Genetic relatedness and diversity of Cryptococcus neoformans strains in the Maltese Islands

PURPOSE: Pilocarpine, a salivary stimulant, has been shown to protect salivary glands from gamma-radiation-induced damage during the radiotherapy of head and neck tumors. This study was performed to determine whether pilocarpine affects the survival of squamous carcinoma cells, line SCC-25, following gamma-radiation treatment. METHODS AND MATERIALS: The survival of squamous carcinoma tumor cells, line SCC-25, following the exposure of cells to pilocarpine at concentration of 0-100 ng/ml given for 0-1 h prior to radiation at dose of 0-20 Gy was determined by an in vitro colony-formation assay. RESULTS: The survival fractions of SCC-25 cells were identical for the control and pilocarpine-treated samples at all tested conditions. Calculated Do and Dq values did not depend on the presence of pilocarpine and were not affected by the time of incubation prior to irradiation. CONCLUSION: Pilocarpine, at clinically relevant concentrations, given to the SCC-25 cells 1 h prior to or at the time of irradiation did not affect survival of SCC-25 cells in vitro. Pilocarpine does not sensitize or protect these tumor cells from the effects of y-radiation, suggesting that this agent should not compromise the tumoricidal effects of radiotherapy.     

Virulence factors of Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast which causes cryptococcosis, a disease typified by an initial pulmonary infection which can disseminate to cause a life threatening meningoencephalitis. Although the disease may occur in individuals who show no evidence of immunosuppression it has had it most significant impact as an infection in patients with AIDS. Research into the potential virulence factors of this yeast has recently attracted particular attention. Capsule synthesis has been the focus of most interest and it is now established as a major virulence determinant. The mechanisms by which the capsule and capsular material effect the immune response have now largely been elucidated, and the genes underlying capsular synthesis are now under investigation. The isolation of mutants incapable of melanogenesis have implicated this process in the pathogenesis of C. neoformans infections, and evidence suggests that the production of melanin protects the yeast against oxidant induced damage. There is also some genetic evidence for the potential involvement of temperature tolerance and mating types in the virulence of this encapsulated yeast. The roles of other potential C. neoformans virulence determinants are more speculative; these include proteinase production, release of polyol metabolites, interaction with hormones, adherence and production of mannoproteins. The involvement of housekeeping enzyme systems in the maintenance of infection by C. neoformans is now also under active investigation.     

Molecular typing of Cryptococcus neoformans: taxonomic and epidemiological aspects

Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, serotype, and killer toxin sensitivity patterns of a wide range of saprobic, clinical, and veterinary isolates of both varieties of Cryptococcus neoformans were examined. C. neoformans var. neoformans and C. neoformans var. gattii differed in chromosomal makeup, RAPD patterns, and killer sensitivity patterns. These results suggest that there are two separate species rather than two varieties. No clear genetic or phenotypic differences were observed among the clinical, saprobic, and veterinary isolates within each taxon. The serotypes differed substantially in their RAPD characteristics. Geographical clustering was observed among the isolates of C. neoformans var. gattii, but not among the isolates of C. neoformans var. neoformans. The isolates of each taxon that originated from restricted geographical areas often had identical or similar karyotypes and RAPD patterns, suggesting that clonal reproduction had occurred. The combination of PFGE and RAPD analysis allowed us to distinguish almost all isolates. This combination of techniques is recommended for further research on epidemiological, ecological, and population issues.     

