Periodontitis and postoperative inflammation in gastric cancer patients: Propensity score analysis.

OBJECTIVE: The objective of this study was to clarify the association between preoperative periodontitis and postoperative systemic inflammation in patients with gastric cancer. SUBJECTS AND METHODS: This retrospective cohort study analyzed data from 140 gastric cancer patients who underwent surgery at Hiroshima University Hospital between May 2019 and May 2022. Periodontal inflamed surface area (PISA) scores were determined to assess periodontitis severity using modified Nesse's methods. Propensity score matching was used to compare patients with high and low PISA scores (> or < the median PISA score of 92.4, respectively). Propensity scores were calculated using a logistic regression model, based on 17 clinical parameters: age, sex, smoking, alcohol consumption, hypertension, diabetes, dyslipidemia, cardiovascular disease, stroke, clinical stage, surgical procedure, surgical approach, neoadjuvant chemotherapy, surgery duration, blood loss during surgery, remaining teeth, and denture use. RESULTS: Thirty-seven patients were propensity-score-matched. Participants with high PISA scores had a higher incidence of surgical site infection (10.5%) than those with low PISA scores (5.3%). Moreover, participants with high PISA scores had significantly higher C-reactive protein levels on postoperative days 1 than those with low PISA scores. CONCLUSION: Preoperative periodontitis may determine the level of postoperative systemic inflammation in patients with gastric cancer.

Association between human papillomavirus and oral cancer: a literature review.

HPV plays a vital role in the development of cervical cancers and oropharyngeal cancers, but it is controversial whether HPV is involved in oral cancer development and to what extent. In this review, the clinical characteristics, diagnosis, treatment, and prognosis of HPV-positive oral cancers are summarized, and the mechanisms of HPV-related oral cancer development are discussed. HPV DNA positivity rates are 20-30% in oral squamous cell carcinoma (OSCC), and HPV16 is the most common high-risk HPV. E6/E7 mRNA positivity rates are 2-6% in OSCC. Detection of both high-risk HPV DNA and E6/E7 mRNA is recommended to determine the presence of active HPV, in agreement with high-risk HPV infection in OSCC. Surgical treatment is the first-line therapy for HPV-positive and -negative oral cancer, but there is no unified view about the prognosis of HPV-positive OSCC patients. HPV16 may play a vital role in malignant transformation in oral epithelial dysplasia, and a model of synergistic carcinogenic impact of HPV and tobacco smoking is predicted. Additionally, it is hypothesized that there are different HPV-associated oral cancers, such as integrated HPV DNA-positive OSCC with stable E6/E7 expression and episomal HPV DNA-positive OSCC. In summary, the role of HPV in oral carcinogenesis seems to be limited because of the low E6/E7 positivity in OSCCs; however, episomal HPV DNA may play a vital role in the malignant transformation of HPV-positive oral premalignant lesions. Further investigation is required to promote new insights into the role of episomal HPV DNA in oral carcinogenesis.

ACE2 expression and spike S1 protein-mediated immune responses in oral mucosal cells.

OBJECTIVES: ACE2, known as a host receptor involved with SARS-CoV-2 infection, binds to viral spike proteins for host cell entry. However, details regarding its induction and function in oral mucosal cells remain unknown. MATERIALS AND METHODS: We examined ACE2 expression and its induction by transfected mimic nucleotides and pro-inflammatory cytokines in oral keratinocytes (RT7) and fibroblasts (GT1). Subsequently, the effects of viral spike S1 protein via ACE2 on CXCL10 expression induced by pro-inflammatory cytokines in both cells were examined. RESULTS: ACE2 was constitutively expressed in RT7 and GT1. Transfected Poly(I:C) and Poly(dA:dT) increased ACE2 expression in those cells, while knockdown of RIG-I decreased ACE2 expression induced by those transfected ds nucleotides. IFN-γ and TNF-α enhanced transfected ds nucleotides-induced ACE2 expression in RT7 but not GT1. S1 protein alone did not affect CXCL10 expression in either cell type, whereas it enhanced IFN-ß-induced CXCL10 in both, while immune responses of IFN-γ- and TNF-α-induced CXCL10 enhanced by S1 protein were different between RT7 and GT1. Finally, knockdown of ACE2 decreased cytokines and S1 protein mediated-CXCL10 levels in both cells. CONCLUSIONS: ACE2 in oral mucosal cells may contribute to development of infection and inflammation in cooperation with pro-inflammatory cytokines following SARS-CoV-2 invasion.

