Expression of Cytokeratin 13, 14, 17, and 19 in 4-nitroquinoline-1-oxide-induced Oral Carcinogenesis in Rat.

The management of epithelial dysplastic spread around an oral squamous cell carci-noma is very important, particularly intraoperatively. Both cytokeratin (CK) 14 and CK19 are believed to be involved in the development of precancerous lesions, and their expression profiles are quite specific in these and early cancer lesions. Here, expression of CK13, 14, 17, and 19 was investigated in a rat model of 4-nitroquinoline-1-oxide-induced tongue cancer during a series of carcinogenetic processes to determine their value in assessing the features of epithelial dysplastic spread around a cancer. Based on tissue conditions, the results showed that expression levels of CK13 and 14 decreased in the order of no change, dysplasia, and cancer, whereas those of CK17 and 19 increased in the same order. Expression of CK13 showed a significant difference among no change, dysplasia, and cancer. This indicates that comparing the immunohistochemical staining profiles of CKs, especially CK13, could help in assessing the characteristics of epithelial dysplastic spread around a cancer.

Expression of Cytokeratin 14 and 19 in Process of Oral Carcinogenesis.

Cytokeratins (CK) are abundant in keratinized cells, particularly CK14 and CK19, which are expressed in stratified squamous epithelial cells. In this study, expression of CK14 and 19 was examined in human epithelial and dysplastic tissues. Surgical specimens from patients with clinically diagnosed oral leukoplakia or early cancer were stained with hematoxylin and eosin and classified into normal, low grade dysplasia (LGD), high grade dysplasia (HGD), or squamous cell carcinoma (SCC). The sections were examined by immunostaining and reverse transcription-polymerase chain reaction (RT-PCR) for CK14 and CK19. Expression and the results of RT-PCR for CK14 showed a decrease in the order of LGD, HGD, and SCC, whereas those of CK19 showed an increase in that order. These results suggest that decreased expression of CK14 and increased expression of CK19 serve as indicators of potential for malignant transformation.

Evaluation of superficial oral squamous cell malignancy based on morphometry and immunoexpression of cytokeratin 13 and cytokeratin 17.

OBJECTIVE: Evaluation of combined morphometry and immunoexpression of cytokeratin 13 (CK13) and cytokeratin 17 (CK17) for cytological identification of superficial oral squamous cells. STUDY DESIGN: Smears from 11 tongue squamous cell carcinoma patients were processed by liquid-based cytology, stained via the Papanicolaou method and divided into multiple specimens by cell transfer. Morphometric indices, including nuclear area, nuclear perimeter, nuclear circular rate, largest-to-smallest dimension ratio of the nucleus and nucleocytoplasmic ratio, were measured using a computerized analysis system. CK13 and CK17 were detected by immunostaining. Morphometric values were compared between cell populations with distinct staining and immunoexpression patterns. RESULTS: Most orange G-stained superficial cells were negative for CK13 (99.4%) and CK17 (98.6%). For light green-stained superficial cells, loss of CK13 was associated with greater cellular atypia in the nuclear area, nuclear perimeter and nucleocytoplasmic ratio (p < 0.01), while expression of CK17 was related to higher-grade cellular atypia in the same parameters (p < 0.01) as well as the nuclear circular rate (p < 0.05). CONCLUSION: Immunoexpression of CK13 and CK17 in light green-stained superficial cells was associated with more severe morphological atypia. Combined morphometry and immunoexpression of CK13 and CK17 might be useful for cytological diagnosis of this cell population.

Ventilator-associated pneumonia risk decreased by use of oral moisture gel in oral health care.

Although oral health care has a preventive effect against ventilator-associated pneumonia (VAP), the most effective method of oral health care in this respect remains to be established. The objective of this single-center, randomized, controlled trial was to investigate the relationship between VAP and various methods of oral health care. All patients included in the study (n=142) were on mechanical ventilation with oral intubation at the intensive care unit of the Tokyo Dental College Ichikawa General Hospital. They were divided into two groups, one receiving standard oral health care (Standard group), and the other receiving oral health care using an oral moisture gel instead of water (Gel group). After removal of the intubation tube, biofilm on cuff of the tube was stained with a disclosing agent to determine the contamination level. Factors investigated included sex, age, number of remaining teeth, intubation time, fever ≥38.5°C, VAP, cuff contamination level, and time required for one oral health care session. No VAP occurred in either group during the study period. The level of cuff contamination was significantly lower in the Gel group than the Standard group, and the time required for one session of oral health care was shorter (p<0.001). Multivariate analysis revealed use of the oral moisture gel as a factor affecting cuff contamination level. Use of an oral moisture gel decreased invasion of the pharynx by bacteria and contaminants together with biofilm formation on the intubation tube cuff. These results suggest that oral health care using an oral moisture gel is effective in preventing cuff contamination.

