Smoking increases oral mucosa susceptibility to Candida albicans infection via the Nrf2 pathway: In vitro and animal studies

Smoking and Candida albicans (C. albicans) infection are risk factors for many oral diseases. Several studies have reported a close relationship between smoking and the occurrence of C. albicans infection. However, the exact underlying mechanism of this relationship remains unclear. We established a rat infection model and a C. albicans-Leuk1 epithelial cell co-culture model with and without smoke exposure to investigate the mechanism by which smoking contributes to C. albicans infection. Oral mucosa samples from healthy individuals and patients with oral leucoplakia were also analysed according to their smoking status. Our results indicated that smoking induced oxidative stress and redox dysfunction in the oral mucosa. Smoking-induced Nrf2 negatively regulated the NLRP3 inflammasome, impaired the oral mucosal defence response and increased the oral mucosa susceptibility to C. albicans. The results suggest that the Nrf2 pathway could be involved in the pathogenesis of oral diseases by mediating an antioxidative response to cigarette smoke exposure and suppressing host immunity against C. albicans.     

Clinical Significance of Keap1 and Nrf2 in Oral Squamous Cell Carcinoma

Oxidative stress has been reported to play an important role in progression and prognostication in various kinds of cancers. However, the role and clinical significance of oxidative stress markers Keap1 and Nrf2 in oral squamous cell carcinoma (OSCC) has not been elucidated. This study aimed to investigate the correlation of oxidative stress markers Keap1 and Nrf2 expression and pathological features in OSCC by using tissue microarray. Tissue microarrays containing 17 normal oral mucosa, 7 oral epithelial dysplasia and 43 OSCC specimens were studied by immunohistochemistry. The association among these proteins and pathological features were analyzed. Expression of oxidative stress markers Keap1, Nrf2, and antioxidants PPIA, Prdx6, as well as CD147 was found to increase consecutively from normal oral mucosa to OSCC, and the Keap1, Nrf2, PPIA, Prdx6, CD147 expression in OSCC were significantly higher when compared to normal oral mucosa. Expression of Keap1, Nrf2 in tumors was not found to be significantly associated with T category, lymph node metastases, and pathological grade. Furthermore, we checked the relationship among these oxidative stress markers and found that Keap1 was significantly correlated with Nrf2, Prdx6 and CD147. Significant relationship between Nrf2 and Prdx6 was also detected. Finally, we found patients with overexpression of Keap1 and Nrf2 had not significantly worse overall survival by Kaplan–Meier analysis. These findings suggest that ROS markers are associated with carcinogenesis and progression of OSCC, which may have prognostic value and could be regarded as potential therapeutic targets in OSCC.     

Detection and identification of non-Candida albicans species in human oral lichen planus

Candida species were detected and identified in samples from the buccal mucosa, dorsal surface of the tongue and supragingival plaque of subjects with oral lichen planus (OLP). The Candida in the samples were cultured on selection agars, and identified by sequence analyses of 18S, 5.8S and 25/28S rRNA. The isolation frequency of Candida was higher in subjects with OLP than in those with healthy oral mucosa. Non-C. albicans were only isolated from people with OLP. These results support the notion that subjects with OLP are more likely to have oral colonization with Candida, and that non-C. albicans are specifically present in subjects with this condition.     

The antilysozyme activity of yeasts in the genus Candida

The fungi of the genus Candida isolated from patients with oral mucosa candidiasis and from candidiasis carriers have been studied for their antilysozyme activity. These fungi (Candida albicans, C. tropicalis, C. krusei, C. quilliermondii) are antilysozyme-active. A high antilysozyme activity of the fungi isolated from patients with oral mucosa candidiasis permits supposing that the presence of this trait may be one of the factors of microorganism pathogenicity. The effective antimycotic therapy (clotrimasole, sanguirhitrin) decreases the antilysozyme activity of fungi of the genus Candida.     

Topical application of a corticosteroid destabilizes the host-parasite relationship in an experimental model of the oral carrier state of Candida albicans

Abstract Using an experimental model in the mouse we have shown that both local and central lines of defense, involving CD4⁺ T cells, participate in a dynamic interaction to maintain a long-term carrier state of Candida albicans in the oral cavity. We have tested the impact of a predisposing factor to oral candidiasis in the form of a topical application of a corticosteroid (Topsyn^® gel) to the oral mucosa for 75 mice twice a day for a 20-day period. Very rapidly after the treatment was initiated, i.e. on day 4, the residual population of Candida increased up to 40-fold and by day 21, the population was 400-fold that of the carrier state. The resident population of intraepithelial CD4⁺ T cells in the oral mucosa virtually disappeared during the treatment. A topical corticosteroid application also resulted in a massive depletion of T cells in the lymph nodes and in the transient abrogation of the DTH reaction to Candida antigens. On cessation of treatment, normal levels of both Candida and intraepithelial CD4⁺ T cells were also quickly restored. These results suggest that resistance to superficial invasion by Candida is linked to the presence of an oral mucosal line of defense and that topical application of corticosteroids may dramatically shift the host-parasite relationship in favor of Candida .     

