正常儿童口腔中白色念珠菌的分布及基因分组

目的 研究以白色念珠菌为代表的念珠菌，以及白色念珠菌基因分组在不同年龄段儿童口腔中的分布情况。方法 4组不同年龄儿童和1组成人作为研究对象，粘膜拭子法取样，CHROMagar Candida鉴定培养基分离鉴定，PCR方法一组引物确认白色念珠菌，一组引物基因分组。结果 不同年龄组儿童口腔中念珠菌的检出率有所不同，除健康青年外，白色念珠菌在检出的念珠菌中均占多数。白色念珠菌3种基因分组的分布也存在差异，以A组为主。结论 不同年龄阶段健康儿童口腔中，A组白色念珠菌组占主导地位。     

Sunflower seed husk agar: a new medium for the differentiation of Candida dubliniensis from Candida albicans

A sunflower (Helianthus annuus) seed husk agar medium has been developed and evaluated for differentiation of Candida dubliniensis from Candida albicans on the basis of colony morphology and chlamydospore production. All C. dubliniensis isolates (n=40) produced rough colonies with hyphal fringes and abundant chlamydospores whereas 101 of 105 (96.2%) C. albicans isolates produced smooth colonies with no evidence of chlamydospore production. Since this medium is free from oil droplets, chlamydospores can be examined with greater clarity by Dalmau plate technique. This medium provides a simple and cost-effective tool for the presumptive differentiation of C. dubliniensis from C. albicans and is particularly suited for clinical microbiology laboratories where biochemical or molecular methods for the differentiation of these two species are not available.     

Study of Candida albicans strains isolated from women with various forms of vaginal candidiasis

Adhesive activity of Candida albicans towards vaginal epitheliocytes is hormone-dependent. Two types of C. albicans adhesins sensitive to polyphenyloxidase and asparaginase were detected. Laser irradiation nonspecifically modulated both adhesin types. Population relationships between fungi and lactobacilli in patients with vaginal candidal infection and in C. albicans carriers were studied. Genodiagnostic method for identification of C. albicans and morphogenesis-associated genes of these yeast-like fungi was approved.     

Interleukin-4 and interleukin-10 inhibit nitric oxide-dependent macrophage killing of Candida albicans

Mouse peritoneal and splenic macrophages treated with interferon-γ (IFN-γ) and infected with the yeast Candida albicans expressed high fungicidal activity in vitro that correlated with increased nitrite concentrations in culture supernatants. Both effects were reduced by an inhibitor of nitric oxide (NO) synthesis which, in vivo, impaired the animals' ability to mount a footpad reaction and clear the fungus from infected organs. Because T helper type-2 (Th2) cytokines in candidiasis are known to limit the expression of protective Th1 functions, we tested the effect of interleukin (IL)-4 and IL-10 on candidacidal activity and NO production of IFN-γ-activated macrophages. Fungal killing and NO secretion were inhibited, in a dose-dependent manner, by the two cytokines either separately or in combination. Impaired candidacidal activity was also demonstrable in the presence of monoiodoacetic acid, an inhibitor of phagocytosis. These data demonstrate that NO is involved in macrophage killing of C. albicans and support the notion that regulation of Th1 effector function by IL-4 and IL-10 might involve modulation of NO synthesis.     

Differential phospholipase gene expression by Candida albicans in artificial media and cultured human oral epithelium

Phospholipases B1, B2, C and D of Candida albicans play a significant role in the host invasive process. Hence we evaluated the in vitro expression of PLB1, PLB2, PLC1 and PLD1 in phospholipase-positive (PL(+)) and -deficient (PL(-)) C. albicans isolates in egg yolk agar (EYA), yeast peptone dextrose broth (YPD), and in a model of oral candidiasis based on reconstituted human oral epithelium (RHOE). The growth of Candida was then determined in YPD and its cellular invasion was investigated using the RHOE model. The PL(+) group demonstrated PLB1, PLB2, PLC1 and PLD1 expression in both EYA and YPD, in contrast to the PL(-) group, which expressed only PLB2 and PLD1. Although PL(+) isolates grew profusely in the RHOE model, they expressed only PLB2, PLC1 and PLD1, and not PLB1. Gene expression investigations could not be carried out with PL(-) isolates due to their inability to grow in the RHOE model. Significant growth differences in YPD medium were also observed within the PL(+) and PL(-) groups. Taken together, these findings indicate that phospholipase gene expression in C. albicans is differentially affected by their growth milieu, and this in turn may modulate the disease outcomes in vivo.     

