguishable from the wild type, whereas acapsular mutants lacked a major capsular antigen and a negatively charged exterior. In genetic analysis, the mutant trait segregated as a Mendelian gene (1:1) when random basidiospores from an outcross were studied, and analysis of products of single meiotic events from outcrossed mutants was likewise consistent with meiotic segregation. Two-factor crosses yielded the expected four classes of progeny, with recombinants equal to parentals. We concluded that chromosomal genes are responsible for synthesis of the cryptococcal capsule and that random basidiospore analysis represents a useful technique for genetic analysis in this species.

Cryptococcal capsular polysaccharide clearance in nonimmune mice.

Clearance of cryptococcal polysaccharide (CP) from tissues and body fluids of nonimmune mice was studied. Mice were injected intravenously only with one mg of purified CP, and serum, urine and tissues were obtained from each animal at various intervals for a period of 84 days. Tissue extracts, serum and urine were tested for CP content by enzyme-linked immunosorbent assay (ELISA) and latex agglutination. High concentrations of CP were detected by both assays one-half hour after injection in blood (serum), liver, spleen, kidney and lung (extracts). The duration of ELISA detectable CP was longest (70 days) in liver and spleen and shortest (14 days) in lung extract. By 14 days after injection, concentration of CP in the blood fell below that found in the liver and spleen. CP remained detectable (titers 32-64) after all other extracts became negative. These results indicate that CP is stored in tissues (binding mechanism and site unknown), and that the liver and spleen possess greater storage capacity than other tissues. Antibody (IgM) to CP appeared in low titer on the 14th day and thereafter.

A microsomal fraction of Cryptococcus neoformans induces lymphocyte blastogenesis in infected guinea pigs.

Differential centrifugation of a homogenate from a mechanically disrupted, acapsular isolate of Cryptococcus neoformans resulted in a 105,000 x g supernatant (105 K) and a microsomal fraction (MS), both of which were capable of eliciting specific delayed cutaneous hypersensitivity and in vitro blastogenesis in infected guinea pigs. Polyacrylamide gel electrophoresis revealed two major proteins in the MS and seven proteins in the 105 K fractions. Electron microscopy of the MS showed both membranes and ribosomes. In vitro lymphocyte blastogenesis elicited by 1 to 10 micrograms/ml of antigens was maximal after 4 days of incubation; the reacting populations were peripheral blood leukocytes (PBL) and peritoneal exudate cells (PEC). Spleen cells of infected animals were unresponsive to in vitro antigenic stimulation. A simplified schedule of priming animals was infection with a single dose of virulent cryptococci. Under these conditions 3 of 6 animals' PBL responded with stimulation ratios of 6.55, 21.1, 35.42 to the MS and 1.41, 9.33, 17.39 to the 105 K antigens at 1 microgram/ml. Four of six animals' PEC response were positive with stimulation ratios of 2.62, 2.72, 4.02 and 7.20 towards MS, and 2.62, 5.13, 5.71, 10.01 to the 105 K antigens at 1 microgram/ml. When small capsule and large capsule isolates were used for infection, the small capsule form was not isolated from the brain, in contrast to its isolation from 2 of 3 animals receiving large capsule forms. Two of three animals in each group responded with blastogenic indices more vigorous in the PBL, and the most potent antigen was MS. There was no obvious difference in lymphocyte reactivity between the two groups.

A DL-DOPA drop test for the identification of Cryptococcus neoformans.

A simple melanin assay using DL.DOPA as the substrate was developed to aid in the identification of Cryptococcus neoformans. The DL-DOPA drop test was simple and efficient. The best results (100% of the C. neoformans isolates were positive) occurred when C. neoformans was grown for two days at room temperature on Sabouraud agar modified. One to three loopfuls of yeast cells were then transferred to a starvation medium for 18-24 hours. Two of three drops of 0.3% DL-DOPA solution was applied to the transferred yeast cells. Only C. neoformans produced a brown or blackgrey pigment within 24 hrs, with 85% of the isolates becoming brown or black-grey within thirty minutes.

New, special stain for histopathological diagnosis of cryptococcosis.

The Masson-Fontana silver stain for melanin was employed for the differentiation of pathogenic fungal species in human or mouse tissues. The fungi studied were Candida albicans, Candida tropicalis, Candida glabrata (Torulopsis glabrata), Cryptococcus neoformans, Cryptococcus bacillisporus, Coccidioides immitis, Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Rhizopus rhizopodiformis, and Aspergillus fumigatus. The tissue sections stained with Masson-Fontana silver stain showed a dark brown to black color in the wall of cryptococci, whereas the walls of remaining fungal species were hyaline, except for those of S. schenckii. The yeastlike cells of S. schenckii showed faint brown pigment on the wall. Cultures of these fungi showed staining characteristics identical to those of the in vivo results. Cultures of four nonpathogenic Cryptococcus species, Cryptococcus uniguttulatus, Cryptococcus laurentii, Cryptococcus terreus, and Cryptococcus luteolus, were also tested for staining by the Masson-Fontana procedure. Of these, only C. laurentii stained positively, and the pigment on the cell wall was as intense as that of the cells of C. neoformans. These results indicate that the Masson-Fontana silver stain can be used as a specific stain in the histological diagnosis of cryptococcosis.