Differences in the microbiota of oral rinse, lesion, and normal site samples from patients with mucosal abnormalities on the tongue.

The oral microbiota associated with mucosal diseases, including oral squamous cell carcinoma and oral potentially malignant disorders, have been extensively analyzed at the phylum and genus levels. However, the details of the oral microbiota remain unclear at the species and operational taxonomic unit (OTU) levels. We aimed to determine differences in the microbiota of oral rinse, lesion and normal site swab samples of patients with mucosal abnormalities on the tongues. Oral samples were obtained from 10 patients with oral mucosal abnormalities. Alpha and beta diversity at the OTU and genus levels of the microbiota samples were analyzed using OTUs clustered with 99.6% similarity based on 16S rRNA gene sequences obtained using the Sanger method. At the OTU level, the microbiota of the lesions were the least diverse but were different from those of the normal site and oral rinse samples. The OTUs corresponding to Streptococcus infantis and Haemophilus parainfluenzae were suggested to contribute to the differences between the microbiota of the lesions and normal sites. At the genus level, no significant differences between these microbiota were observed. In conclusion, strict OTU-level microbiota analysis might be able to discriminate lesions from normal sites of patients with mucosal abnormalities.

A Case of Non-hereditary White Sponge Nevus in the Oral Cavity Successfully Treated with Tetracycline Ointment and Oral Multivitamins.

White spongiform nevus is an autosomal dominant inherited disorder first reported by Cannon in 1935. It is a rare disease in which the oral mucosa thickens into an edematous and spongy state and is often accompanied by difficult to recognize subjective symptoms. We report a case of multiple non-hereditary white cavernous nevi in the oral mucosa. The subject was a 22-year-old man with a chief complaint of white lesions in his oral cavity. Examination revealed thick edematous and sponge-like white lesions on the bilateral buccal mucosa, upper and lower lip mucosa, and bilateral lingual margins. There was no history of similar lesions in his family or among his relatives. We diagnosed the case as non-hereditary white sponge nevus, based on clinical and histopathological findings. Although difficult to treat, the lesions disappeared with tetracycline ointment application and oral intake of multiple vitamin supplements. No recurrence of the lesion was observed thereafter.

Poor oral hygiene is associated with the detection of obligate anaerobes in pneumonia.

BACKGROUND: Oral cavity is a reservoir of various respiratory pathogens, and poor oral hygiene is associated with an increase in anaerobic bacteria in oral cavity. In addition, it positively relates higher risk of developing pneumonia and increased pneumonia-related mortality. However, the association between poor oral hygiene and increase in obligate anaerobes in the lungs of pneumonia patients is unclear. METHODS: A total of 39 patients with pneumonia in whom bronchoscopic examination and oral hygiene evaluation were performed were prospectively enrolled. The microbiota of the bronchoalveolar lavage fluid (BALF) directly obtained from the pneumonia lesion was analysed by the clone library analysis. In addition, oral hygiene evaluations were performed using oral hygiene index (OHI), tongue coating score, oral dryness, and community periodontal index of treatment needs (CPITN). The association between the detection of oral streptococci and obligate anaerobes and oral hygiene status was evaluated. RESULTS: Using the clone library analysis of BALF, the phylotypes of oral streptococci and obligate anaerobes were detected in 31 (79.5%) and 26 (66.7%) patients, respectively. Increased oral dryness, OHI, and CPITN, but not the tongue coating score, significantly correlated with higher rate of detection of obligate anaerobes, although no significant associations between the detection of oral streptococci in the lungs and each oral hygiene evaluation were observed. Significantly higher number of obligate anaerobes were detected in the lungs in patients with total oral hygiene score of ≥ 5 (P = 0.008). CONCLUSION: Poor oral hygiene is associated with increased obligate anaerobes in the lungs of patients with pneumonia.

Chronic mandibular osteomyelitis caused by Granulicatella adiacens in an immunocompetent child.

We report a pediatric case aged 10 years with Granulicatella adiacens-associated chronic mandibular osteomyelitis. The causative pathogen was uncertain because polymicrobial species were detected from the bacterial culture in bone marrow fluid. In contrast, G. adiacens was predominantly identified in the clone library analysis of the bacterial 16S rRNA gene sequence. Vancomycin to which G. adiacens was reported to be susceptible was not administrated sufficiently to this patient because of its adverse event, whereas linezolid and ciprofloxacin was alternatively effective for the treatment of chronic mandibular osteomyelitis.

[Treatment of Mandibular Fracture-Associated Infectious Pseudarthrosis Accompanied by Osteomyelitis in a Peritoneal Dialysis Patient].