Mononuclear phagocyte system in mice with intrauterine Candida albicans infection and postnatal experimental tuberculosis

Intrauterine Candida albicans infection in mouse fetuses affected the type of granulomatous inflammation induced by BCG vaccine during the postnatal period. It manifested in increased formation of granulomas and variations in their cellular composition. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 1, pp. 103–106, January, 2006     

老年人口腔白色念珠菌的分布与基因分型研究

目的研究健康老年人口腔白色念珠菌的分布、基因型特点及基因型特点与白色念珠菌分布的相关性。方法来自成都地区212例老年人分为4组：A1（男性，有义齿）；A2（男性，无义齿）；B1（女性，有义齿）和B2（女性，无义齿）。CHROMagar Candida^TM鉴定培养基、碳水化合物同化反应鉴定体系（API 20C AUX）和随机扩增片段多态性分析（randomly amplified polymorphic DNA assays，RAPD）鉴定白色念珠菌。RAPD分析健康老年人口腔白色念珠菌分离株的基因型特征。统计学分析比较不同分组健康老年人口腔白色念珠菌基因型之间的差异。结果212例受检个体中检出89株白色念珠菌（42％），A1、A2、B1、B2白色念珠菌检出率分别为56．2％、21．3％、56．6％、38％。A1组与A2组白色念珠菌检出率差异有统计学意义，P〈0．01。以JWFP和NA为随机引物的RAPD分析将白色念珠菌分为5型。RAPD分析基因型构成在A1组与A2组之间比较差异有统计学意义，P〈0．05，A1组主要以A型为主。结论健康老年人口腔白色念珠菌的检出率与义齿修复相关。义齿修复可能促进具有特定基因型特点的白色念珠菌检出率增高。     

Candida glabrata,an emerging fungal pathogen,exhibits superior relative cell surface hydrophobicity and adhesion to denture acrylic surfaces compared with Candida albicans

Oral candidosis is a common opportunistic infection in debilitated individuals and Candida glabrata is the second most frequently isolated species from this condition, after Candida albicans. Candidal adherence to various biological or non-biological surfaces is considered a prerequisite for colonization, and pathogenesis of candidal infections, and their relative cell surface hydrophobicity (CSH) is likely to be a possible contributory force involved in this process. Whereas a large body of data on the latter features of C. albicans is available, there is surprisingly little information on C. glabrata. As a comprehensive database on the relative adhesion and CSH of Candida spp. is instructive and useful, we investigated in vitro the latter attributes of 34 oral isolates of C. glabrata and 15 isolates of C albicans. There were remarkable intraspecies differences in both the CSH and the adhesive ability of C. glabrata strains (p < 0.001). Compared with C. albicans, C glabrata demonstrated a four-fold greater CSH value (30.63 +/- 11.20% vs 7.23+/-3.56%, p < 0.0001) and a two-fold greater tendency to adhere to denture acrylic surfaces (75.18 +/- 39.96 vs 30.36+/-9.21, p < 0.0001). A significant positive correlation between CSH and adhesion was also noted for both C. glabrata (r=0.674, p < 0.0001) and C. albicans ( r = 0.636, p < 0.05). When the effect of different incubation conditions on the relative CSH and adherence of C. glabrata was examined, CSH and the adherence to acrylic surfaces of four of six C. glabrata isolates were significantly affected by a reduction of the culture temperature (from 37 degrees C to 25 degrees C). A positive relationship also emerged when the temperature-induced variations in the adherence values were correlated with their relative CSH. These data provide hitherto unavailable archival information on important pathogenic attributes of the two most common oral Candida species that may help explain their predominance in this milieu.     

Candida albicans suppresses nitric oxide (NO) production by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages

We examined the in vitro effect of Candida albicans on NO production by macrophages. Candida albicans suppressed not only NO production but also expression of inducible NO synthase (iNOS) mRNA by murine IFN-gamma and bacterial LPS-stimulated peritoneal macrophages. The suppression was not associated with inhibition but rather stimulation of IL-1 beta production. This effect was observed when more than 1 x 10(3)/ml of Candida albicans were added to macrophage cultures (1 x 10(6) cells/ml) and reached a maximal level at 1 x 10(6)/ml. The NO inhibitory effect of Candida albicans was mediated predominantly by as yet unidentified soluble factor(s) and to a lesser extent by direct contact. In addition, heat- or paraformaldehyde-killed Candida albicans did not show this inhibitory activity. Culture supernatant of Candida albicans also inhibited NO production by activated macrophages in a dose-dependent manner, and increased IL-1 beta production. Finally, the inhibitory effect was not mediated by IL-10 and transforming growth factor-beta (TGF-beta), since neutralizing antibodies to these cytokines did not influence Candida albicans-induced reduction in macrophage NO production. Our results suggest that Candida albicans may evade host defence mechanism(s) through a soluble factor-mediated suppression of NO production by stimulated macrophages, and that the effect is independent of production of immunosuppressive cytokines such as IL-10 and TGF-beta.     