Head and neck contrast-enhanced CT for identification of internal carotid artery stenosis progression on the affected side after treatment for oral squamous cell carcinoma.

OBJECTIVES: To determine the incidence of progressive internal carotid artery (ICA) stenosis by head and neck contrast-enhanced computed tomography (CT) in 82 patients who underwent surgery, chemotherapy, or combination therapy for oral squamous cell carcinoma (OSCC). METHODS: The study included 82 patients who underwent head and neck contrast-enhanced CT after surgery alone or combined surgery and chemotherapy for OSCC at the Department of Oral and Maxillofacial Surgery of Ichikawa General Hospital, Tokyo Dental College, or Tokyo Dental College Oral Cancer Center between December 2002 and March 2010. RESULTS: Comparison with previously obtained head and neck contrast-enhanced CT images revealed progressive arterial stenosis of the ICA in five patients with a mean age of 62.0 years. All five patients were male, and their OSCC sites were the tongue in two, the floor of the mouth in two, and the mandibular gingiva in one. Tumor resection and neck dissection were performed for four patients and tumor resection alone for one patient. Four patients underwent chemotherapy. ICA stenosis occurred on the same side as the tumor in all five patients. CONCLUSIONS: The results of this study suggest that, given the possibility of post-treatment vascular events, attention must be paid to subsequent changes in the ICA over time. The results also indicate the usefulness of head and neck contrast-enhanced CT in identifying such problems.

Relationship between hyposalivation and acute respiratory infection in dental outpatients.

BACKGROUND: Hyposalivation may affect respiratory disease because the mouth serves as the entrance to the respiratory apparatus, as well as to the digestive tract. Patients with acute respiratory infection generally have a favorable prognosis and a short natural course. However, in cases in which the host has lowered resistance, such as in elderly patients, the infection may develop into pneumonia. OBJECTIVES: A prospective study was performed to examine the relationship between hyposalivation, which is common in elderly patients, and acute respiratory infection, which tends to become severe in elderly patients. METHODS: The subjects were 323 male and female patients ≥40 years old who lived in Utsunomiya City and surrounding areas and regularly visited the Department of Dentistry and Oral Surgery, Tochigi National Hospital. A 6-month follow-up survey was performed to examine development of acute respiratory infection. Age, sex, and known risk factors were also investigated. Hyposalivation was defined as a saliva production (saliva secretion rate) of ≤0.6 ml/min. Multivariate analysis adjusted for age and sex was performed to examine potential risk factors associated with the development of acute respiratory infection. RESULTS: Data were analyzed for 278 subjects who completed the follow-up survey. The incidence of acute respiratory infection was 60.4%, while hyposalivation was present in 96 subjects (35.5%). Multivariate analysis showed that the incidence of acute respiratory infection was higher in subjects with hyposalivation than in those without hyposalivation (adjusted odds ratio 1.761, p = 0.048). CONCLUSION: The results of this study suggest that hyposalivation may be a risk factor for acute respiratory infection. This also suggests that improvement of hyposalivation might prevent acute respiratory infection.

Visual and auditory stimuli associated with swallowing activate mirror neurons: a magnetoencephalography study.

