Exosome-Derived microRNAs from Mouthrinse Have the Potential to Be Novel Biomarkers for Sjögren Syndrome.

Sjögren syndrome (SS) is diagnosed based on invasive tissue biopsies and blood sampling. Therefore, a novel non-invasive and simple inspection diagnostic marker of SS is required. Here, we identified exosome-derived microRNAs (miRNAs) as biomarkers for SS using non-invasive mouthrinse samples collected from patients with SS and healthy volunteers. We compared miRNAs derived from exosomes in mouthrinse samples from the two groups using microarrays and real-time polymerase chain reaction (PCR) and identified 12 miRNAs as biomarker candidates. The expression ratios of four miRNAs were significantly increased in the SS group compared to the control group. Logistic regression analysis revealed a more significant influence of miR-1290 and let-7b-5p in the SS group than that in the control group. We combined these miRNAs to create a diagnostic prediction formula using logistic regression analysis. The combination of miR-1290 and let-7b-5p distinguished SS from the control samples with an AUC, sensitivity, specificity, positive predictive value, and negative predictive value of 0.856, 91.7%, 83.3%, 84.6%, and 90.9%, respectively. These results indicated that an increased ratio of these miRNAs could serve as a novel and non-invasive diagnostic marker for SS. This is the first report of diagnosis and screening of SS by adopting a non-invasive method using mouthrinse.

In vitro and in vivo removal of oral Candida from the denture base.

OBJECTIVES: To clarify the effectiveness of ultrasonic cleaning for removing Candida lodged in the denture base. MATERIALS AND METHODS: In vitro - Specimens of acrylic resin for denture plates contaminated with Candida cells were ultrasonically cleaned for 30 min. Washings were sampled every 5 min and cultured to investigate residual contamination, measured as colony forming units/ml, and the surfaces of the specimens were subjected to low-vacuum scanning electron microscopy (LV-SEM). In vivo - A total of 24 maxillary denture bases were brushed for 2 min under running tap water, then ultrasonically cleaned for 30 min. The washings were sampled every 5 min and cultured to investigate residual contamination. RESULTS: In vitro - Maximum removal was achieved during the first 5 min of cleaning, with the mean CFU/ml counts significantly declining over time. More than 85% of all Candida was removed within the first 15 min in specimens inoculated with phosphate-buffered saline suspensions of Candida albicans and Candida glabrata. LV-SEM revealed a decreased number of Candida on the surface of the specimens after 30 min of ultrasonic cleaning. In vivo - Maximum removal was achieved during the first 5 min of cleaning, then the mean CFU/ml count significantly declined during the first 10 min. Ultrasonic cleaning for 15 min removed 88.4% of Candida species from the denture base. CONCLUSIONS: Ultrasonic cleaning is a reliable and simple method for removing Candida lodged in the denture base.

Use of Candida-specific chicken egg yolk antibodies to inhibit the adhering of Candida to denture base materials: prevention of denture stomatitis.

OBJECTIVES: Polyclonal anti-Candida chicken egg yolk antibodies (anti-IgY) were used to investigate the prevention of adherence of Candida species to denture base material in vitro. BACKGROUND: Candida is a potential virulence factor that can cause systemic infection and even death in immunocompromised individuals. Because long-term antifungal treatment may lead to the emergence of drug-resistant strains, it is necessary to develop novel preventive measures and treatments for candidiasis. MATERIALS AND METHODS: Three types of chicken egg yolk antibodies were used in this study: non-specific antibody (control IgY), Candida albicans-specific antibody (anti-C.a.IgY) and Candida glabrata-specific antibody (anti-C.g.IgY). A mixture of different dilutions of each antibody with a suspension of Candida species and denture base material was incubated for 3 h, and then the colony-forming units of Candida on the denture base material were counted. RESULTS: Compared with control IgY, anti-C.a.IgY and anti-C.g.IgY significantly inhibited the adherence of C. albicans, but anti-C.a.IgY tended to be more potent than anti-C.g.IgY. The adherence of C. glabrata was also inhibited significantly by anti-C.a.IgY and anti-C.g.IgY with almost equivalent potency, indicating that their actions against C. glabrata were comparable. CONCLUSIONS: This study revealed the inhibitory effects of anti-C.a.IgY and anti-C.g.IgY against the adherence of C. albicans and C. glabrata to denture base material. This finding indicates the possibility of a beneficial effect of IgYs for the prevention of denture stomatitis and candidiasis in clinical settings.

Combination of MUC1 and MUC4 expression predicts clinical outcome in patients with oral squamous cell carcinoma.