Mice lacking both G-CSF and IL-6 are more susceptible to Candida albicans infection: Critical role of neutrophils in defense against Candida albicans

Neutrophils play an important role in the host's defense against infection with various pathogenic organisms. Granulocyte colony stimulating factor (G-CSF) is regarded as a major regulator of neutrophil production and function. Mice lacking G-CSF or its receptor are neutropenic. IL-6 is another cytokine that has been shown to promote neutrophil production and modulate the function of many types of immune cells. We have analyzed G-CSF/IL-6 double deficient (G-CSF^{? / ?} /IL-6^{? / ?} ) mice to gain an insight into the possible contribution of IL-6 to the residual granulopoiesis in G-CSF-deficient (G-CSF^{? / ?} ) mice. Furthermore, we have evaluated the ability of G-CSF^{? / ?} /IL-6^{? / ?} mice to combat an experimental infection with Candida albicans. Our data shows that IL-6 plays a role in granulopoiesis during early post natal period but it is dispensable for steady-state granulopoiesis in adult mice. However, adult G-CSF^{? / ?} /IL-6^{? / ?} mice are more susceptible to Candida infection than similarly infected G-CSF^{? / ?} mice. Although, the candidacidal function of neutrophils of G-CSF^{? / ?} /IL-6^{? / ?} mice is deficient, the ability to produce IFN-γ and TNF-α in response to Candida infection is not compromised. Similarly, nitric oxide production by peritoneal macrophages from G-CSF^{? / ?} /IL-6^{? / ?} mice in response to Candida is comparable to G-CSF^{? / ?} mice.     

A crossreactivity at the immunoglobulin E level of the cell wall mannoproteins of Candida albicans with other pathogenic Candida and airborne yeast species

Background Candida albicans crossreacts with Saccharomyces cerevisiae or Pityrosporum ovate at the IgE level. However, the extent of crossreactivity of C. alhicans with other yeast species is not known. Objective The crossreactivity at the immunoglobulin E (IgE) level of Candida alhicans with other pathogenic Candida species and to the airborne yeast species Cryptococcus and Rhodotorula was studied by immunoblot analysis. Methods Crude antigens, designated as heat extract, were prepared from 13 different yeast species and a dot blot test was performed to detect IgE antibodies against each of the heat extracts in 349 patients with allergies who were positive for IgE antibodies against C alhicans in a CAP system. Results In the dot blot test, most of the sera reacted with the heat extracts of not only C. albicans but also those prepared from the other yeast species. The sera of 41 of the 349 patients (11.7%) reacted with the heat extracts of all 13 yeast species. The extent of the binding of IgE antibodies to multiple yeast species correlated with both the fluorescence intensities measured in the CAP system and the intensities of dots generated by the heat extract of C. alhicans in the dot blot test. In an inhibition dot blot test, mannoproteins. but not proteins, of C. albicans strongly inhibited the subsequent binding of IgE antibodies to all yeast species. Conclusion Our data suggest that the C. albicans mannoproteins are responsible for the crossreactivity among these yeast species at the IgE level.     

桔梗皂苷D防御口腔黏膜上皮细胞感染白色念珠菌的作用

目的:探讨桔梗皂苷D对白色念珠菌黏附口腔黏膜上皮细胞的影响。方法:MTT法检测不同桔梗皂苷D对口腔上皮KB细胞存活率的影响;将白色念珠菌、KB细胞以及不同浓度的桔梗皂苷D共同培养,革兰染色检测白色念珠菌黏附数,台盼蓝排斥实验检测念珠菌活力和菌丝转换率;ELISA法检测上清液中IL-18与人β-防御素2(HBD-2)的蛋白含量;实时荧光定量PCR和Western blot法分别检测KB细胞中HBD-2 mRNA与蛋白表达的变化。结果:桔梗皂苷D对KB细胞存活率无影响;随着桔梗皂苷D浓度的增加,白色念珠菌的黏附数、菌活力和菌丝转换率逐渐下降,上清液中IL-18与HBD-2的蛋白含量以及KB细胞中HBD-2 mRNA表达与蛋白水平逐渐降低。结论:桔梗皂苷D具有抑菌作用,并参与了口腔黏膜上皮细胞防御白色念珠菌感染的免疫反应。     

