靛玉红、蛇床子素抗外阴阴道念珠菌病混合菌生物膜作用的研究

目的研究中药有效成分靛玉红、蛇床子素抗外阴阴道念珠菌病混合菌生物膜的作用。方法体外建立白念珠菌（Candidaalbicans）铜绿假单胞菌（Pseudomonasaeruginosa,P．a）混合菌生物膜（Biofilm,BF）,XTT减低法及形态学观察白念珠菌混合茵生物膜的形成过程;形态学观察、活菌计数法评价中药有效成分靛玉红（indirubin）、蛇床子素（Ostho）对白念珠菌混合菌生物膜的最小抑膜浓度（SMIC）,并经扫描电镜确认。结果白念珠菌混合菌48h能形成成熟的生物膜;62．5mg／L浓度的靛玉红能抑制白念珠菌混合菌生物膜的形成。500mg／L浓度的蛇床子素未见有抑制白念珠菌混合菌生物膜的作用。结论靛玉红由于具有抗生物膜的作用,可用于预防外阴阴道念珠菌病的复发。     

Karyotyping of Candida albicans and Candida glabrata isolates from recurrent vaginal infections by pulsed-field gel electrophoresis

In the present study, 16 women with recurrent vulvovaginal candidiasis (RVVC) due to Candida albicans and Candida (Torulopsis) glabrata were followed for a period of 4 to 12 months, and 36 vaginal isolates were evaluated by pulsed-field gel electrophoresis (PFGE). Eleven women were infected by C. albicans and 5 by C. glabrata. Three electrophoretic karyotypes of C. albicans and 3 of C. glabrata were identified throughout the follow-up. All patients but one was infected with the same karyotype of C. albicans or C. glabrata during the follow-up period. Two different karyotypes of C. glabrata were identified in one patient in the course of 12 months. The results confirmed the diversity of the karyotypes of C. albicans and C. glabrata causing vulvovaginitis, and demonstrated the persistence of colonization with the same strain over different periods of time despite therapy (15/16 women).     

Host defense against oropharyngeal and vaginal candidiasis: Site-specific differences

Mucosal candidiasis is extremely common in immunocompromised patients. However, the prevalence of site-specific infection (i.e., oropharyngeal, vaginal, and esophageal candidiasis) can be quite variable depending on the immune status of the host. While vulvovaginal candidiasis is common in normal healthy women, oropharyngeal and esophageal candidiasis are more frequently encountered under immunocompromised states. Candida albicans, the causative agent in most cases of candidiasis, is a commensal organism of the gastrointestinal and lower female reproductive tracts. Thus, most healthy individuals have demonstrable Candida-specific immunity in the peripheral circulation. The pathogenic state is often precipitated by a deficiency or dysfunction in this immunity. Studies from animal models, women with recurrent vulvovaginal candidiasis, and HIV-infected individuals, however, suggest that distinct host defense mechanisms may function against oropharyngeal and vulvovaginal candidiasis. While cell-mediated immunity (CMI) appears important for protection against oropharyngeal candidiasis (OPC), there is little evidence to indicate that T cell-mediated immunity is protective against vulvovaginal candidiasis (VVC). Furthermore, whereas both local and systemically derived immune defenses appear important for protection against OPC, host defenses that protect against VVC appear limited to the local tissue and possibly restricted to innate mechanisms. Thus, current evidence suggests that VVC, unlike OPC, may not represent a strict opportunistic infection.     