Head and neck cancer patients show poor oral health as compared to those with other types of cancer.

PURPOSE: Several studies have found associations between periodontitis and various types of cancer. Since the site of head and neck cancer (HNC) has contiguity or proximity to the oral cavity, it may be particularly influenced by oral inflammation. This study aimed to determine whether HNC patients have poor oral health as compared to those with other types of cancer. METHODS: This study retrospectively examined oral environmental factors including periodontal inflamed surface area (PISA), a new periodontal inflammatory parameter. A total of 1030 cancer patients were divided into the HNC (n = 142) and other cancer (n = 888) groups. Furthermore, the HNC group was divided into high (n = 71) and low (n = 71) PISA subgroups, and independent risk factors affecting a high PISA value were investigated. RESULTS: Multivariate logistic regression analysis showed that number of missing teeth (odds ratio 1.72, 95% CI 1.15-2.56, P < 0.01), PISA (odds ratio 1.06, 95% CI 1.03-1.06, P < 0.05), and oral bacterial count (odds ratio 1.02, 95% CI 1.01-1.03, P < 0.01) were independent factors related to HNC. In addition, multivariate logistic regression analysis indicated that current smoker (odds ratio 7.51, 95% CI 1.63-34.71, P < 0.01) and presence of untreated dental caries (odds ratio 3.33, 95% CI 1.23-9.00, P < 0.05) were independent risk factors affecting high PISA values in HNC patients. CONCLUSION: HNC patients have higher levels of gingival inflammation and poor oral health as compared to patients with other types of cancer, indicating that prompt oral assessment and an effective oral hygiene management plan are needed at the time of HNC diagnosis.

Melatonin enhances cisplatin-induced cell death through inhibition of DERL1 in mesenchymal-like CD44high OSCC cells.

BACKGROUND: Melatonin is a hormone that is primarily produced in the pineal gland and is involved in wide range of biological functions. However, the impact of melatonin on chemotherapy-induced cell death remains to be elucidated in oral squamous cell carcinoma (OSCC) cells. The objective of this study was to clarify the role of melatonin in cisplatin-induced cytotoxicity in CD44high OSCC cells. METHODS: CD44high OSCC cells were cultured on fibronectin-coated hydrogel. A lactate dehydrogenase cytotoxicity assay was performed to evaluate cisplatin-induced cell death. The effect of melatonin on cisplatin-induced cell death and Derlin-1 (DERL1) endoplasmic reticulum membrane protein expression was investigated. RESULTS: CD44high OSCC cells exhibited mesenchymal-like features when cultured on fibronectin-coated hydrogel. Mesenchymal-like CD44high OSCC cells demonstrated strong resistance to cisplatin-induced cell death compared with epithelial-like CD44high OSCC cells. DERL1 mRNA and DERL1 protein expression levels were significantly higher in mesenchymal-like CD44high cells compared with epithelial-like CD44high cells. Cisplatin-induced cell death was significantly enhanced after DERL1 siRNA knockdown, suggesting that DERL1 is involved in resistance to cisplatin-induced cell death. Melatonin significantly inhibited DERL1 expression and enhanced cisplatin-induced cell death in mesenchymal-like CD44high cells. miR-181c-5p expression was significantly upregulated in the presence of melatonin. Furthermore, melatonin-inhibited DERL1 expression was significantly recovered by miR-181c-5p inhibitor. In addition, melatoninenhanced cisplatin-induced cell death was attenuated by miR-181c-5p inhibitor. These results suggest that melatonin-induced miR-181c-5p enhances cisplatin-induced cell death through inhibition of DERL1 in mesenchymal-like CD44high cells. CONCLUSIONS: Melatonin plays a vital role in promoting cisplatin-induced cytotoxicity in mesenchymal-like CD44high OSCC cells.

Immune response to cytosolic DNA via intercellular receptor modulation in oral keratinocytes and fibroblasts.