Isolation of specific DNA probes from Cryptococcus neoformans

OBJECTIVE: To construct plasmid library and screen specific DNA probes for Cryptococcus neoformans. METHODS: Serotype A Cryptococcus neoformans was used as the study strain, plasmid pUC18 as vector, and Escherichia coli JM103 as host cell. The plasmid library of cryptococcus neoformans was constructed (pCN). Other pathogenes causing affection diseases which should be distinguished from cryptococcusis clinically, and other fungi similar to Cryptococcus neoformans with physiological and biochemical characteristics were used as a distinguishing system, specific colonies were screened by hibridization in double steps. RESULTS: The inserts of the library were 280 to 1800 base pairs and 580 base pairs in average length. Repeated sequence was 32.43% and single copy sequence was 67.57% in genome of cryptococcus neoformans respectively. Three specific colonies were isolated from the library. Colony pCNII A6 was serotype A specific, pCNII B5 species specific and pCNIII G1-specific for var. neoformans. CONCLUSION: A rapid diagnosis of Cryptococcus neoformans infection at early stage can be made by using species-specific probe, and serotype and variaty of neoformans and gattii be distinguished in epidemic study.     

Effects of Aspergillus sulphureus mycotoxins on Cryptococcus neoformans

Many studies have evaluated the toxicity of mycotoxins to mammals, but there is little information on their action against fungal cells, even although mycotoxins are frequently active against fungi in nature. A crude extract of Aspergillus sulphureus was tested for its growth-inhibitory effect on Cryptococcus neoformans. The reduction in cell growth of Cr. neoformans caused by the extract was dose dependent. Using a liquid medium containing 2% A. sulphureus extract, the RNA content of Cryptococcus amounted to about 60% of that of non-treated cells. Capsule thickening, demonstrated biochemically and with cytological stains, occurred at doses that had minimal effect on cell growth and RNA content. Our results suggest that the virulence of Cr. neoformans may increase in cases of coenobiosis with A. sulphureus, which is theoretically possible in places where corn-fed pigeons are numerous.     

Signal transduction pathways regulating differentiation and pathogenicity of Cryptococcus neoformans

There are no known standardized instruments that encompass all of the essential elements of a streamlined outpatient geriatric assessment. A process of arriving at a consensus on essential elements of streamlined geriatric assessment in 3 domains was initiated and described. Two rounds of the Delphi technique were conducted to identify social, environmental and economic variables essential for such an assessment. Consensus among an expert panel was achieved and a list of 27 variables was reduced to 16. This result is discussed in terms of assessment context and standardizing assessment, using case examples.     

Pathogenesis of pulmonary Cryptococcus neoformans infection in the rat

The pathogenesis of Cryptococcus neoformans pulmonary infection in the rat was studied after intratracheal inoculation. Lungs were examined at various times following infection for histopathology in conjunction with macrophage markers, proliferating cell nuclear antigen (PCNA), and capsular glucuronoxylomannan (GXM) antigen. Serum GXM, immunoglobulin M (IgM) and IgG titers and organ fungal burden were compared with pathological findings. C. neoformans organisms were in the lung parenchyma 2 h postinoculation, and GXM antigen was present in surrounding tissues shortly thereafter. Extrapulmonary dissemination occurred early in infection. Two phases of host cellular inflammatory response were discernible: early local macrophage recruitment at 2 to 4 days followed by granulomatous inflammation, which reached maximum intensity 14 days after infection. The granulomatous phase was preceded by lymphocyte influx with macrophage proliferation and maturation into epithelioid histiocytes; this was paralleled by a shift of yeasts from extracellular to intracellular spaces. Tissue IgG deposits, serum IgG to GXM, and localization of tissue GXM immunoreactivity to epithelioid cells were noted at 2 to 4 weeks. A 10-fold decrease in lung fungal burden occurred 25 days postinfection and was associated with resolving granulomas, fewer proliferating cells, and decreased tissue GXM. The present study demonstrates that (i) C. neoformans penetrates the lung parenchyma shortly after infection; (ii) immunocompetent rats control pulmonary cryptococcosis efficiently, with minimal extrapulmonary dissemination and low levels of serum GXM; and (iii) macrophage activation is likely to play a crucial role in limiting C. neoformans infection in the rat lung.     