Peritoneal dialysis can be performed at home, and the transfer of solutes in the blood and other body fluids is slow compared to hemodialysis, reducing the load on the circulatory organs and lessening the frequency of hospital visits. We encountered a male patient in his 70s on peritoneal dialysis for end-stage renal failure who developed obsolete mandibular fracture-associated pseudarthrosis accompanied by osteomyelitis, which was treated with noninvasive reduction and fixation using circumferential wiring after the resolution of inflammation. The inflammation was resolved by an intravenous drip infusion of ampicillin and lavage of the local region through the fistulated region during hospitalization, and sequestrum was removed under local anesthesia. After the disappearance of drainage from the fistula, the mandibular fracture was fixed with circumferential wiring (noninvasive reduction and fixation) using a mandibular resin base (occlusion is possible). For noninvasive reduction and fixation of a midline fracture, a 6-week fixation period is usually necessary after surgery, but in this case it was fixed for 3 months after surgery because of the presence of infection and bone defect. In jaw bone infection in patients on long-term dialysis, high sensitivity to infection and incomplete cure occur due to a decline in cell-mediated immunity, renal osteodystrophy (ROD), and chronic kidney disease (CKD)-mineral and bone disorder. In the present patient, infection complicated the odontogenic source of infection and fracture, which may have protracted the condition. When jaw bone infection is noted in a patient on long-term dialysis, it is important to closely cooperate with the dialysis physician and select the administration method and dose corresponding to the route of administration and metabolism of antimicrobial agents in order to minimize the influence on the renal function. For the local region, infection control by oral hygiene management and cleaning is important, targeting treatment and management while avoiding the use of any antimicrobial agent.

Strong expression of polypeptide N-acetylgalactosaminyltransferase 3 independently predicts shortened disease-free survival in patients with early stage oral squamous cell carcinoma.

The polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts) family of enzymes regulates the critical initial steps of mucin-type O-glycosylation. Among GalNAc-Ts that may significantly influence cancer biology, thus affecting cell differentiation, adhesion, invasion, and/or metastasis, GalNAc-T3 exhibits a high expression in several human cancers, closely associated with tumor progression and a poor prognosis. However, the expression pattern of GalNAc-T3 in oral squamous cell carcinoma (OSCC) remains obscure. Since postoperative recurrence of even early stage OSCC (ESOSCC) occurs at an early phase, significantly affecting their clinical course and worse outcome, the identification of clinically significant accurate biomarkers is needed. Therefore, we investigated the correlation between the immunohistochemical GalNAc-T3 expression and various clinicopathological characteristics and recurrence using 110 paraffin-embedded tumor samples obtained from patients with surgically resected ESOSCC (T1-2N0). Recurrence was recognized in 37 of 110 (33.6 %) patients. The GalNAc-T3 expression was considered to be strongly positive when 20 % or more of the cancer cells showed positive cytoplasmic staining. Consequently, a strong expression of GalNAc-T3 was observed in 40 patients (36.4 %), showing a close relationship to poor differentiation, the presence of lymphatic and vascular invasion, and recurrence. Univariate and multivariate analyses further demonstrated that the patients with a strong GalNAc-T3+ status had markedly lower disease-free survival (DFS) rates, especially within the first 2 years postoperatively. Therefore, GalNAc-T3 might play a role in the pathogenesis of ESOSCC recurrence, and its immunohistochemical detection potentially predicts a shorter DFS and may be a useful parameter for providing clinical management against ESOSCC in the early postoperative phase.

Intraoperative frozen section histological analysis of resection samples is useful for the control of primary lesions in patients with oral squamous cell carcinoma.

To ensure reliable surgical margins, intraoperative frozen section histological analysis (FS) has been performed since October, 2005 as follows: i) the orientation at the anatomical position and extent of the tumor are shared between oral pathologists and oral surgeons using imaging evaluations and pathological pictures and the planned site of sampling for intraoperative FS is confirmed; ii) a tumor team is organized and the team marks the tumor area and sets the resection range to correct the setting errors of the resection range among operators; iii) vital Lugol staining is applied to the lesion prior to tumor resection, the surgical margin is set based on the non-stained region and the extent of the tumor is macroscopically confirmed in the maximum cross-sectional surface of the resected specimen; and iv) FS is performed using samples from resected specimens to confirm the mucoepithelium and safety margin of the deep stump. The aim of this study was to evaluate the usefulness of our FS method. The treatment outcomes of oral squamous cell carcinoma were retrospectively investigated in patients treated prior to (Group 1) and after (Group 2) the introduction of our FS method. The recurrence rate of the primary lesions was high (17.3%) in Group 1, but decreased significantly in Group 2 (6.9%). Regarding clinicopathological factors, the condition of the surgical margins was associated with recurrence of the primary lesion in Group 1, but not in Group 2. In conclusion, our FS method appears to be useful for resecting tumors with reliable safety margins.