Candida albicans abrogates the expression of interferon-gamma-inducible protein-10 in human keratinocytes

Candida albicans is the predominant causative agent of human cutaneous candidiasis. Epidermal keratinocytes play an important role in the cutaneous immune response through the production of cytokines and chemokines, including IFN-gamma-inducible protein 10 (IP-10). Here, we investigated the influence of C. albicans infection on IP-10 production by normal human epidermal keratinocytes (NHEK) in vitro. Our results showed that IFN-gamma-stimulated NHEK showed enhanced IP-10 mRNA and protein expression; this expression was downregulated by C. albicans infection. Candida tropicalis also impaired IFN-gamma-induced IP-10 expression, but Candida glabrata did not. Heat-killed C. albicans did not impair IFN-gamma-induced IP-10 expression. We found that coincubation of NHEK with live C. albicans without cell-to-fungi contact impaired IFN-gamma-induced IP-10 mRNA and protein expression in NHEK, suggesting the role of soluble factors derived from live C. albicans in this impairment. Enzyme-linked immunosorbent assay analysis revealed that C. albicans and C. tropicalis could produce marked levels of prostaglandin (PG) E(2), while C. glabrata produced low levels of this prostaglandin. Treatment with E-series prostaglandin receptor antagonists, AH6809 and AH23848, restored IFN-gamma-induced IP-10 expression in C. albicans-infected NHEK. Thus, Candida-derived PGE(2) may impair IFN-gamma-induced IP-10 expression in human keratinocytes and may play a role in the pathogenesis of cutaneous candidiasis.     

Identification and susceptibility against fluconazole and albaconazole of 100 yeasts' strains isolated from vaginal discharge

Vulvovaginal candidiasis is a condition that affects a great number of fertile women. It is considered the second cause of genital infection after vaginosis due to GAM complex. Candida albicans is the most frequent isolated species from vaginal discharge. However, sometimes more than one yeast species could be found in the same clinical sample that are more resistant to antifungal drugs. Nowadays, it is necessary to identify properly up to species level the isolated microorganism and to determine the antifungal susceptibility profile. One hundred strains obtained from vaginal discharge of 94 patients suffering acute vulvovaginal candidiasis were studied. The identification of the isolates showed: C. albicans 86%, Candida glabrata 6%, Candida inconspicua 3%, Candida krusei 2% and Candida intermedia, Candida holmii and Trichosporon asahii one case each. Minimal inhibitory concentrations (MIC) of all the yeasts against fluconazole and albaconazole were performed. C. glabrata, C. krusei and C. inconspicua were the most resistant against fluconazole, on the other hand albicans was susceptible to this drug. All the isolates presented MIC against albaconazole much lower than fluconazole.     

复发性外阴阴道念珠菌病的致病菌种鉴定及药敏分析

目的：探究复发性外阴阴道念珠菌病致病菌的菌种分布及耐药情况,指导临床治疗。方法：收集210例念珠菌性外阴阴道炎患者阴道分泌物标本,其中复发性外阴阴道念珠菌病（RVVC）组76例,外阴阴道念珠菌病（VVC）组134例。运用科玛嘉显色培养基法进行菌种鉴定和药物敏感试验。结果：在210例病例中,RVVC组中白色念珠菌54株（71.05%）,光滑念珠菌15株（19.74%）,热带念珠菌4株（5.26%）,克柔念珠菌2株（2.63%）,近平滑念珠菌1株（1.32%）。非白色念珠菌中以光滑念珠菌为主,在RVVC组中的比例明显高于VVC组,有显著性差异（P〈0.05）。药敏试验显示,RVVC的念珠菌株中仅8株对7种药物全部敏感,23株敏感药物〈5种,其中1株仅对制霉菌素敏感;对药物的敏感率为制霉菌素（97.37%）〉克霉唑（60.52%）〉酮康唑（51.32%）〉伊曲康唑（36.84%）〉咪康唑（35.53%）〉氟康唑（23.68%）〉特比奈芬（10.53%）。结论：白色念珠菌仍是RVVC的主要致病菌,非白色念珠菌比例在RVVC组中显著高于VVC组,其中以光滑念珠菌为主。念珠菌对制霉菌素敏感率最高,对唑类药物耐药性有增加趋势。     

Biotherapeutic effects of Bifidobacterium spp. on orogastric and systemic candidiasis in immunodeficient mice