Antigen-induced protective and nonprotective cell-mediated immune components against Cryptococcus neoformans

Mice immunized with two different cryptococcal antigen preparations, one a soluble culture filtrate antigen (CneF) in complete Freund's adjuvant (CFA) and the other heat-killed Cryptococcus neoformans cells (HKC), develop two different profiles of activated T cells. CneF-CFA induces CD4+ T cells responsible for delayed-type hypersensitivity (DTH) reactivity and for amplification of the anticryptococcal DTH response, whereas HKC induce CD4+ and CD8+ T cells involved in anticryptococcal DTH reactivity and activated T cells which directly kill C. neoformans cells. The main purpose of this study was to assess the level of protection afforded by each of the two different T-cell profiles against challenge with viable C. neoformans cells, thereby identifying which activated T-cell profile provides better protection. CBA/J mice immunized with CneF-CFA had significantly better protective responses, based on better clearance of C. neoformans from tissues, on longer survival times, and on fewer and smaller lesions in the brain, than HKC-immunized mice or control mice similarly infected with C. neoformans. Both immunization protocols induced an anticryptococcal DTH response, but neither induced serum antibodies to glucuronoxylmannan, so the protection observed in the CneF-CFA immunized mice was due to the activated T-cell profile induced by that protocol. HKC-immunized mice, which displayed no greater protection than controls, did not have the amplifier cells. Based on our findings, we propose that the protective anticryptococcal T cells are the CD4+ T cells which have been shown to be responsible for DTH reactivity and/or the CD4+ T cells which amplify the DTH response and which have been previously shown to produce high levels of gamma interferon and interleukin 2. Our results imply that there are protective and nonprotective cell-mediated immune responses and highlight the complexity of the immune response to C. neoformans antigens.     

Specific activities of dolastatin 10 and peptide derivatives against Cryptococcus neoformans

The biosynthetic peptide dolastatin 10 is currently in phase I and II cancer clinical trials. We evaluated the antifungal spectrum of dolastatin 10 and four structural modifications. In broth macrodilution assays, the peptides were fungicidal for American Type Culture Collection strains and clinical isolates (including fluconazole-resistant strains) of Cryptococcus neoformans but no other yeasts or filamentous fungi examined. Specificity for C. neoformans was also demonstrated in the solid-phase disk diffusion assay, and fungicidal activity was confirmed in time-kill experiments. For a methyl ester modification, the MICs at which 50 and 90% of 19 clinical isolates were inhibited (MIC50 and MIC90, respectively) were 0.195 and 0.39 microg/ml, respectively. The MFC50 (50% minimum fungicidal concentration) for this peptide was 0.39 microg/ml, and the MFC90 was 0.78 microg/ml. MICs and MFCs were identical or lower in the presence of human serum but increased with lowered pH. These peptides should be pursued as potential chemotherapeutics for C. neoformans, a leading cause of infection and mortality in immunocompromised patients.     

Structural characterization of the galactoxylomannan of Cryptococcus neoformans Cap67

The galactoxylomannan (GalXM) obtained from the culture supernatant of an acapsular mutant of Cryptococcus neoformans Cap67 was purified by Concanavalin A affinity, ion-exchange, and gel-filtration chromatographies. The structure of GalXM was determined by methylation analysis and by 1D and 2D NMR spectroscopic studies of the intact polysaccharide and of the oligosaccharide fragments generated by Smith degradation and by acetolysis. GalXM is a complex polysaccharide with an alpha-(1-->6) -galactan backbone. The polysaccharide is branched at c-3 of alternate Gal units of the backbone. C-3 is the point of attachment of the oligosaccharide side chains comprised of alpha-D-Man- (1-->3)-alpha-D-Man-(1-->4)- beta-D-Gal-substituted with zero to three terminal beta-Xyl residues as shown in the following structure: [formula: see text].     