The effect of oral bacteria on Candida albicans germ-tube formation

A total of eight bacterial isolates belonging to six species, and a select group of 12 oral Candida albicans isolates, were used to study the effect of bacteria on germ-tube formation. Briefly, each bacterial suspension (10(5-6) cells/ml) was mixed with a C. albicans suspension (10(7) cells/ml) and incubated at 37 degrees C for 90 min with bovine serum, and the percentage germ-tube-positive Candida cells was quantified using a haemocytometer, under light microscopy. In general, out of eight bacteria, Streptococcus sanguis SK21A, Streptococcus salivarius SK56, Escherichia coli ATCC 25922, and S. salivarius OBU3 suppressed germ-tube formation to varying degrees, with different C albicans isolates. Porphyromonas gingivalis Pg 50, Lactobacillus casei ATCC 7469 and Prevotella intermedia OBU4 elicited significant enhancement of germ-tube formation, whereas S. sanguis OBU 2 had no effect. E. coli ATCC 25922 was the only organism to show statistically significant suppression of germ-tube formation (p=0.0312). A significant increase in the germ tube production of C. albicans isolated from HIV-infected compared with HIV-free individuals was also noted. The current results tend to suggest that commensal and transient oral bacterial populations may selectively influence the differential expression of germ-tube-forming ability of C. albicans isolates.     

Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome

In a recent report, we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, C. albicans Sap2 and Sap6 potently induced IL‐1β, TNF‐α, and IL‐6 production. Here, we demonstrate that Sap2 and Sap6 proteins trigger IL‐1β and IL‐18 production through inflammasome activation. This occurs via NLRP3 and caspase‐1 activation, which cleaves pro‐IL‐1β into secreted bioactive IL‐1β, a cytokine that was induced by Saps in monocytes, in monocyte‐derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL‐1β. Inflammasome activation required Sap internalization via a clathrin‐dependent mechanism, intracellular induction of K⁺ efflux, and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS‐ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.     

Molecular heterogeneity of fluconazole-resistant and -susceptible oral Candida albicans isolates within a single geographic locale

The emergence of drug-resistant Candida albicans in immunocompromised patients is common. A disconcerting aspect of this phenomenon is the rapid emergence of C. albicans strains that are resistant to a widely used azole drug, fluconazole (FLZ). To understand the origin of FLZ-resistant yeast isolates, we investigated molecular profiles of 20 geographically related oral C. albicans isolates using three genotyping methods: randomly amplified polymorphic DNA-PCR, with six different primers (OBU1, OBU2, OBU3 RSD6, RSD11 and RSD12); electrophoretic karyotyping by pulsed-field gel electrophoresis; and HinfI restriction fragment analysis. Of the 20 isolates studied, 10 were FLZ- resistant and originated from patients with oral candidosis with a history of FLZ therapy, and the remainder were FLZ susceptible from individuals with oral candidosis, but without a history of FLZ therapy. A composite genotype was generated for each strain by combining molecular types derived from the three independent molecular methods. The composite profiles indicated genetic diversity amongst both the FLZ-resistant as well as -sensitive isolates, and no specific features emerged distinguishing the drug-resistant and -sensitive groups. These observations cast doubt on the theory of a clonal origin of FLZ-resistant C. albicans isolates.     

Differential role of NK cells against Candida albicans infection in immunocompetent or immunocompromised mice

Little is known regarding the role of NK cells during primary and secondary disseminated Candida albicans infection. We assessed the role of NK cells for host defense against candidiasis in immunocompetent, as well as immunodeficient, hosts. Surprisingly, depletion of NK cells in immunocompetent WT mice did not increase susceptibility to systemic candidiasis, suggesting that NK cells are redundant for antifungal defense in otherwise immunocompetent hosts. NK‐cell‐depleted mice were found to be protected as a consequence of attenuation of systemic inflammation. In contrast, the absence of NK cells in T/B/NK‐cell‐deficient NSG (NOD SCID gamma) mice led to an increased susceptibility to both primary and secondary systemic C. albicans infections compared with T/B‐cell‐deficient SCID mice. In conclusion, this study demonstrates that NK cells are an essential and nonredundant component of anti‐C. albicans host defense in immunosuppressed hosts with defective T/B‐lymphocyte immunity, while contributing to hyperinflammation in immunocompetent hosts. The discovery of the importance of NK cells in hosts with severe defects of adaptive immunity might have important consequences for the design of adjunctive immunotherapeutic approaches in systemic C. albicans infections targeting NK‐cell function.     