OBJECTIVE: Double-strand (ds) DNA-enveloped viruses can cause oral infection. Our aim is to investigate whether oral mucosal cells participate in immune response against cytosolic dsDNA invasion. METHODS: We examined the response to transfected herpes simplex virus (HSV) dsDNA via intracellular receptors in oral keratinocytes (RT7) and fibroblasts (GT1), and the effect of TNF-α on those responses. RESULTS: Transfected dsDNA increased CXCL10 expression via NF-κB activation in both cell types, while those responses were inhibited by knockdown of RIG-I, an RNA sensor. Although IFI16, a DNA sensor, was expressed in the nuclei of both types, its knockdown decreased transfected dsDNA-induced CXCL10 expression in GT1 but not RT7 cells. IFI16 in GT1 cells was translocated into cytoplasm from nuclei, which was attributed to immune response to cytosolic dsDNA. TNF-α enhanced transfected dsDNA-induced CXCL10, and knockdown of IFI16 decreased TNF-α and dsDNA-driven CXCL10 expression in both RT7 and GT1 cells. Finally, the combination of TNF-α and transfected dsDNA resulted in translocation of IFI16 from nuclei to cytoplasm in RT7 cells. CONCLUSION: RIG-I and IFI16 in oral mucosal cells may play important roles in host immune response against DNA viral infection, while TNF-α contributes to development of an antiviral system via those intracellular receptors.

Sunitinib promotes apoptosis via p38 MAPK activation and STAT3 downregulation in oral keratinocytes.

OBJECTIVE: Sunitinib, a targeted cancer drug, inhibits tyrosine kinases receptors and is widely used as first-line treatment for metastatic renal cell carcinoma. Patients undergoing chemotherapy with sunitinib frequently have oral mucosal complications, such as oral stomatitis, though cytotoxic effects of the drug on oral keratinocytes remain unknown. METHODS: The effects of sunitinib on immortalized oral keratinocytes, RT7 cells, in regard to cell injury and apoptosis, as well as apoptosis-mediated signaling pathways were investigated. RESULTS: Sunitinib treatment caused a significant increase in lactate dehydrogenase (LDH) in RT7 cells and primary oral keratinocytes. Additionally, the drug induced apoptosis-related events, such as DNA fragmentation, decreased anti-apoptotic Bcl-2 protein expression, and induction of cleaved PARP and caspase 3/9 in RT7 cells. Furthermore, phosphorylation of p38 MAPK, but not of ERK or JNK, was increased. On the contrary, constitutive phosphorylated STAT3 was decreased by sunitinib treatment, which was recovered by exposure to SB203580, a p38 MAPK inhibitor. Finally, SB203580 was found to reduce sunitinib-induced cell injury and apoptosis. CONCLUSION: The present results indicate that sunitinib promotes cell injury and apoptosis in oral keratinocytes via p38 activation and STAT3 downregulation. Sunitinib-mediated oral complications may be associated with cytotoxic effects of the drug on oral keratinocytes.

Co-Infection of Oral Candida albicans and Porphyromonas gingivalis Is Associated with Active Periodontitis in Middle-Aged and Older Japanese People.

Background and Objectives: Candida albicans can be detected in subgingival sites of patients with periodontitis. However, the association between oral Candida albicans and periodontitis has not been fully elucidated in Japanese adults. The aim of this study is to clarify the relationship between oral Candida albicans infection/co-infection of oral C. albicans and Porphyromonas gingivalis and periodontitis among middle-aged and older Japanese people. Materials and Methods: Eighty-six patients (mean age 70.4 years) who visited the Hiroshima University Hospital from April to September 2021 were investigated in this study. Oral swab samples were collected from the tongue surface. C. albicans and P. gingivalis DNA was detected by real-time PCR using specific DNA primer sets. C. albicans-positive participants were classified into two groups according to the presence or absence of intron insertion of C. albicans DNA by PCR analysis. Results: C. albicans was detected in 22 (25.6%) of the 86 patients. Patients in their 80s recorded a higher C. albicans-positive rate (35.3%) compared with other participants. However, there was no significant association between the C. albicans positivity rate and clinical parameters such as sex, age, systemic disease, denture use, or oral health status. Of the 22 C. albicans-positive participants, 10 participants (45.5%) had C. albicans with intron insertion; 70% of participants who had C. albicans with intron insertion exhibited ≥6 mm probing depth. C. albicans/P. gingivalis co-infection was found in 12 patients (14%). Importantly, binomial logistic regression analysis revealed that C. albicans/P. gingivalis co-infection was significantly associated with ≥6 mm periodontal pockets with bleeding on probing (p = 0.02). Conclusions: Co-infection of C. albicans and P. gingivalis is involved in active periodontitis in middle-aged and older people.

TGF-ß1 induces amoeboid-to-mesenchymal transition of CD44high oral squamous cell carcinoma cells via miR-422a downregulation through ERK activation and Cofilin-1 phosphorylation.