Two commercially available Bifidobacterium spp. (Bifidobacterium infantis and Bifidobacterium lactis) were compared for their capacities to protect immunodeficient bg/bg-nu/nuand bg/bg-nu/+mice from orogastric and lethal candidiasis. Both Bifidobacterium spp. prolonged the survival of Candida albicans-colonized adult and neonatal bg/bg-nu/numice. The bifidobacteria affected the production of antibodies to C. albicans, inhibited disseminated candidiasis, suppressed weight loss associated with C. albicans infection, inhibited the growth of C. albicans in the alimentary tract, inhibited systemic candidiasis of endogenous origin, and decreased the severity of gastric candidiasis in both mouse strains. B. infantis inhibited systemic candidiasis of endogenous origin better than B. lactis; however, B. lactis was significantly more effective at inhibiting C. albicans colonization of the alimentary tract, suppressing gastric candidiasis, and protecting bg/bg-nu/numice from lethal candidiasis than B. infantis. These results show that Bifidobacterium spp. can protect immunodeficient mice from candidiasis but different species manifest quantitative and qualitative differences in their probiotic and biotherapeutic effects.     

Genotyping and Drug Resistance Profile of Clinical Isolates of Candida albicans from Vulvovaginal Candidiasis in the Eastern China

Mycopathologia - A total of 244 Candida albicans isolates recovered from vulvovaginal candidiasis (VVC) patients in Suzhou, Eastern China, were investigated. According to CLSI documents M27-A4 and...     

Protective effects of farnesol against oral candidiasis in mice

Farnesol is known as a quorum-sensing molecule for Candida albicans and is recognized to play pathogenic roles in Candida infection. To assess the possible role of farnesol in mucosal C. albicans infection, the effects of farnesol treatment against experimental oral candidiasis in mice were examined. Prednisolone-pretreated ICR mice were orally infected with C. albicans and 3, 24 and 30 hr later the animals were orally given farnesol. Forty-eight hr later they were killed for observation. Farnesol treatment in a dose ranging between 1.125 and 9 micromol/mouse showed a protective effect against oral candidiasis in a dose-dependent manner, at least as estimated by symptom scores of tongues. At 9 micromol/mouse it decreased bodyweight loss. Histological studies of 2.25 micromol/mouse farnesol-treated animals indicated that farnesol suppressed mycelial growth of C. albicans on the surface of tongues, but microbiological study did not prevent the change of CFU of C. albicans cells not only on tongues but also in feces, kidneys and livers. These results suggest that farnesol has very characteristic roles in protection against mucosal candidiasis.     

Microbiological and immunological features of oral candidiasis

Candida albicans(C. albicans) is the major infectious agent of oral candidiasis, and both innate immunity and cell-mediated immune response participate in the control of the fungal infections. The aim of this study was to correlate the clinical forms of oral candidiasis with the number of colony forming units (CFU) of C. albicans in saliva and to characterize T cell response in patients with oral candidiasis. Participants included 75 subjects: 36 with lesions of candidiasis and 39 without lesions of oral candidiasis. A 2-ml sample of saliva was collected from all subjects for microbiological analysis. Cytokine levels were determined by ELISA in supernatants of peripheral blood mononuclear cells of 25 patients with oral candidiasis, after in vitro stimulation with C. albicans antigens. In 48% of patients, no association was observed with denture use. C. albicans was detected in the saliva of 91.7% of patients with oral candidiasis, and there was an association between the number of CFU and the presence of oral lesions. A type Th1 immune response was observed in supernatants of peripheral blood mononuclear cells stimulated with C. albicans antigens. In contrast, IL-5 and IL-10 levels were very low or undetectable. Together, this study shows an association between clinical forms of oral candidiasis and the number of colonies of C. albicans in saliva, and that a systemic immune response characterized by the production of TNF-alpha and IFN-gamma is observed in patients with oral candidiasis.     