Genotypic profiles and virulence attributes of Candida albicans isolates from patients with oral lichen planus

Candida albicans carriage has been found to be increased in patients with oral lichen planus. In the present work we have investigated the genotypic profiles of 112 C. albicans strains isolated from patients with erosive or nonerosive OLP, and from healthy controls. The virulence attributes of the isolated strains were compared to elucidate the pathogenetic mechanisms through which C. albicans may cause OLP. We have characterized the genotypic profiles of these isolated strains using a randomly amplified polymorphic DNA assay. In addition, we used assays to measure adhesion to buccal epithelial cells and phospholipase activity to evaluate the virulence attributes of these isolates. Our RAPD analyses revealed four different genotypes, named type A to D, among all isolates, and identified statistically significant associations with disease conditions. Specifically, type A (58.1%) and C (29.0%) were primarily found in erosive OLP, while type A (33.3%) and D (58.3%) were identified in nonerosive OLP. However, the healthy group seemed to have type B (38.5%) and D (61.5%). Using adhesion to BEC assay, we demonstrated that the isolates with genotype A had the strongest adherence among the four genotypes (P=0.000<0.05). The phospholipase activity of the isolates with genotype A and C was higher than for those with genotype B and D (P=0.000<0.05). In conclusion, some C. albicans isolates with special genotypic profiles and virulence attributes may contribute to the pathogenesis of OLP.     

An in vitro and in vivo study of the effect of incorporation of chlorhexidine into autopolymerizing acrylic resin plates upon the growth of Candida albicans

Chlorhexidine acetate was incorporated into autopolymerizing acrylic resin and, after studying its ability to diffuse out in vitro, an investigation was made into the potential of the mixture to treat palatal candidosis in the rat. Chlorhexidine was found to diffuse out of acrylic in fungicidal concentrations for up to three weeks when mixed with the acrylic powder in the proportion of 7.5% (w/w). At this concentration it was found that palatal candidosis as produced by the technique of Shakir et al. was cured or prevented. However, rats fitted with chlorhexidine supplemented plates were found not to take sufficient food during the experimental period to maintain their body weight.     

Detection of phospholipase activity of Candida albicans and non albicans isolated from women of reproductive age with vulvovaginal candidiasis in rural area

Background: Vulvovaginal candidiasis (VVC) is most common accounting for 17 to 39% of symptomatic women. Both Candida albicans and non albicans Candida species are involved in VVC. Amongst various virulence factors proposed for Candida, extracellular phospholipases is one of the virulence factor implicated in its pathogenicity. With this background the present study was carried out to find the prevalence of different Candida species and to detect phospholipase producing strains isolated from symptomatic women with VVC. Materials and Methods: At least two vaginal swabs from 156 women of reproductive age with abnormal vaginal discharge were collected. Direct microscopy and Gram’s stained smear examined for presence of budding yeast and pseudo mycelia followed by isolation and identification of Candida species. Extracellular phospholipase activity was studied by inoculating all isolates on Sabouraud’s dextrose egg yolk agar (SDA) medium. Results: Of the 156 women with curdy white discharge alone or in combination with other signs, 59 (37.82%) women showed laboratory evidence of VVC. A total of 31 (52.54%) women had curdy white discharge followed by 12 (20.33%) with other signs and symptoms. C. albicans (62.59%) and non albicans Candida (37.28%) in a ratio of 1.68:1 were isolated. Of the 37 strains of C. albians 30 (81.08%) showed the enzyme activity. Seventeen (56.66%) strains showed higher Pz value of < 0.70 (++++). Conclusion: Although there may be typical clinical presentation of Candidiasis. all the patients did not show laboratory evidence of infection. Pregnancy was found to be major risk factor for development of VVC. C. albicans was prevalent species but non albicans species were also frequently isolated. Extracellular phospholipase activity was seen in C. albicans and not in non albicans Candida isolates.     

白色念珠菌性阴道炎对人精子顶体反应影响机制的初步探讨

目的探讨白色念珠菌性阴道炎对人精子顶体反应（AR）的影响机制。方法以ELISA和硝酸还原酶法分别检测阴道分泌物中的IL-8与NO,采用BAEE/ADH法测定顶体酶的活性,并通过FITC-PSA法检测AR。结果无症状的白色念珠菌携带者阴道分泌物中IL-8及NO水平无显著变化,但在白色念珠菌性阴道炎患者阴道分泌物中,其水平显著升高,且二者水平呈正相关;IL-8对精子顶体酶活性和AR均无影响;低浓度的NO可诱导精子顶体酶活性,同时促进AR,而高浓度的NO对其起抑制作用。结论白色念珠菌性阴道炎患者阴道分泌物IL-8和NO水平升高,可直接或/和间接抑制精子顶体酶活性和AR,其中高浓度的NO起着直接的抑制作用,而IL-8可能间接参与了此作用。