In the present study, we evaluated activated areas of the cerebral cortex with regard to the mirror neuron system during swallowing. To identify the activated areas, we used magnetoencephalography. Subjects were ten consenting volunteers. Swallowing-related stimuli comprised an animated image of the left profile of a person swallowing water with laryngeal elevation as a visual swallowing trigger stimulus and a swallowing sound as an auditory swallowing trigger stimulus. As control stimuli, a still frame image of the left profile without an additional trigger was shown, and an artificial sound as a false auditory trigger was provided. Triggers were presented at 3,000 ms after the start of image presentation. The stimuli were combined and presented and the areas activated were identified for each stimulus. With animation and still-frame stimuli, the visual association area (Brodmann area (BA) 18) was activated at the start of image presentation, while with the swallowing sound and artificial sound stimuli, the auditory areas BA 41 and BA 42 were activated at the time of trigger presentation. However, with animation stimuli (animation stimulus, animation + swallowing sound stimuli, and animation + artificial sound stimuli), activation in BA 6 and BA 40, corresponding to mirror neurons, was observed between 620 and 720 ms before the trigger. Besides, there were also significant differences in latency time and peak intensity between animation stimulus and animation + swallowing sound stimuli. Our results suggest that mirror neurons are activated by swallowing-related visual and auditory stimuli.

Expression of myelin and lymphocyte protein (MAL) in oral carcinogenesis.

In the pathological diagnosis of oral squamous cell carcinoma, we often confront the difficulty of determining whether it is invasive carcinoma or epithelial dysplasia. Recently, myelin and lymphocyte protein (MAL; T-cell differentiation-related gene) has been reported to be a candidate gene suppressed in esophageal carcinoma. When we performed cDNA microarray analysis, we found that gene expression of MAL was significantly downregulated in oral squamous cell carcinoma (OSCC). We evaluated the expression of the MAL gene by laser microdissection and real-time PCR methods and protein localization by immunohistochemistry. The gene expression of MAL was significantly decreased in OSCC compared with normal epithelium (P < 0.05). Furthermore, protein expression of MAL disappeared gradually in proportion to malignancy. The results suggest that MAL plays an important role during oral carcinogenesis and that the gene may have potential as a biomarker target for OSCC.

Upper gastrointestinal tract cancers as double-cancers in elderly patients with oral squamous cell carcinoma.

Against a background of a rapidly aging society, the number of patients with oral cancers in Japan is increasing yearly. The number of double-cancers with oral cancer as the first malignancy is also reportedly on the rise. Esophageal and gastric cancers are the most common second malignancies. At our institution, our policy is to proactively perform upper gastrointestinal (GI) fiberscopy (GIF) in patients with oral cancer. In anticipation of a probable further increase in the number of patients with double-cancers consisting of oral and GI tract malignancies, we retrospectively analyzed the occurrence of upper GI tract cancers in patients with oral squamous cell carcinoma (OSCC). The cohort consisted of 171 patients in whom OSCC had been diagnosed and who had undergone upper GIF between March 1996 and August 2008. Multivariate analysis was performed. Upper GIF identified 8 patients (7 men, 1 woman, totaling 4.7% of 171 patients) with double-cancer in the upper GI tract. One patient had a triple malignancy consisting of oral, esophageal and gastric cancers. Seven patients had esophageal cancer, while two had gastric cancer. An age of over 65 years was significantly higher in patients with double-cancers including esophageal cancer than in patients without esophageal cancer (OR=10.454, 95% CI=1.143-95.621). None of the other analyzed patient factors (sex, smoking habit, drinking habit, site of OSCC, TNM classification, staging results) showed a significant difference. These results indicate that, when treating elderly patients with oral cancers, physicians need to devise suitable treatment plans which take into account the possibility of upper GI tract cancer, particularly esophageal cancer, as a double-cancer.

Two patients requiring surgical management for leakage of calcium hydroxide paste from root canal into infraorbital space.

Two patients requiring surgical management for leakage of calcium hydroxide paste from a root canal into the infraorbital space are reported. A paste root canal treatment material used at the time of maxillary root canal treatment had leaked out of the root canal in both patients. Computed tomography confirmed displacement of the root canal treatment material into the soft tissue, with extension into the infraorbital space. In both cases, foreign body removal was performed. Root canal treatment using a calcium hydroxide paste should be performed carefully without strong pressure.

Risk factors for postoperative dysphagia in oral cancer.