妊娠期外阴阴道假丝酵母菌病发病机制的研究概述

外阴阴道假丝酵母菌病(vulvovaginal candidiasis,VVC)是由假丝酵母菌引起的常见外阴阴道炎症。VVC在妊娠期有较高的发病率。妊娠期VVC的常见病原体主要是白色假丝酵母菌,其发病机制包括菌体本身的致病因素、妊娠期激素的升高及免疫功能下降。菌体的致病因素包括粘附力、细胞外酶及形态转换。同时VVC对妊娠有一定影响,主要导致宫内感染、胎膜早破、早产、低体质量儿出生、流产、死胎、假丝酵母菌性肺炎等疾病。本文就国内外妊娠期外阴阴道假丝酵母菌病的发病机制进行综述。     

Sciadonic acid modulates prostaglandin E2 production by epithelial cells during infection with C. albicans and C. dubliniensis

Candida albicans is an important opportunistic pathogen in humans. During infection, arachidonic acid (ω6) is released from host phospholipids, leading to the production of host and yeast derived prostaglandin E(2) (PGE(2)). This stimulates yeast hyphal formation, is immunomodulatory and causes cell damage during infection. Although supplementation of mammalian cells with ω3 fatty acids has received attention due to their immunomodulatory and anti-inflammatory activities, increased production of ω3 fatty acid metabolites could lower the host's ability to combat infections. Since mammalian cells cannot produce PGE(2) from sciadonic acid (SA), a non-methylene interrupted ω6 fatty acid (NMIFA), supplementation of cells with SA may decrease the production of PGE(2) without increasing levels of ω3 fatty acid metabolites. Our study evaluated PGE(2) production by SA supplemented epithelial cells in response to Candida albicans and C. dubliniensis. We show that PGE(2) production during infection can be modulated by incorporation of SA into host lipids and that this does not influence the levels of ω3 fatty acids in the epithelial cells.     

Prevalence of specific and phylogenetically closely related genotypes in the population of Candida albicans associated with genital candidiasis in China

Genitourinary candidiasis, which is most frequently caused by Candida albicans, is a common problem worldwide. The pathogenesis of the infection, especially recurrence of the infection, remains to be elucidated. This study analyzed 199 independent Chinese C. albicans isolates using multilocus sequence typing (MLST) and microsatellite typing, with the focus on the isolates associated with vulvovaginal candidiasis (VVC) of Chinese women. MLST data of 221 vaginal isolates from other countries available from the consensus MLST database of C. albicans were retrieved for comparison. A total of 124 diploid sequence types (DSTs) were recognized from the Chinese C. albicans isolates, among which, 98 (79.0%) have not been reported in the MLST database of the species. The majority of the VVC (71.6%) and balanitis (92.3%) isolates from China were located in clade 1 of C. albicans; while only 40.6% of the vaginal isolates and 7.8% of the oral isolates from healthy volunteers were found in the same clade. Furthermore, 69.1% of the VVC and 84.5% of the balanitis isolates concentrated in a cluster of clade 1 with DST 79 as the primary founder. The isolates in this cluster possessed microsatellite genotypes CAI 30-45, CAI 32-46 and their close derivatives. Interestingly, a remarkable difference in genotype distribution patterns between Chinese and non-Chinese vaginal isolates of C. albicans was observed. Only 11.3% of the non-Chinese vaginal isolates compared were located in the cluster concentrated with Chinese VVC isolates. The results suggest significant association of specific and genetically similar genotypes with genital infections in China.     

