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[Pulmonary pseudotumor caused by Cryptococcus neoformans].

Since 1923, eight cases of cryptococcosis have been described in Norway, all with meningeal affection. A distinct, solitary infiltration in the upper lobe of the right lung was discovered in a 68 year-old woman. The lobe was extirpated. Microbiological and histological investigations showed infection with Cryptococcus neoformans. No antimycotic treatment was given. Four months later the patient developed osteomyelitis of the skull due to cryptococcosis, and was treated successfully with amphotericin B and flucytosin. Further investigation of the patient revealed a defect in the T-lymphocyte immune system, but it is uncertain whether this was of any significance for the development of the cryptococcus infection.

Aged

[CSF examination in cryptococcus meningitis].

CSF studies of 14 cases of cryptococcus meningitis revealed: 1. Direct discerning of yeast cells in the blood cell counting chamber, by Indian ink stain, and by cytological examination based on Sayk's technic, all were highly positive in repeated examinations. 2. Morphology of cryptococcus and inflammatory cellular reactions in CSF were investigated, and were quite characteristic. 3. Suppression and destruction of yeast cells were attainable only when doses of amphotericin B were sufficient.

Adult

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

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

Animals

Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neoformans.

Murine natural killer (NK) cell-mediated inhibition of growth of a yeast-like target cell, Cryptococcus neoformans, was completely abrogated by blocking the effector cell secretory process with monensin. Therefore, further studies were performed to determine the ability of various cytoplasmic fractions of NK cells to mediate inhibition of cryptococcal growth. Percoll-fractionated homogenates of rat LGL tumor cells demonstrated that the granule-containing fractions plus three additional sets of less dense cytoplasmic fractions displayed anti-cryptococcal activity; whereas only the cytoplasmic granule-containing fractions had cytotoxic activity against YAC-1 tumor cell and sheep erythrocyte targets. Maximal cryptococcal growth inhibition induced by LGL granules occurred after a 1 h incubation, required the presence of Ca2+ (1.0 mM) or Mg2+ (0.5 mM or 5.0 mM), and was completely abrogated in the presence of rabbit anti-LGL granule IgG. Cytolysin, the granule component which mediates tumor cell and sheep erythrocyte lysis, effectively limited the growth of cryptococci. Since Percoll gradient fractionation of the LGL homogenates demonstrated three separate peaks of anti-cryptococcal activity other than the granule peak, it is possible that the cytolysin-containing granules are not the only subcellular component of NK cells playing a role in inhibition of C. neoformans growth.

Animals

Evaluation of a fluorescent method (fluorescein diacetate and ethidium bromide solution) in the study of the viability Cryptococcus neoformans strains.

The effectiveness of the fluorescent viability test (fluorescein diacetate-FDA and ethidium bromide-EB-solution) compared to the plaque counting test (Miles & Misra M & M) was performed on 10 samples of Cryptococcus neoformans cultivated in Sabouraud dextrose agar at 25 degrees C. The optimum incubation period of 50 minutes was determined. Growth curves of the fungal strains studied based on the mean cell number were drawn for both the FDA & EB and M & M methods. The statistical evaluation (Student's T test) of the average sum of the viable cell counts showed that the FDA-EB method is more sensitive than the M & M test for the studied species. The growth curves of the samples usually followed a homogeneous pattern comparable to other non-dimorphic fungi.

Animals

Clinical and environmental isolates of Cryptococcus neoformans in Bangkok (Thailand).

Cryptococcus neoformans was isolated from 13 patients (7 females and 6 males) suffering from systemic cryptococcosis. Eight patients were suffering from central nervous system cryptococcosis and 5 were suffering from disseminated cryptococcosis. Systemic lupus erythematosus was found to be the common underlying disease in 3 patients. The results of treatment depended on the underlying diseases (7 improved, 6 died). Also, 13 isolates of C. neoformans were obtained from feces of 30 pet birds. All 26 isolates of C. neoformans were cultured in glycine cycloheximide medium and were found to be of serotypes A and D.

Adolescent

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.

Antigens, Fungal

Ultrastructure of Cryptococcus neoformans in the cerebrospinal fluid of a patient with cryptococcal meningitis.

The ultrastructure of Cryptococcus neoformans in the cerebrospinal fluid, which was obtained from a patient suffering from systemic lupus erythematosus and cryptococcal meningitis, before treatment and on the 10th and 20th day after the start of treatment was studied. Numerous cryptococci were detected in their cerebrospinal fluid before treatment. However, in most of them their biological activity was low and their organelles were not clear. A few yeasts preserved well their organelles. Such cells showed a tendency to develop vesicular membrane structures (lomasomes) touching the internal part of the cell wall. In the cerebrospinal fluid on the 20th day all of the yeasts were dead, even though numerous yeasts were observed by an India-ink method. At this time no colonies were recovered from the fluid.