BACKGROUND: Both MUC1 and MUC4 are high molecular weight glycoproteins and are independent indicators of worse prognosis in many human epithelial cancers including oral squamous cell carcinoma (OSCC). However, there has been no investigation of the clinical importance of the co-expression of MUC1 and MUC4 in OSCC. The aim of this study was to evaluate the co-expression profile of MUC1/MUC4 and analyze the prognostic significance in OSCC. METHODS: We examined the expression profile of MUC1 and MUC4 in OSCC tissues from 206 patients using immunohistochemistry. The co-expression profile of MUC1/MUC4 and its prognostic significance in OSCC was statistically analyzed. RESULTS: MUC1 and MUC4 overexpression were strongly correlated with each other (p < 0.0001) and a combination of both MUC1 and MUC4 expression was a powerful indicator for tumor aggressiveness such as tumor size (p = 0.014), lymph node metastasis (0.0001), tumor stage (p = 0.006), diffuse invasion (p = 0.028), and vascular invasion (p = 0.014). The MUC1/MUC4 double-positive patients showed the poorest overall and disease-free survival. Multivariate analysis revealed that MUC1/MUC4 double-positivity was the strong independent prognostic factor for overall and disease-free survival (p = 0.007 and (p = 0.0019), in addition to regional recurrence (p = 0.0025). CONCLUSIONS: Taken together, these observations indicate that the use of a combination of MUC1/MUC4 can predict outcomes for patients with OSCC. This combination is also a useful marker for predicting regional recurrence. MUC1 and MUC4 may be attractive targets for the selection of treatment methods in OSCC.

Factors associated with the presence of atrophic tongue in patients with dry mouth.

PURPOSE: This study aimed to identify factors associated with atrophic tongue in patients with dry mouth. METHODS: Discriminant analysis was performed in 1265 patients with dry mouth to identify factors that might influence the risk of developing atrophic tongue. The dependent variable was the presence of atrophic tongue, while patient age, resting saliva flow rate, stimulated saliva flow rate and Candida colony-forming units (CFU) were used as the independent variables. RESULTS: The standardised linear discriminant coefficients showed that Candida CFU, stimulated saliva flow rate and age were significantly associated with the presence of atrophic tongue. The following linear discriminant function was obtained: z = 0.024 × age - 0.63 × (resting saliva flow rate) - 0.81 × (stimulated saliva flow rate) + 0.002 × Candida CFU - 0.611. CONCLUSION: High Candida CFU, low stimulated saliva flow rate and advanced age were identified as closely associated factors for the risk of development of atrophic tongue.

Frequency of clinically isolated strains of oral Candida species at Kagoshima University Hospital, Japan, and their susceptibility to antifungal drugs in 2006-2007 and 2012-2013.

BACKGROUND: The isolation frequency and susceptibility to antifungal agents of oral Candida isolates from patients with oral candidiasis (OC) were compared between studies conducted in 2006-2007 and 2012-2013. METHODS: A total158 strains was isolated from 112 patients who visited Kagoshima University Hospital for the treatment of OC during the 14-month period from February 2012 and March 2013, and evaluated on the isolation frequency of each Candida strain and the susceptibility against antifungal drugs as compared to those evaluated in 2006-2007. RESULTS: There was a higher frequency of xerostomia as a chief complaint and of autoimmune disease in the 2012-2013 study than in the 2006-2007 study. More than 95% of Candida isolates were C. albicans and C. glabrata. In addition, the proportion of the latter increased from 12.3% in the 2006-2007 study to 23.4% in the 2012-2013 study, while the proportion of the former decreased from 86.2% to 72.8%, respectively. C. albicans was isolated in almost all patients, while C. glabrata was only isolated concomitantly with C. albicans. Minimal inhibitory concentrations (MICs) were not significantly different between groups with a few exceptions. Candida isolates, of which MICs surpassed break points, apparently increased for miconazole and itraconazole against C. glabrata in the 2012-2013 study, but this was not statistically significant. As a result, more cases of autoimmune disease, a greater number of C. glabrata isolates, and higher resistance to azoles were seen in the 2012-2013 study than in the 2006-2007 study. CONCLUSION: These data indicate that with recent increases in C. glabrata infection, a causative fungus of OC, and in C. glabrata resistance to azoles, caution is needed in the selection of antifungal drugs for the treatment of OC.

DF3 epitope expression on MUC1 mucin is associated with tumor aggressiveness, subsequent lymph node metastasis, and poor prognosis in patients with oral squamous cell carcinoma.