Candida albicans internalization by host cells is mediated by a clathrin-dependent mechanism

Candida albicans is a major cause of oropharyngeal, vulvovaginal and haematogenously disseminated candidiasis. Endocytosis of C. albicans hyphae by host cells is a prerequisite for tissue invasion. This internalization involves interactions between the fungal invasin Als3 and host E- or N-cadherin. Als3 shares some structural similarity with InlA, a major invasion protein of the bacterium Listeria monocytogenes . InlA mediates entry of L. monocytogenes into host cells through binding to E-cadherin. A role in internalization, for a non-classical stimulation of the clathrin-dependent endocytosis machinery, was recently highlighted. Based on the similarities between the C. albicans and L. monocytogenes invasion proteins, we studied the role of clathrin in the internalization of C. albicans . Using live-cell imaging and indirect immunofluorescence of epithelial cells infected with C. albicans , we observed that host E-cadherin, clathrin, dynamin and cortactin accumulated at sites of C. albicans internalization. Similarly, in endothelial cells, host N-cadherin, clathrin and cortactin accumulated at sites of fungal endocytosis. Furthermore, clathrin, dynamin or cortactin depletion strongly inhibited C. albicans internalization by epithelial cells. Finally, beads coated with Als3 were internalized in a clathrin-dependent manner. These data indicate that C. albicans , like L. monocytogenes, hijacks the clathrin-dependent endocytic machinery to invade host cells.     

Plant production of anti-β-glucan antibodies for immunotherapy of fungal infections in humans

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.     

Variable biotherapeutic effects of Lactobacillus acidophilus isolates on orogastric and systemic candidiasis in immunodeficient mice

Two commercially available isolates of Lactobacillus acidophilus (NCFM and LA-1) were compared for their capacities to protect immunodeficient bg/bg-nu/un and bg/bg-nu/+ mice from candidiasis. L. acidophilus NCFM prolonged survival of adult and neonatal bg/bg-nu/nu mice, inhibited disseminated candidiasis in both mouse strains, suppressed weight loss associated with Candida albicans infection in bg/bg-nu/nu females, but did not decrease the severity or the incidente of orogastric candidiasis in gnotobiotic mice. L. acidophilus LA-1 suppressed numbers of C. albicans in the alimentary tracts of bg/bg-nu/+ mice and reduced the severity of mucosal candidiasis in bg/bg-nu/nuand bg/bg-nu/+ mice; however, L. acidophilus LA-1 did not improve the survival of bg/bg-nu/nu mice after oral challenge (colonization) with C. albicansand it was associated with lethality in gnotobiotic adult female bg/bg-nu/nu mice. These results demonstrate that the two isolates of L. acidophilus differed in their capacity to protect immunodeficient mice from candidiasis.     

Inhibitory effect of prostaglandin E2, forskolin, and dibutyryl cAMP on arachidonic acid release and inositol phospholipid metabolism in guinea pig neutrophils

The effect of prostaglandin E2 (PGE2), forskolin, and dibutyryl cAMP on arachidonic acid release, inositol phospholipid metabolism, and Ca2+ mobilization was investigated. The chemotactic tripeptide (formylmethionyl-leucyl-phenylalanine (fMLP))-induced arachidonic acid release in neutrophils was significantly inhibited by PGE2, forskolin, and dibutyryl cAMP. Among them, PGE2 was found to be the most potent inhibitor. However, when neutrophils were stimulated by Ca2+ ionophore A23187, such inhibitory effect by these agents was less marked. PGE2 also suppressed the enhanced incorporation of [32P]Pi into phosphatidic acid (PA) and phosphatidylinositol in a dose-dependent manner in fMLP-stimulated neutrophils. Also in this case, Ca2+ ionophore-induced alterations were hardly inhibited by PGE2. As well, PGE2 inhibited the fMLP-induced decrease of [3H]arachidonic acid in phosphatidylcholine and phosphatidylinositol and the increase in PA very significantly. But the inhibitory effect by PGE2 was found to be weak in Ca2+ ionophore-stimulated neutrophils. These results suggest that a certain step from receptor activation to Ca2+ influx is mainly inhibited by PGE2. Concerning polyphosphoinositide breakdown, PGE2 did not affect the fMLP-induced decrease of [32P]phosphatidylinositol 4,5-bisphosphate which occurred within 10 s but inhibited the subsequent loss of [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol, suggesting that the compensatory resynthesis of phosphatidylinositol 4,5-bisphosphate was inhibited. On the other hand, fMLP-induced diacylglycerol formation was suppressed for the early period until 1 min, but with further incubation, diacylglycerol formation was rather accelerated by PGE2. Moreover, the inhibition of PA formation by PGE2 became evident after a 30-s time lag, suggesting that the conversion of diacylglycerol to PA is inhibited by PGE2. The formation of water-soluble products of inositol phospholipid degradation by phospholipase C, such as inositol phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, was also suppressed by PGE2 treatment. However, the inhibition was not so marked as that of arachidonic acid release and PA formation. Thus, PGE2 appeared to inhibit not only initial events such as polyphosphoinositide breakdown but also turnover of inositol phospholipids. PGE2, forskolin, and dibutyryl cAMP did not block the rapid elevation of intracellular Ca2+ which was observed within 10 s in fMLP-stimulated neutrophils. However, subsequent increase in intracellular Ca2+ which was caused from 10 s to 3 min after stimulation was inhibited by PGE2, forskolin, and dibutyryl cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protease and phospholipase activities of Candida albicans isolated from vaginal secretions with different pH values