Adult

Persistence of infection in mice inoculated intranasally with Cryptococcus neoformans.

Cryptococcus neoformans was instilled intranasally into mice which were periodically sacrificed to determine the course of infection. Cryptococci persisted within the nasal passages throughout the 90 day study. Extranasal dissemination began 14-28 days after instillation and was still demonstrable 90 days post-exposure. Ten percent mortality was observed in mice receiving 10(6) cryptococci, while no mortality was observed in mice exposed to 10(3) or 10(4) cryptococci. Our research suggests that nasal colonization with C. neoformans can precede pulmonary and systemic cryptococcosis by weeks or months.

Animals

Cryptococcus neoformans: a central nervous system isolate from an AIDS patient that is rhinotropic in a normal mouse model.

A strain of Cryptococcus neoformans that was isolated from the cerebrospinal fluid of a human diagnosed as having acquired immunodeficiency syndrome (AIDS), and that produced cutaneous lesions in experimentally infected, normal mice is described. Although no unusual cutaneous manifestations were noted in the patient's records, this isolate of C. neoformans proved to be dermotropic when injected intravenously into CD-1 mice. The LD50 at 28 days post infection ranged from 3.6-7.5 X 10(5) cells per mouse, and in vitro growth rate studies demonstrated that this isolate grew well at 35 degrees C and at 37 degrees C, but did not grow at 40 degrees C and higher. This isolate was rhinotropic producing large granulomatous lesions in the nasal tissues. Other cutaneous tissues affected were the periocular tissues, ears, feet and tail, although the granulomas were nodular in structure and less necrotic than the nasal lesions. The brain, lungs, liver, kidneys and spleen also were culture positive for C. neoformans. Histopathologically, each affected tissue examined had large densities of yeast cells and a chronic, granulomatous host response. Animals surviving the infection appeared to develop a commensal-type relationship with the infective yeast. This is the first report of an isolate of C. neoformans from an AIDS patient that has caused cutaneous manifestations in an animal model. The model described in this report may be useful for elucidating pathogenic mechanisms of cryptococcosis, particularly cutaneous manifestations of the disease.

Acquired Immunodeficiency Syndrome

Virulence and antifungal susceptibility of environmental and clinical isolates of Cryptococcus neoformans from Puerto Rico.

Studies on the distribution, epidemiology and pathogenesis of Cryptococcus neoformans on the island of Puerto Rico are few. We have studied mouse virulence and in vitro antifungal susceptibility of 133 isolates of C. neoformans: 121 environmental and 12 clinical (9 from AIDS patients), that were isolated in Puerto Rico. In experimental CD-1 mice infected intravenously, the mean lethal dose 50% values (28 days) were greater than 5.2 x 10(6) and 1.1 x 10(5) cells/mouse for environmental and clinical isolates, respectively. Using an agar dilution assay, the minimum inhibitory concentrations of amphotericin B, ketoconazole and 5-fluorocytosine were comparable for environmental and clinical isolates in both yeast nitrogen dextrose base agar and Kimming's agar. These data suggest a difference in lethality for mice, but no difference in antifungal susceptibility of environmental and clinical isolates of C. neoformans obtained in Puerto Rico.

Animals

Isolation of saprophytic Cryptococcus neoformans from Puerto Rico: distribution and variety.

Until the present decade, no studies had been conducted in Puerto Rico on the saprophytic distribution and variety of Cryptococcus neoformans. Samples (522) of pigeon droppings from 14 western towns were tested for the presence of C. neoformans. The yeast was recovered from 24.7% (129 isolates) of the samples, representing 10 of the 14 towns studied. All environmental isolates were identified as C. neoformans var. neoformans using canavanine-glycine-bromthymol blue (CGB) agar. The yeast was isolated from 79.4% of the samples in one town, Isabela. The average number of yeast cells isolated from sites within this municipality was 5.1 x 10(5) per gram of pigeon droppings. This was 2.6 times the average number of yeast cells of C. neoformans isolated from sites in other towns. In addition, the yeast was isolated from four patients with the acquired immune deficiency syndrome (AIDS), each of whom died of cryptococcal meningitis. Each of these poorly encapsulated isolates was identified as C. neoformans var. neoformans using CGB agar. The results of this investigation demonstrate that C. neoformans var. neoformans is prevalent in Puerto Rico.