With the founding of its Oral Cancer Center at the Ichikawa General Hospital, Tokyo Dental College established a support system for patients and family members that not only provides surgery and other conventional cancer-oriented treatments, but also palliative care, nutritional support, rehabilitation, and discharge support. With this in mind, the present study sought to examine the nature of support for oral cancer patients with postoperative eating and swallowing disorders by investigating these disorders and identifying their risk factors. The study population comprised 75 surviving oral cancer patients (46 men and 29 women) discharged from the Tokyo Dental College Oral Cancer Center following treatment over a 2-year period from April 2009 to March 2011. Risk factors affecting eating and swallowing function were identified by statistical analysis. Mean age of the patients was 67.3±13.7 years. Fifteen patients had stage I cancer, while 25 had stage II, 13 had stage III, and 22 had stage IV. The feeding route at the time of discharge was oral feeding in 74 patients and a combination of oral and gastrostomy tube feeding in 1 patient. The Tokyo Dental College Ichikawa General Hospital has standardized the expert evaluation and rehabilitation of oral cancer patients with eating and swallowing disorders by establishing a multidisciplinary support system from the preoperative stage onwards. In this context, the results of our analysis of factors influencing the ability of oral cancer patients to orally ingest food after treatment suggest that preoperative cancer stage classification, neck dissection, and tracheotomy are all influential factors. Patients affected by these factors require further multidisciplinary treatment, which in turn necessitates more extensive coordination with other medical professionals and community health care providers.

Effect of maxillomandibular advancement on morphology of velopharyngeal space.

PURPOSE: The objectives of the present study were to assess the changes in upper airway morphology and function in response to advancement of the maxilla and mandible. PATIENTS AND METHODS: Orthognathic surgery was performed. During the surgery, the maxilla and mandible were each advanced as a maxillomandibular advancement simulation. A total of 18 patients with a chief complaint of malocclusion were studied. The distance in jaw advancement and the anteroposterior and left-right diameters of the velopharyngeal space before and after jaw advancement were measured. After the anteroposterior and left-right dilation rates and area enlargement rates were calculated, we compared advancement of the maxilla with that of the mandible. RESULTS: Each of the jaw advancements resulted in statistically significant increases in the anteroposterior and left-right diameters of the velopharyngeal space, and the area was significantly enlarged. The anteroposterior dilation rate was significantly greater after advancement of the maxilla, and the left-right dilation rate was significantly greater after advancement of the mandible. The velopharyngeal space area enlargement rate was significantly greater with advancement of the maxilla. CONCLUSIONS: These data suggest that the mode of dilation of the velopharyngeal space differs between maxillary advancement and mandibular advancement. Jaw advancement affects the soft palate muscles, and the velopharyngeal space is expanded 3-dimensionally by each of those muscles. The difference in the pattern of expansion of the velopharyngeal space was related to differences in the functions of the soft palate muscles.

Th1/Th2 balance in mouse delayed-type hypersensitivity model with mercuric chloride via skin and oral mucosa.

In order to compare delayed-type hypersensitivity (DTH) among different exposure sites, we evaluated the sensitization potency of mercuric chloride (HgCl(2)) via exposure to the skin, or oral or esophageal mucosa using the mouse ear swelling test. Furthermore, we investigated in vitro splenocyte proliferation reaction and cytokine profile in HgCl(2)-exposed and control mice. Sensitization with HgCl(2) was established via the skin and oral mucosa but not via the esophageal mucosa. The splenocyte proliferation reaction was significantly enhanced to a similar degree in skin and oral mucosa-sensitized mice compared with in the control mice. IL-10 levels from cultured splenocytes were significantly increased in skin and oral mucosa-sensitized mice compared with those in control mice, whilst IFN-γ significantly increased only in splenocytes from skin-sensitized mice. These results suggest that exposure of the skin or oral mucosa to HgCl(2) can induce DTH, but that Th1/Th2 balance differs according to the site of antigen exposure.

Splenocyte cytokine profile in mouse with oral mucosa-sensitization and oral-tolerization by NiSO(4).