影响正常口腔念珠菌检出率的方法学研究

目的:研究不同检测方法对正常口腔念珠菌检出率的影响,寻找一种比较简便可靠的检测方法.方法:以健康的平均年龄7.4岁的儿童为检测人群,比较不同的取样部位,取样方法,检测方法,被检人群口腔中白色念珠菌以及其他念珠菌的检出率.结果:取样和检测方法对检出率有不同程度的影响,PCR检测方法的检出率显著高于培养法.结论:黏膜拭子加离心,和CHROMagar CandidaTM鉴定培养基相结合的方法是一种简便理想的分离培养方法,PCR方法则敏感度更高.     

Nuclear erythroid 2 p45-related factor–2 Nrf2 ameliorates cigarette smoking-induced mucus overproduction in airway epithelium and mouse lungs

Background and objectiveNuclear erythroid 2 p45-related factor–2 (Nrf2) is known to play important roles in airway disorders, whereas little has been investigated about its direct role in airway mucus hypersecretion. The aim of this study is to determine whether this factor could protect pulmonary epithelium and mouse airway from cigarette-induced mucus overproduction.MethodsUsing genetic approaches, the role of Nrf2 on cigarette smoking extracts (CSE) induced MUC5AC expression was investigated in lung A549 cells. Nrf2 deficiency mice were smoked for various periods, and the airway inflammation and mucus production was characterized.ResultsAcute smoking exposure induced expression of MUC5AC and Nrf2 in both A549 cells and mouse lungs. Genetic ablation of Nrf2 augmented, whereas overexpression of this molecule ameliorated CSE-induced expression of MUC5AC. Nrf2 knockout mice, after exposure to cigarette smoking, displayed enhanced airway inflammation and mucus production.ConclusionNrf2 negatively regulated smoking-induced mucus production in vitro and in vivo, suggesting therapeutic potentials of this factor in airway diseases with hypersecreted mucus.     

口腔念珠菌基因多态性检测

目的检测正常人群口腔念珠菌基因型,以了解口腔念珠菌基因多态性。方法采集162例正常人群口腔样本,用念珠菌显色培养基(CHROM agar)进行菌种分离培养鉴定,用玉米Tween80培养基进行真菌孢子形态学检查鉴定,随机抽取45个菌株样本进行内含子转录间隔区(internal transcribed spacer region,ITS)序列检测,并比对同源性,建立进化树,观察菌株进化分支情况。结果分离培养出念珠菌菌株57株(57/162,35.2%),其中白色念珠菌(Candida albicans)36株(36/57,63.1%)。进行ITS序列检测的45个菌株申请GenBank序列注册号为FJ697166-GQ280292。结论正常人群口腔念珠菌基因具有多态性。     

Adhesion of Candida to the obturator and oral mucosa as a cause of the presence of inflammation in patients treated surgically for neoplasia

The aim of the investigation was to prove the relationship between Candida species, silicone and the oral mucosa as well as its effect on mutual reinfections of the mucosa and the obturator. Twenty-five patients with post-surgical prostheses in the maxilla were examined. All the prostheses had an obturator with the external part made of Mucopren silicone manufactured by Kettenbach, Germany. The presence of Candida species was investigated by means of electron microscopy visualization of the silicone samples prior to and after disinfection. The microscopic pictures confirmed a strong adherence of Candida species to Mucopren silicone in 90% of cases, which should be considered as the primary reason of unsuccessful elimination of the fungi by means of disinfection.     

DJ-1 plays an important role in caffeic acid-mediated protection of the gastrointestinal mucosa against ketoprofen-induced oxidative damage