Acquired Immunodeficiency Syndrome

Effect of temperature on growth and macromolecular biosynthesis in Cryptococcus species.

Cryptococcus neoformans, a pathogenic yeast, grows at temperatures between 25 and 37 degrees C. However, the closely related non-pathogen C. albidus exhibits restricted growth at temperatures above ambient with little or no growth at 37 degrees C. The inhibition of growth of the non-pathogen, as measured by turbidity, cell number, and per cent budding, is reversible after 48 hr at the non-permissive temperature (37 degrees C). Growth cessation at 37 degrees C is accompanied by a corresponding decrease in DNA synthesis, which is not observed in C. neoformans. RNA and protein synthesis in C. albidus and C. neoformans are only slightly affected at the elevated temperature. Degradation by nucleases does not seem to account for the differences found in this cumulative DNA synthesis in C. albidus at 25 and 37 degrees C. These facts suggest that C. albidus may possess a thermo-sensitive defect in the machinery responsible for the initiation of DNA replication.

Cryptococcus

Factors affecting experimental infection with Cryptococcus neoformans in mice with special reference to an endotoxic substance of C. neoformans.

A close correlation was observed between body weight and length of the survival time of mice inoculated intravenously (i.v.) with Cryptococcus neoformans (p less than 0.001). An endotoxic substance of C. neoformans (Cr-ET) increased the susceptibility of mice to i.v. infection of C. neoformans only when more than 50 mug of Cr-ET was injected i.v. 24 hours before infection. Intraperitoneal (i.p.) administration of dimethyl sulfoxide which is found to enhance bacterial infection did not enhance death rate of mice infected i.p. with C. neoformans.

Animals

Immunization of mice with an avirulent pseudohyphal form of Cryptococcus neoformans.

Mice were immunized with a viable, avirulent strain of Cryptococcus neoformans. Lymphocyte blastogenic assays showed a 10-fold increase in reactivity of sensitized spleen cells, and histopathologic examination revealed marked splenic hyperplasia. Thirty-two days after intravenous inoculation with a virulent strain of C. neoformans, none of the control animals survived whereas 60 percent of the immunized mice were alive with no clinical evidence of disease. This animal model shows that protective immunity can be established, and once developed, provide a better model for the study of important aspects of immunity in fungal disease.

Animals

Cytochemical and biochemical identification of lysosomes in Cryptococcus neoformans.

Normaski optics, fluorescence and electron microscopy were employed to demonstrate the occurrence of lysosomes in capsulated, enztmatically decapsulated, and dewalled cells of a human isolate of Cryptococcus neoformans. Fluorescent studies, using acridine orange as a lysosomal indicator, revealed the presence of variously sized, spherical, reddish-orange fluorescing bodies. Electron microscopy studies demonstrated the presence of acid phosphatase (AP), a lysosome marker enzyme, in single-membrane bound organelles. Lysosomes were removed from dewalled cells and separated by differential centrifugation on ficoll gradients. That fraction indicating the highest assay for AP was centrifuged at high speed, and the resulting pellet was fixed for electron microscopy and stained by the Gomori procedure for AP. Sections of the pellets revealed AP stained vesicles of the same size range as those within intact cells.

Cryptococcus

A rapid pigmentation test for identification of Cryptococcus neoformans.

A rapid pigmentation test for identification of Cryptococcus neoformans is described. The method is based on the formation of a characteristic, mouse-grey to violaceous-black pigment when shake cultures of C. neoformans in a phosphate-buffered, l-DOPA - ferric citrate medium are incubated at 37 C for one hour.

Catechol Oxidase

The in vivo incorporation of [32P]-labeled orthophosphate into pyrophosphatidic acid and other phospholipids of Cryptococcus neoformans through cell growth.

Cryptococcus neoformans was cultured in a liquid medium containing [32P]-orthophosphate and harvested at various stages of cell growth. An aliquot of the [32P]-labeled cells was transferred to a nonradioactive medium, and the culture was continued again for some hours. The [32P]-radioactivity composition and the phosphorus composition of individual phospholipids relative to the total phospholipid through the incubation periods were estimated. Although levels of major phospholipids remained constant throughout the cell growth, the distribution pattern of the [32P]-radioactivity of individual phospholipids changed remarkably along with the progress of cell growth. The changing patterns of the specific radioactivities of individual phospholipids through the growth phase demonstrated that phosphatidic acid was one of the most active metabolites in phospholipids and that pyrophosphatidic acid was also metabolically active.

Cryptococcus