Metals used in the oral cavity have been reported to cause various allergic diseases of the skin and mucosa. Skin manifestations due to dental restorations appear not only in the oral cavity, but also on the hands, feet or the whole body, as in the cases of pustulosis palmoplantaris and lichen planus. These phenomena implicate different pathogeneses from that of conventional skin sensitization and tolerance. Therefore, we compared skin and oral mucosa sensitization with nickel and oral tolerance for nickel in a mouse model. Female C57BL/6J mice were sensitized by injection of NiSO(4) into the skin or oral mucosa. Allergic reactions were evaluated by the mouse ear swelling test and splenocyte proliferation and cytokine profiles. Skin and oral mucosa sensitization succeeded in all mice. Ear swelling was significantly greater in the skin- than in the oral mucosa-sensitized mice at 48 hr after challenge. Ear swelling was also suppressed by single oral administration of NiSO(4) in both the skin- and oral mucosa-sensitized mice to the level of that in nonsensitized mice. Splenocytes from skin-sensitized mice proliferated similarly to those from oral mucosa-sensitized mice. Splenocytes from orally-tolerized mice also showed similar proliferation activity to those from skin and oral mucosa-sensitized mice. In the challenge phase, IL-2, IFN-γ, and IL-10 production was induced in splenocytes from both skin- and oral mucosa-sensitized mice. However, IL-4 was induced only in those from skin-sensitized mice. In addition, IL-4 in splenocytes from oral mucosa-sensitized mice was up-regulated to the level in those from skin-sensitized mice by oral tolerance. These results suggest that sensitization sites in mice influence not only the degree of excitation, but also Th-1 and Th-2 balance in the challenge phase and oral tolerance.

A case of drug-induced hypersensitivity syndrome due to carbamazepine.

The patient was a 51-year-old man who had been prescribed carbamazepine for right third-branch trigeminal neuralgia. He had stopped taking the medication after the neuralgia resolved. When the neuralgia recurred, he resumed medication, and about 1 month later he developed fever, fatigue, cervical lymphadenopathy, generalized skin flushing, facial edema and perioral vesicles, and was admitted to Ichikawa General Hospital, Tokyo Dental College. Oral findings showed reddening and erosion of the buccal mucosa. Routine laboratory examination revealed leukocytosis and hepatic dysfunction. Human herpesvirus 6 antibody titer remarkably increased during development of eruptions. These findings led to a diagnosis of drug-induced hypersensitivity syndrome. Carbamazepine was discontinued, and prednisolone (30 mg/day) was started and tapered based on improvement of symptoms. Because skin symptoms recurred after he was discharged 15 days after admission, the dose of prednisolone was increased and the symptoms finally disappeared. The patient has experienced no further recurrence.

Dental-care utilization patterns and factors associated with regular dental check-ups in elderly.

The purpose of this study was to investigate dental care utilization patterns in the elderly and to evaluate factors associated with regular dental check-ups in this age group. A total of 211 elderly people (50 men, 161 women) aged between 60 and 98 years were investigated. A cross-sectional survey by questionnaire was carried out on visitors to the Mihama Ikiiki Plaza (an institution for the elderly) in the city of Chiba, Japan between July and September, 2008. Items on the questionnaire included self-reported oral status, use of dentures, use of regular medication, oral problems, cleaning of teeth/dentures and dental care utilization patterns. The results showed that 135 (64.0%) of the responders had visited a dentist in the past year, 185 (87.7%) had visited the same dentist and 85 (40.3%) had had regular dental check-ups. A stepwise multiple logistic regression analysis adjusting for age and sex revealed that the factors associated with regular dental check-ups were significantly higher ADLs (odds ratio (OR)=0.439 in the partially insured and 0.192 in the fully insured in comparison with healthy subjects, p=0.0012), visiting the same dentist (OR=11.978 in comparison with not visiting the same dentist, p=0.0183) and cleaning teeth/dentures three or more times per day (OR=1.962 in comparison with cleaning them two or fewer times per day, p=0.0368). In this study, a higher ADL, visiting the same dentist and cleaning teeth/dentures three or more times per day were associated with having regular dental check-ups in the elderly. The best predictive factor for regular dental check-ups was a high ADL in the elderly.

Activation of PI3K-AKT pathway in oral epithelial dysplasia and early cancer of tongue.