BACKGROUND: DF3/MUC1 mucin is expressed in various cancer tissues, and many in vitro studies have suggested that it may play a role in the aggressive behavior of malignant tumors. However, to the best of the authors' knowledge, the relation between DF3/MUC1 expression and outcome has not yet been investigated in patients with oral squamous cell carcinoma (OSCC). The objective of the current study was to evaluate the prognostic significance of DF3/MUC1 expression in patients with OSCC. METHODS: The expression profile of DF3/MUC1 in OSCC tissues from 206 patients was examined using immunohistochemistry. Its prognostic significance in OSCC was statistically analyzed on the basis of detailed clinicopathologic factors. RESULTS: DF3/MUC1 expression was found to be significantly correlated with tumor aggressiveness, such as pathologic lymph node metastasis (P = .002), advanced tumor stage (P = .02), diffuse invasion of cancer cells (P = .03), and vascular invasion (P = .01). Respectively, the overall survival (OS)and disease-free survival (DFS) rates were significantly worse for patients with DF3/MUC1 expression compared with those without DF3/MUC1 expression (P = .001 and P = .0003, respectively). Multivariate analysis demonstrated that DF3/MUC1 expression was an independent prognostic factor for both OS and DFS (P = .04 for both). In addition, DF3/MUC1 expression was found to be an independent risk factor for subsequent regional lymph node metastasis (P = .03). CONCLUSIONS: Aberrant expression of DF3/MUC1 is an independent prognostic factor indicating poor prognosis in patients with OSCC. DF3/MUC1 expression is a risk factor for subsequent lymph node metastasis in patients with OSCC and therefore may represent an indication for elective neck dissection. Patients with OSCC demonstrating positive expression of DF3/MUC1 should be followed carefully.

Aberrant DNA methylation of tumor-related genes in oral rinse: a noninvasive method for detection of oral squamous cell carcinoma.

BACKGROUND: The early detection of oral squamous cell carcinoma (OSCC) is important, and a screening test with high sensitivity and specificity is urgently needed. Therefore, in this study, the authors investigated the methylation status of tumor-related genes with the objective of establishing a noninvasive method for the detection of OSCC. METHODS: Oral rinse samples were obtained from 34 patients with OSCC and from 24 healthy individuals (controls). The methylation status of 13 genes was determined by using methylation-specific polymerase chain reaction analysis and was quantified using a microchip electrophoresis system. Promoter methylation in each participant was screened by receiver operating characteristic analysis, and the utility of each gene's methylation status, alone and in combination with other genes, was evaluated as a tool for oral cancer detection. RESULTS: Eight of the 13 genes had significantly higher levels of DNA methylation in samples from patients with OSCC than in controls. The genes E-cadherin (ECAD), transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains 2 (TMEFF2), retinoic acid receptor beta (RARß), and O-6 methylguanine DNA methyltransferase (MGMT) had high sensitivity (>75%) and specificity for the detection of oral cancer. OSCC was detected with 100% sensitivity and 87.5% specificity using a combination of ECAD, TMEFF2, RARß, and MGMT and with 97.1% sensitivity and 91.7% specificity using a combination of ECAD, TMEFF2, and MGMT. CONCLUSIONS: The aberrant methylation of a combination of marker genes present in oral rinse samples was used to detect OSCC with >90% sensitivity and specificity. The detection of methylated marker genes from oral rinse samples has great potential for the noninvasive detection of OSCC.

Comprehensive epigenetic analysis using oral rinse samples: a pilot study.

PURPOSE: To prove that chromatin immunoprecipitation assay can be performed with oral rinse samples and to develop a protocol for comprehensive analysis of functional interactions among DNA methylation, histone modification, and gene expression using such samples. MATERIALS AND METHODS: Eleven cancer cell lines and oral rinse samples from 10 patients with oral squamous cell carcinoma and 3 healthy subjects were examined. The expression of CDKN2A, a tumor suppressor gene, was determined by reverse transcription/polymerase chain reaction and immunohistochemistry. Promoter DNA methylation was assessed by methylation-specific polymerase chain reaction. Chromatin modifications were analyzed by a chromatin immunoprecipitation assay using antibodies for dimethylation and acetylation of lysine 9 of histone H3. RESULTS: Epigenetic control of CDK2NA was observed in vitro in 11 cancer cell lines. Using the present protocol, comprehensive epigenetic analysis could be successfully performed with oral rinse samples. All patients were comfortable using the prescribed amount (16 mL) of normal saline to rinse their mouths. Nine patients (90%) and 1 healthy subject (33%) showed dimethylation of lysine 9 of histone H3. Moreover, 8 patients (80%) showed hypoacetylation of lysine 9 of histone H3, which was not observed in healthy subjects. CONCLUSIONS: The present study showed for the first time that chromatin modifications can be analyzed using oral rinse samples by chromatin immunoprecipitation analysis. To evaluate the contribution of histone modifications for carcinogenesis of oral squamous cell carcinoma, studies including a larger number of subjects should be conducted in the future.