Notch signaling induces EMT in OSCC cell lines in a hypoxic environment.

Epithelial-mesenchymal transition (EMT) is an early step in the acquisition of invasiveness by malignant tumors. It has been clarified that the tumor microenvironment affects malignancy in a number of different carcinomas, in particular, that a hypoxic environment induces EMT. Activation of Notch signaling induces EMT, but it remains unclear how the Notch pathway is involved in oral squamous cell carcinoma (OSCC) under hypoxia. Three OSCC cell lines were cultured for examination under hypoxic (1% O2) and normoxic (21% O2) conditions. Expression of E-cadherin was investigated as a hallmark of EMT by immunohistochemical examination. Cell motility and invasion were examined by wound-healing and invasion assays, respectively. The expression of Notch pathway molecules was analyzed by qPCR. Hypoxia increased the mRNA expression of Notch receptors, ligands and target genes, and Snail. Hypoxia decreased the expression of E-cadherin, and increased the motility and invasiveness of OSCC cell lines. γ-secretase inhibitor, a Notch-specific inhibitor, prevented these effects caused by h-ypoxia. These findings suggest that hypoxia induces EMT in OSCC cell lines via activation of Notch signaling, and inhibition of the Notch signaling pathway to suppress EMT may be a useful approach for the treatment of OSCC.

A novel ameloblastoma cell line (AM-3) secretes MMP-9 in response to Wnt-3a and induces osteoclastogenesis.

OBJECTIVE: Ameloblastoma has a high risk of bone invasion and local recurrence. However, the mechanisms of bone invasion in ameloblastoma remain unclear. In this study, we established an experimental model for matrix metalloproteinase (MMP) induction and osteoclastogenesis using ameloblastoma-derived cells. STUDY DESIGN: We established an ameloblastoma-derived cell line without viral genes and analyzed the expression of all Wnt and Frizzled members and MMPs by real-time reverse transcription-polymerase chain reaction, and analyzed the activity of MMP-2 and MMP-9 by the in-gel-gelatinase assay. RESULTS: AM-3, newly established ameloblastoma-derived cells retained the morphology of primary-cultured ameloblastoma cells. AM-3 cells overexpressed the messenger RNA of Wnt-5a, Frizzled-2, MMP-2, and MMP-9 and showed the potential of osteoclastogenesis. In addition, Wnt-3a-treatment induced expression and activation of MMP-9 in AM-3 cells. CONCLUSIONS: Our study suggests that AM-3 cells retained the characteristics of ameloblastoma, without acquiring typical features of cancer cells. Furthermore, Wnt signaling induced MMP-9 in ameloblastoma cells.

The transcription factor Snail enhanced the degradation of E-cadherin and desmoglein 2 in oral squamous cell carcinoma cells.

Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity as well as the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail is a regulator of EMT that represses E-cadherin transcription through its interaction with proximal E-boxes in the promoter region of target genes. To investigate the role of Snail in EMT, we generated stable Snail transfectants using the oral squamous cell carcinoma cell line HSC-4 (Snail/HSC-4). Snail/HSC-4 cells had a spindle-shaped mesenchymal morphology, and enhanced migration and invasiveness relative to control cells. Consistent with these EMT changes, the downregulation of epithelial marker proteins, E-cadherin and desmoglein 2, and the upregulation of mesenchymal marker proteins, vimentin and N-cadherin were detected. Despite these observations, the mRNA levels of E-cadherin and desmoglein 2 did not decrease significantly. Although E-cadherin and desmoglein 2 proteins were stable in parental HSC-4 cells, these proteins were rapidly degraded in Snail/HSC-4 cells. The degradation of E-cadherin, but not desmoglein 2, was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis. Therefore, in HSC-4 cells Snail regulates levels of these proteins both transcriptionally and post-translationally.

Analysis of the outcome of concurrent neoadjuvant chemoradiotherapy with S-1 compared to super-selective intra-arterial infusion for oral squamous cell carcinoma.

We introduced concurrent neoadjuvant chemoradiotherapy (CCRT) with S-1, an oral fluoropyrimidine, as treatment for oral squamous cell carcinoma (OSCC) from October 2005. The clinical usefulness and medical safety of CCRT with S-1 (S-1 group) for OSCC were analyzed and compared with CCRT using super-selective intra-arterial infusion (AI group). The subjects in the S-1 group underwent external irradiation, at a total dose of 30 Gy, with S-1 chemotherapy. The AI group received cisplatin (CDDP) or carboplatin (CBDCA) combined with daily radiotherapy at a total dose of 40 Gy. The histological effects and disease-specific survival rates were almost equivalent in the S-1 and AI groups. Adverse events were less frequent in the S-1 group, while hematological toxicity, including anemia, thrombopenia and pharyngeal edema, was observed in the AI group. The results of this study indicate that CCRT combined with S-1 is a more effective and safer treatment for OSCC than AI.