Increasingly, cancers are being diagnosed at an early stage. Leukoplakia, a precancerous lesion, progresses to cancer in 5-10% of cases. We performed genetic analysis using cDNA microarray and immunohistochemistry in 6 patients, 3 with precancerous lesions and 3 with early tongue cancer, to evaluate the usefulness of these methods in the diagnosis of precancerous lesions and early cancer. Samples of normal epithelium, epithelial dysplasia, and cancer tissues were collected by laser microdissection, RNA was extracted, and the signals converted to numerical values. Immunohistochemical analysis was performed using antibody against phospho AKT (p-AKT), a component of the phosphoinositide 3-kinase (PI3K) signal pathway. Five genes showed a 2 times or greater level of increase in expression in epithelial tissue in comparison with in normal tissue, while 4 genes showed a 2 times or greater increase in early cancer tissues. In cancer tissues and epithelial dysplasia tissues, PI3K class III was expressed at 2.5 times and 11 times the level of that found in normal tissue, respectively. Histochemistry using p-AKT antibody revealed no positive cells in normal tissue. Positive cells were noted in the basal and parabasal cell layers, and partially in the spinous layer of epithelial dysplasia tissues, and in the spinous layer of early cancer tissues. These findings suggest that activation of the PI3K-AKT signal pathway is associated with oral carcinogenesis.

Visual and auditory stimuli associated with swallowing: an FMRI study.

We focused on brain areas activated by audiovisual stimuli related to swallowing motions. In this study, three kinds of stimuli related to human swallowing movement (auditory stimuli alone, visual stimuli alone, or audiovisual stimuli) were presented to the subjects, and activated brain areas were measured using fMRI and analyzed. When auditory stimuli alone were presented, the supplementary motor area was activated. When visual stimuli alone were presented, the premotor and primary motor areas of the left and right hemispheres and prefrontal area of the left hemisphere were activated. When audiovisual stimuli were presented, the prefrontal and premotor areas of the left and right hemispheres were activated. Activation of Broca's area, which would have been characteristic of mirror neuron system activation on presentation of motion images, was not observed; however, activation of brain areas related to swallowing motion programming and performance was verified for auditory, visual and audiovisual stimuli related to swallowing motion. These results suggest that audiovisual stimuli related to swallowing motion could be applied to the treatment of patients with dysphagia.

Barrier function and cytologic features of the ocular surface epithelium after autologous cultivated oral mucosal epithelial transplantation.

OBJECTIVE: To determine the barrier function and cytologic features of ocular surface epithelium after autologous cultivated oral mucosal epithelial transplantation in a prospective observational study. METHODS: The status of the epithelium in 4 eyes with limbal stem cell deficiency was studied preoperatively and postoperatively. We used an impression method to determine the cytologic features and anterior fluorophotometry to determine barrier function. RESULTS: Impression cytology showed nonkeratinized, squamous, polygonal, cohesive cells with a low nuclear to cytoplasmic cell ratio and no goblet cells, corresponding to cultivated oral mucosal epithelium, at up to 16 months after surgery. In some cases, the epithelium displayed a mixture of oral mucosal and conjunctival epithelium, especially in cases with a longer postoperative period. Central epithelial permeability remained persistently high throughout the follow-up period, regardless of the epithelial phenotype. CONCLUSIONS: Cultivated oral mucosal epithelial cells were observed to survive for more than 1 year after transplantation, with gradual replacement by conjunctival epithelium in some cases. Decreased barrier function of the transplanted epithelium may have prognostic implications, suggesting the presence of oral mucosal epithelium long after surgery.

Peroxynitrite formation in radiation-induced salivary gland dysfunction in mice.

Xerostomia frequently arises in patients with head and neck malignancies that are treated by radiation. However, the mechanisms responsible for the destruction of the salivary gland remain unknown. We previously established a xerostomia model of mice and identified the pathway through which nitric oxide (NO) affects the pathogenesis of radiation-induced salivary gland dysfunction. Although the toxicity of NO alone is modest, NO with superoxide anion (O2(*-)) rapidly forms peroxynitrite (ONOO), a more powerful toxic oxidant. In this study, we used the experimental model to examine: 1) when NO and O2(*-) production is maximum in the salivary gland after irradiation;2) whether peroxynitrite, as assessed by nitrotyrosine production, is responsible for salivary gland dysfunction; and 3) the effect of the iNOS selective inhibitor, aminoguanidine (AG), on nitrotyrosine formation. The increases in production of NO and O2(*-) in the salivary gland peaked on day 7 after irradiation. Nitrotyrosine detected immunohistochemically was significantly reduced by AG in the salivary gland. On the basis of these results, we concluded that NO together with O2(*-) forms the more reactive ONOO, which might be an important pathogenic factor in radiation-induced salivary gland dysfunction.