Enhanced activity of antifungal drugs by lysozyme against Cryptococcus neoformans

The in vitro susceptibility of 16 isolates of Cryptococcus neoformans to three antifungal drugs and lysozyme in combination was determined using an urea broth microdilution method. The antifungal activities of each drug alone against 16 isolates of Cr. neoformans were determined as mean minimal inhibitory concentrations (MICs). MICs of fluconazole, itraconazole and terbinafine were 2.0 micrograms ml-1, 0.004 microgram ml-1 and 0.25 microgram ml-1, respectively. Lysozyme alone inhibited the growth of Cr. neoformans in a dose-dependent manner, although the lysozyme was unable to kill the cells of Cr. neoformans at the highest concentration of 20 micrograms ml-1. The mean MICs of fluconazole, itraconazole and terbinafine in combination with lysozyme were 0.13 microgram ml-1, 0.004 microgram ml-1 and 0.03 microgram ml-1 respectively. The antifungal activity of fluconazole and terbinafine in combination with lysozyme against Cr. neoformans was greatly enhanced compared with that of each drug alone. Itraconazole was unable to enhance the antifungal activity, as it demonstrated higher activity against Cr. neoformans when alone rather than in combination. Lysozyme was confirmed to enhance the antifungal activity of fluconazole and terbinafine in vitro.     

A rapid urease test for presumptive identification of Cryptococcus neoformans

Extra-contractual referrals (ECRs) can be a cause of considerable anxiety to purchasing authorities, mainly because of their potential to generate unexpected expenditure. But ECRs can also be used as a tool for monitoring the demand for, and quality of, clinical services. ECRs were studied in the Darlington Health Authority district using a variety of methods including inter-disciplinary meetings, a series of interviews with local GPs, and a questionnaire to general practitioners on 230 consecutive ECRs. The methods and results of the questionnaire study are presented. The commonest reasons for making ECRs included the mistaken belief that a contract existed with the ECR provider, patient dissatisfaction with the local provider, and referral to benefit from shorter waiting lists. ECRs for bone-mass densitometry, orthopaedics, and ear nose and throat services were over-represented. Questionnaire results were validated by comparison with an interview study of all GPs in the district. We conclude that trends in ECRs can be monitored as a convenient "early warning system' to alert purchasing authorities to changes in demand or perceived problems with local provider units. ECR data must be interpreted in the context of further local background information from sources such as GPs and public health physicians. In the case of Darlington, scrutiny of ECRs has led to changes in services and contracts.     

Binding of Cryptococcus neoformans to heterologously expressed human complement receptors

Previously, we demonstrated that monoclonal antibodies (MAb) directed against any of the three defined complement receptors (CR) for the third component of complement (CR1, CR3, and CR4) profoundly inhibited the binding of serum-opsonized Cryptococcus neoformans to monocyte-derived macrophages. These studies suggested either that a synergistic interaction between multiple CR was required for optimal binding of C. neoformans or that the MAb were exerting nonspecific effects (such as receptor coassociation). In the present studies, we took a novel approach to dissecting out the contributions of individual receptors to binding of a microbial pathogen. Chinese hamster ovary (CHO) cells stably transfected with human CR1, CR3, or CR4 were challenged with serum-opsonized C. neoformans. We found that CHO cells transfected with any of the three receptors bound C. neoformans, with the avidity of binding to CR3 being the greatest followed in decreasing order by CR1 and CR4. Following binding of C. neoformans to transfected CHO cells, most organisms remained surface attached only, although for each receptor a significant percentage (18.5 to 27.3%) of C. neoformans was internalized. Both C. neoformans and sheep erythrocytes that were selectively opsonized with the fragments of the third component of complement, C3b and iC3b, were bound preferentially by CHO cells transfected with CR1 and CR3, respectively. These data establish CR1, CR3, and CR4 as receptors independently capable of binding C. neoformans opsonized with fragments of C3. Moreover, our study demonstrates the usefulness of transfected cell lines as a powerful tool for identifying the contribution of individual receptors to the binding of a microbial pathogen.     

Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2',7'-difluorofluorescein (Oregon Green). These probes have pKas of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 microM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.     