SUVmax of FDG-PET correlates with the effects of neoadjuvant chemoradiotherapy for oral squamous cell carcinoma.

The aim of this study was to analyze the relationship between the maximum standardized uptake value (SUVmax) of 18F-fluoro-2-deoxyglucose-positron emission tomography (FDG-PET) and the effects of neoadjuvant chemoradiotherapy in oral squamous cell carcinoma (OSCC), and to identify the possible biological background of this association. Thirty-seven patients with OSCC, who underwent preoperative FDG-PET followed by cancer treatment with neoadjuvant chemoradiotherapy, were enrolled in this study. The various histological effects following neoadjuvant chemoradiotherapy were compared to the SUVmax in the primary OSCC. These effects were also compared to the immunohistochemical staining score of hypoxia-inducible factor-1alpha (HIF-1alpha), glucose membrane transporter (GLUT)-1 and vascular endothelial growth factor (VEGF) in the biopsy specimen. Furthermore, we analyzed the chemosensitivity of KB-3-1 cells to cisplatin under hypoxic conditions using the MTT assay. A negative correlation was observed between the SUVmax and the histological effects following neoadjuvant chemoradiotherapy (p<0.01). The SUVmax was also correlated with the staining score of HIF-1alpha (p<0.03), but not with GLUT-1 and VEGF. The mean staining score of HIF-1alpha in the highly effective group was 2.7+/-1.1, which was significantly lower than that (3.7+/-0.9) of the poorly effective group (p<0.05). The cell chemosensitivity assay revealed chemoresistant effects under a hypoxic condition in OSCC. In conclusion, the SUVmax is correlated with the effectiveness of neoadjuvant chemoradiotherapy in OSCC. Our clinical and experimental analyses further suggest a possible association of the upregulation of HIF-1alpha with chemoradiosensitivity in SCC cells.

Upregulation of Notch pathway molecules in oral squamous cell carcinoma.

The constitutive activation of the Notch pathway has been demonstrated in various types of malignancies. However, it remains unclear how the Notch pathway is involved in the pathogenesis of oral squamous cell carcinoma (OSCC). We investigated the expression of Notch pathway molecules in OSCC cell lines and biopsy specimens and examined the effect of Notch pathway inhibition. Reverse transcription-polymerase chain reaction revealed upregulation of Notch1, Notch2, Jagged1, HES1 and HEY1 in both OSCC cell lines and biopsy specimens. Immunohistochemical examination showed that the Notch intracellular domain accumulates in the nucleus of cells in OSCC cell lines and biopsy specimens. In addition, Jagged1 is expressed in the cytoplasm of cells in OSCC cell lines and biopsy specimens. Furthermore, Notch pathway inhibition using a gamma-secretase inhibitor prevented the growth of OSCC in vitro. These findings suggest that inhibition of the Notch pathway suppresses OSCC growth and may be a useful approach for the treatment of patients with OSCC.

Hyperosmotic stress up-regulates the expression of major vault protein in SW620 human colon cancer cells.

The major vault protein (MVP) is the major constituent of the vault particle, the largest ribonuclear protein complex described to date and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several normal tissues, little is known about its physiological role. MVP played a protective role against some xenobiotics and other stresses. We thus investigated the effect of osmotic stress on MVP expression by treating human colon cancer SW620 cells with sucrose or NaCl. The expression level of both MVP protein and MVP mRNA was increased by the osmostress. Sucrose or sodium chloride could also enhance MVP promoter activity. Inhibition of p38 MAPK in SW620 cells by SB203580 inhibited the expression of MVP under hyperosmotic stress. These findings suggested that osmotic stress up-regulated the MVP expression through p38 MAPK pathway. Down-regulation of MVP expression by MVP interfering RNA (RNAi) in SW620 cells increased the sensitivity of the cells to hyperosmotic stress and enhanced apoptosis. Furthermore, MVP RNAi prevented the osmotic stress-induced, time-dependent increase in phosphorylated Akt. These findings suggest that the PI3K/Akt pathway might be implicated in the cytoprotective effect of MVP. Our data demonstrate that exposure of cells to hyperosmotic stress induces MVP that might play an important role in the protection of the cells from the adverse effects of osmotic stress.