Ketoprofen is widely used to alleviate pain and inflammation in clinical medicine; however, this drug may cause oxidative stress and lead to gastrointestinal (GI) ulcers. We previously reported that nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in protecting cells against reactive oxygen species, and it facilitates the prevention of ketoprofen-induced GI mucosal ulcers. Recent reports suggested that Nrf2 becomes unstable in the absence of DJ-1/PARK7, attenuating the activity of Nrf2-regulated downstream antioxidant enzymes. Thus, increasing Nrf2 translocation by DJ-1 may represent a novel means for GI protection. In vitro, caffeic acid increases the nuclear/cytosolic Nrf2 ratio and the mRNA expression of the downstream antioxidant enzymes, _ϒ-glutamyl cysteine synthetase, glutathione peroxidase, glutathione reductase, and heme oxygenase-1, by activating the JNK/p38 pathway in Int-407 cells. Moreover, knockdown of DJ-1 also reversed caffeic acid-induced nuclear Nrf2 protein expression in a JNK/p38-dependent manner. Our results also indicated that treatment of Sprague–Dawley rats with caffeic acid prior to the administration of ketoprofen inhibited oxidative damage and reversed the inhibitory effects of ketoprofen on the antioxidant system and DJ-1 protein expression in the GI mucosa. Our observations suggest that DJ-1 plays an important role in caffeic acid-mediated protection against ketoprofen-induced oxidative damage in the GI mucosa.     

Application of the micronucleus test to exfoliated epithelial cells from the oral cavity of beedi smokers, a high-risk group for oral cancer

The primary sites for occurrence of oral cancer include the buccal mucosa, tongue, alveolus, palate, lip and the floor of the mouth. In this study, an attempt was made to estimate the cytogenetic damage in different regions of the oral mucosa in people habituated to smoking beedi,which is one of the major forms of tobacco consumption in India and believed to be a major risk factor for oral cancer. By using the micronucleus assay on exfoliated cells from the buccal mucosa, palate and tongue of beedi smokers, we examined an early cellular response to the effect of beedi smoking. A total number of 50 randomly selected male subjects were included in the study. Case and control groups (smokers and non-smokers, respectively) comprised 25 subjects each. The difference in mean micronucleated cell count between cases and controls was significant (P <0.01) for buccal mucosa and palate, but not for tongue. The correlation between age and micronucleus cell count was weak for both cases (r=0.27) and controls (r=0.36).     

成人口腔黏膜念珠菌病的多因素分析

为探讨影响成人口腔黏膜念珠菌病发生的因素,选取口腔黏膜念珠菌病、复发性阿弗他溃疡、扁平苔藓、舍格伦综合征、白斑、天疱疮、类天疱疮患者及口腔黏膜健康者为研究对象,详细记录731个样本的年龄、性别、吸烟、口腔卫生情况、抗生素使用、义齿、口腔黏膜疾病、全身疾病,对结果进行多元Logistic回归分析,得到影响口腔黏膜念珠菌病发生的因素有4类,分别为发病前使用抗生素、口腔卫生情况、口腔黏膜疾病、全身疾病,即多种因素的作用导致口腔黏膜念珠菌病的发生。     

白色念珠菌对人口腔黏膜上皮角质细胞凋亡和增殖的影响

目的:探索白色念珠菌对人口腔黏膜上皮角质细胞(KB细胞)凋亡和增殖的影响。方法:分别培养孢子型白色念珠菌和菌丝型白色念珠菌,取健康志愿者的颊粘膜制备KB细胞,分别加入孢子型白色念珠菌(孢子组)和菌丝型白色念珠菌(菌丝组),另外取单纯KB细胞作为对照组。对比分析各组细胞凋亡率及各个周期的细胞数,统计分析各组细胞增殖指数(PI)。结果:菌丝组凋亡率显著高于对照组及孢子组(P0.05);菌丝组在G0/G1期的细胞比例均显著低于对照组及孢子组(P0.05);菌丝组在S期和G2/M期的细胞比例均显著高于对照组及孢子组(P0.05);菌丝组的PI显著高于孢子组和对照组(P0.05)。结论:菌丝型白色念珠菌可诱导KB细胞凋亡率的上升及改变KB细胞周期变化,并引起KB细胞的PI升高,对于临床上治疗口腔念珠菌病具有重要的指导意义。     

Denture contamination by yeasts in the elderly

Objectives: The aim of this study was to investigate yeast carriage in healthy denture wearers by swabbing and to evaluate the effect of denture hygiene habits. Materials and methods: Denture wearers (n = 87) without evidence of denture stomatitis or any other oral disease were investigated by separately swabbing the fitting surface of the upper denture and the corresponding palatal mucosa in contact with the appliance. In a group of volunteers, a gel without any active compound was spread on the palatal side of the denture once in every morning for 2 weeks. Results: Screening showed Candida colonisation of upper prosthesis in 75.9% of individuals. The most frequent species isolated were Candida albicans (77.9% of the positive cultures), Candida glabrata (44.1%) and Candida tropicalis (19.1%). Carriage of more than one yeast species was found in 48.5% of the contaminated dentures. There was a statistically significant association between denture contamination and palatal mucosa colonisation (chi‐squared test: p < 0.0001). Repeated swabbings after 1 week as well as during a weekly follow‐up for 1 month confirmed the denture contamination and its degree of severity. A daily gel application produced a yeast‐count decrease to 10% of the initial value after 2 weeks (chi‐squared test: p = 0.0134 and p = 0.2841 for prosthesis and palatal mucosa, respectively). Conclusion: This study documented the reliability of oral swabbing when investigating yeast carriage in healthy denture wearers. Moreover, just a diagnostic tool, sampling upper dentures for Candida could be the opportunity to verify the patient’s compliance to hygiene advice.     