Phenotypic and genotypic differentiation of several human and avian isolates of Cryptococcus neoformans

The Cryptococcus neoformans strains isolated from two human cases could be diagnosed as Cr. neoformans var. neoformans by differentiation on the basis of their characteristics determined by proline, canavanine and EDTA urease tests. The results of the serovar assignment were: for the isolate from the meningoencephalitis patient with lethal outcome, serovar A; for the strain isolated from the osteomyelitis patient with benign course, serovar D. Also, the PCR fingerprinting using primers (GACA)4, (CAC)5 and FM 1 resulted in a clear and reproducible assignment of the Cr. neoformans strains to the varieties neoformans and gattii, respectively, and, in addition, it confirmed the serovar assignment. No statistically confirmed differences in virulence between the osteomyelitis and the meningoencephalitis strain could be established by i.v. testing in mice, nor did the PCR with several primers provide any clues to a genetically determined higher virulence of the meningoencephalitis strain. The different classification as serovars A and D does not allow any conclusions concerning different virulence. It was not possible to retrospectively establish the sources of infection of the two Cr. neoformans infections, but pigeon faeces may well have played a role as a reservoir for one of the illnesses.     

Susceptibility testing of Cryptococcus neoformans using the urea broth microdilution method

An urea broth microdilution method to assay the susceptibility of Cryptococcus neoformans to antifungal drugs was newly developed. Using this method, urease activity of the fungus was measured instead of the viability by checking colony development. The urease activities were indicated by colour changes in optical density at 545 nm. The end point in this assay was considered as 99% inhibitory concentration. When we measured antifungal activities of the three drugs against 16 isolates of Cr. neoformans using this assay method, mean minimum-inhibitory concentrations (MICs) of fluconazole, itraconazole and terbinafine were 2.0 micrograms ml-1, 0.008 microgram ml-1 and 0.25 microgram ml-1 respectively. This assay method resulted in higher sensitivity in MICs of the three antifungal drugs than the broth microdilution method recommended by the Committee for Laboratory Standards of the Japanese Society for Medical Mycology. The results obtained using this assay method support the more effective evaluation of antifungal substances in susceptibility testing of Cr. neoformans.     

Identification by polymerase chain reaction fingerprinting of Cryptococcus neoformans serotype AD

Seventy-three Cryptococcus neoformans isolates and eight other yeast strains were studied. Fingerprints produced by priming with (GACA)4 differentiated C. neoformans from all other yeasts tested and identified the five C. neoformans serotypes. Four major bands of molecular size 800, 540, 475 and 410 bp were recognized for serotypes A, AD and D. Two of them were specific for serotype A and the other two for serotype D isolates. Serotype AD strains were identified by five different genotypic patterns in which at least one of the two bands specific for serotype A and D were present in different combinations. On repeated and simultaneously performed genotype and serotype testing of nine strains, the genotypic pattern did not change, whereas serotyping was unstable in three cases. PCR-fingerprinting using (GACA)4 as a primer proved more stable than serology in discriminating among C. neoformans serotypes A, D and AD and was able to distinguish among serotype AD strains.     

Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies

Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.     

Cryptococcus neoformans and Candida albicans regulate CD4 expression on human monocytes

This study examined the capability of Candida albicans and Cryptococcus neoformans to modulate CD4 expression on human monocytes. C. albicans and an acapsular strain of C. neoformans induced higher levels of CD4 expression than encapsulated strains. Purified glucuronoxylomannan did not regulate CD4 expression on monocytes, but down-regulation of CD4 expression compared with stimulation by acapsular C. neoformans alone was observed when glucuronoxylomannan was used in combination with acapsular C. neoformans. The ability of opsonic factors to facilitate fungal-mediated CD4 overexpression suggests that binding or internalization (or both) of the yeast cells is a critical event. Protein synthesis was required, excluding redistribution of the intracellular pool of CD4 receptors to the cellular surface as the sole possible mechanism. Results demonstrate a new effect of fungi on professional phagocytic cells and raise the possibility that modulation of CD4 could influence gp120-mediated human immunodeficiency virus entry.