MUC4: a novel prognostic factor of oral squamous cell carcinoma.

MUC4 mucin is now known to be expressed in various normal and cancer tissues. We have previously reported that MUC4 expression is a novel prognostic factor in several malignant tumors; however, it has not been investigated in oral squamous cell carcinoma (OSCC). The aim of our study is to evaluate the prognostic significance of MUC4 expression in OSCC. We examined the expression profile of MUC4 in OSCC tissues from 150 patients using immunohistochemistry. Its prognostic significance in OSCC was statistically analyzed. MUC4 was expressed in 61 of the 150 patients with OSCC. MUC4 expression was significantly correlated with higher T classification (p = 0.0004), positive nodal metastasis (p = 0.049), advanced tumor stage (p = 0.002), diffuse invasion of cancer cells (p = 0.004) and patient's death (p = 0.004) in OSCC. Multivariate analysis showed that MUC4 expression (p = 0.011), tumor location (p = 0.032) and diffuse invasion (p = 0.009) were statistically significant risk factors. Backward stepwise multivariate analysis demonstrated MUC4 expression (p = 0.0015) and diffuse invasion (p = 0.018) to be statistically significant independent risk factors of poor survival in OSCC. The disease-free and overall survival of patients with MUC4 expression was significantly worse than those without MUC4 expression (p < 0.0001 and p = 0.0001). In addition, the MUC4 expression was a significant risk factor for local recurrence and subsequent nodal metastasis in OSCC (p = 0.017 and p = 0.0001). We first report MUC4 overexpression is an independent factor for poor prognosis of patients with OSCC; therefore, patients with OSCC showing positive MUC4 expression should be followed up carefully.

The assessment of cell proliferation during 9,10-dimethyl-1,2-benzanthracene-induced hamster tongue carcinogenesis by means of histone H3 mRNA in situ hybridization.

The aim of this study was to investigate cell kinetics and ultrastructural changes during carcinogenesis using a hamster 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tongue cancer model. Five squamous cell carcinomas, five dysplastic epithelia, seven hyperplastic epithelia, and four normal epithelia were obtained from 21 hamster tongues by applying 1.0% acetone solution of DMBA on the left lingual mucosa after scratching with a root canal broach. Ultrastructural examination revealed that the number of microvilli increased, whereas that of desmosomes decreased during carcinogenesis. Cell proliferation was analyzed by means of 5-bromodeoxyuridine (BrdU) immunohistochemistry and in situ hybridization (ISH) for histone H3 mRNA. The BrdU and histone H3 mRNA labeling indices (LIs) were lowest for normal epithelium, higher for hyperplastic and dysplastic epithelia, and highest for squamous cell carcinoma. Cytoplasmic histone H3 mRNA and nuclear BrdU were localized in virtually identical areas of serial sections. The correlation coefficient for the relationship between these two LIs was 0.97 ( P << 0.001). These results suggest that the assessment of cell proliferation using H3 mRNA ISH will be a useful technique for investigating biological behavior during carcinogenesis.

Histochemical, immunohistochemical, and ultrastructural studies of gingival fibromatosis: a case report.

Gingival fibromatosis is a rare disease characterized by enlargement of the gingiva. The purpose of this study was to analyze a case of idiopathic gingival fibromatosis, using histochemical and immunohistochemical staining and transmission electron microscopy. The patient was a 39-year-old Japanese man, in whom the gingiva was enlarged throughout the entire mandible and maxilla. Specimens of gingival fibromatosis exhibited epithelial hyperplasia and increased amounts of collagen fiber bundles in the connective tissue light-microscopically. Well-developed collagen bundles were strongly stained with Azan and Masson trichrome staining. Immunohistochemically, the gingival connective tissue was specifically stained by type I collagen and vimentin antibodies. Ultrastructurally, the lesion consisted of fibroblasts and mature collagen fibers running in all directions. No myofibroblasts were detected histochemically, immunohistochemically, or ultrastructurally. These findings suggested that this disease may be the result of an increase in collagen synthesis by the fibroblasts and/or that it may be associated with one of the findings of histologic heterogeneity.