Induction of NAD(P)H:quinone oxidoreductase 1 expression by cysteine via Nrf2 activation in human intestinal epithelial LS180 cells

In this study, we examined the effects of 20 amino acids on the expression level of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human intestinal LS180 cells. Five amino acids were associated with significant increases in NQO1 mRNA expression; the most substantial increase was induced by cysteine, which markedly increased the NQO1 mRNA level in a time- and dose-dependent manner. Cysteine also increased the protein level of NQO1 and its enzymatic activity in LS180 cells. Furthermore, cysteine significantly up-regulated NQO1 promoter activity, and this induction was completely abolished by mutation of the antioxidant response element, a binding site of the nuclear factor erythroid 2-related factor 2 (Nrf2). Knockdown experiment using siRNA against Nrf2 showed the involvement of Nrf2 on cysteine-induced increase in NQO1 mRNA expression. Further, cysteine treatment increased the amount of Nrf2 protein in the nucleus and decreased the amount of Kelch-like ECH-associated protein 1 (a suppressor protein of Nrf2) in the cytosol, suggesting that Nrf2 was activated by cysteine. Oral administration of cysteine to mice significantly increased NQO1 mRNA levels in the mouse intestinal mucosa. These findings show that cysteine induces NQO1 expression in both in vitro and in vivo systems and also suggest that Nrf2 activation is involved in this induction.     

The oral yeast flora of 10-year-old schoolchildren

The yeast flora of the hard palate mucosa and pooled saliva of 122 schoolchildren (average age 9 years 10 months, 63 males, 59 females) has been studied in detail. Of the population sampled, 71.3% carried oral yeasts. The carriage was approximately 3 times greater in females than in males. The yeast isolates were Candida albicans 71% Saccharomyces spp. 19.7% and Candida tropicalis 8.6%. No isolates of Torulopsis spp. or Rhodotorula spp. were recovered. 71.5% of the Candida albicans isolates were serotype A and 26.3% were serotype B.     

Fungispecificity of fluconazole against Candida albicans

Candida albicans is an opportunistic pathogen of human mucosal surfaces. Colonization of oral and vaginal mucosa by this yeast is antagonized by the resident normal bacterial population. However, antibacterial therapy can alter the normal flora to allow fungal cells to attach, grow and invade host tissues. We studied the antimicrobic activity of fluconazole against clinical isolates of oral and vaginal bacteria and Candida albicans in vitro and in vivo by scanning and transmission electron microscopy; we also compared the bactericidal activity of fluconazole with clotrimazole in vitro by microbiologie assay. Fluconazole lysed fungi but did not change the ultrastructure of bacteria. Clotrimazole, but not fluconazole, was bactericidal against lactobacillus and streptococcus, the principal species of the oral and vaginal cavities. We conclude that Candida albicans, but not oral and vaginal bacteria, is susceptible to fluconazole. These observations help explain the antimycotic specificity of fluconazole and its efficacy against candidiasis in humans.     

慢性阻塞性肺疾病并念珠菌性口炎患者病原学及危险因素分析

目的 探讨入院慢性阻塞性肺疾病(COPD)并念珠菌性口炎患者的病原学特征以及相关危险因素.方法 采用病例研究,对2007年4月1日至2011年1月31日入院的82例COPD并念珠菌性口炎患者和82例无念珠菌性口炎COPD患者进行匹配,应用SPSS 17.0统计软件行条件logistic回归模型分析COPD患者念珠菌性口炎发生的危险因素.结果 (1)从念珠菌性口炎患者假膜培养共分离出念珠菌83株,以白念珠菌(90.4％)为最多,其次为光滑念珠菌(3.6％)、克柔念珠菌(2.4％)、热带念珠菌(2.4％)、近平滑念珠菌(1.2％);(2)统计学分析结果显示全身应用、吸入糖皮质激素是入院患者口腔念珠菌感染的独立危险因素.结论 白色念珠菌是COPD患者口腔部念珠菌感染的主要病原菌,规范使用全身糖皮质激素、正确吸入糖皮质激素是预防COPD患者口腔念珠菌感染的关键.