Even though vulvovaginal candidiasis and bacterial vaginosis are seldom simultaneously found, we have detected this association at an above average frequency. Thus, we set out to study the activity of proteinases and phospholipases, virulence factors of Candida albicans, to assess their role in the above mentioned association. Of a total of 70 Candida isolates were retrieved from samples of vaginal secretions analyzed at our Diagnostic Service, 65 were identified as C. albicans (a group of n=26 obtained from clinical samples of pH>4.5 and a group of n=39 from clinical samples of pH=or<4.5). The evaluation of phospholipases activity was performed on malt agar and Sabouraud dextrose agar with the addition of egg yolk as substrate. The proteolytic activity was detected on plates of agar base medium with the addition of bovine albumin serum as substrate as sole nitrogen source. Phospholipases activity was essentially the same in both groups of samples (p=0.2003). Proteolytic activity was detected in 61.5% of the isolates from the group with pH=or<4.5 and in 96.2% in the group with pH>4.5; being the former much higher than the latter (p=0.0001). Based on these results we postulate that the simultaneous occurrence of bacterial vaginosis and vulvovaginal candidiasis could be related to the proteolytic activity but unrelated to phospholipases activity.     

Effect of Streptococcus salivarius K12 on the in vitro growth of Candida albicans and its protective effect in an oral candidiasis model

Oral candidiasis is often accompanied by severe inflammation, resulting in a decline in the quality of life of immunosuppressed individuals and elderly people. To develop a new oral therapeutic option for candidiasis, a nonpathogenic commensal oral probiotic microorganism, Streptococcus salivarius K12, was evaluated for its ability to modulate Candida albicans growth in vitro, and its therapeutic activity in an experimental oral candidiasis model was tested. In vitro inhibition of mycelial growth of C. albicans was determined by plate assay and fluorescence microscopy. Addition of S. salivarius K12 to modified RPMI 1640 culture medium inhibited the adherence of C. albicans to the plastic petri dish in a dose-dependent manner. Preculture of S. salivarius K12 potentiated its inhibitory activity for adherence of C. albicans. Interestingly, S. salivarius K12 was not directly fungicidal but appeared to inhibit Candida adhesion to the substratum by preferentially binding to hyphae rather than yeast. To determine the potentially anti-infective attributes of S. salivarius K12 in oral candidiasis, the probiotic was administered to mice with orally induced candidiasis. Oral treatment with S. salivarius K12 significantly protected the mice from severe candidiasis. These findings suggest that S. salivarius K12 may inhibit the process of invasion of C. albicans into mucous surfaces or its adhesion to denture acrylic resins by mechanisms not associated with the antimicrobial activity of the bacteriocin. S. salivarius K12 may be useful as a probiotic as a protective tool for oral care, especially with regard to candidiasis.