BACKGROUND: The objective of this study was to clarify the molecular mechanism of amoeboid-to-mesenchymal transition (AMT) of CD44high oral squamous cell carcinoma (OSCC) cells. METHODS: Morphology and expression of mesenchymal genes were investigated in CD44high OSCC cells (CD44high OM-1 cells) cultured on laminin-coated soft silicone gel. Additionally, microarray analysis was performed to investigate microRNA (miRNA) expression inhibited by transforming growth factor-ß1 (TGF-ß1) in CD44high OM-1 cells. RESULTS: When CD44high OM-1 cells were cultured on 2.0-kPa laminin-coated silicone gel, the cells exhibited an amoeboid-like round morphology. Cofilin-1 expression was found in the nucleus and cytoplasm of amoeboid-like CD44high OM-1 cells. The invasive capacity was significantly reduced after Cofilin-1 knockdown. Additionally, Cofilin-1 knockdown cells had an irregularly extended shape. Phosphorylated Cofilin-1 was significantly upregulated by TGF-ß1. Additionally, TGF-ß1 enhanced N-cadherin and Snail mRNA expression and induced a spindle-shaped morphology. ERK1/2 phosphorylation was induced by TGF-ß1. Microarray analysis revealed that miR-422a exhibited the greatest downregulation (fold change: 0.22) in the presence of TGF-ß1. Importantly, TGF-ß1-inhibited miR-422a expression was recovered by the ERK inhibitor or ERK1/2 knockdown. Additionally, miR-422a inhibitor-transfected CD44high OM-1 cells exhibited high N-cadherin and Snail mRNA expression. Furthermore, Cofilin-1 knockdown and miR-422a inhibition induced a spindle cell morphology. CONCLUSION: Cofilin-1 is involved in the invasive ability of CD44high OSCC cells. TGF-ß1 contributes to AMT by downregulation of miR-422a via ERK activation and Cofilin-1 phosphorylation. Our findings suggest that miR-422a and Cofilin-1 play major roles in the maintenance of amoeboid-like CD44high cells.

Effects of miR-224-5p-enhanced downregulation of pannexin-1 on docetaxel-induced apoptosis in amoeboid-like CD44high oral cancer cells.

We previously found that microRNAs play major roles in the maintenance of amoeboid-like oral squamous cell carcinoma (OSCC) cells with high expression of CD44 (CD44high ). However, the roles of microRNAs in chemotherapeutic resistance exhibited by CD44high amoeboid-like OSCC cells are unclear. Here, docetaxel-induced apoptosis was examined in CD44high OSCC cells (CD44high OM-1 cells) cultured on laminin-coated silicone gel. Amoeboid-like CD44high OSCC cells exhibited robust resistance to docetaxel-induced apoptosis and significant upregulation of miR-224-5p expression compared with epithelial-like CD44high OSCC cells and mesenchymal-like CD44high OSCC cells. The expression of pannexin-1 (PANX1), a channel-forming protein that regulates the release of ATP, was significantly upregulated following transfection of amoeboid-like CD44high OSCC cells with an miR-224-5p inhibitor. These results suggest that miR-224-5p inhibits PANX1 expression. Furthermore, miR-224-5p inhibitor-transfected amoeboid-like CD44high OSCC cells exhibited significant enhancement of the proportion of apoptotic cells; however, this effect was significantly inhibited by knockdown of PANX1 with PANX1 small interfering RNA. Additionally, the miR-224-5p inhibitor-enhanced extracellular ATP levels were significantly reduced by PANX1 knockdown. These findings imply that miR-224-5p plays a vital role in the resistance to docetaxel-induced apoptosis by attenuating PANX1-induced ATP discharge. Moreover, amoeboid-like CD44high OSCC cells may be involved in chemotherapeutic resistance of OSCC.

Effects of perioperative oral care on postoperative inflammation following heart valve surgery.

OBJECTIVE: Whether oral health care during the perioperative period can lead to a better outcome after heart valve surgery has not been adequately elucidated. We examined the effects of perioperative oral care on postoperative inflammation response in patients who underwent heart valve surgery. MATERIALS AND METHODS: In this retrospective cohort study, 223 patients scheduled for single valve heart surgery were divided into the oral care, who underwent professional teeth cleaning or scaling within 3 days prior to surgery, and also following surgery at least twice a week (n = 111), and non-oral care (n = 112) groups. After propensity score matching, records of both groups (80:80) were examined after surgery to evaluate inflammation markers (white blood cell count [WBC], neutrophil/white blood cell ratio [NWR], C-reactive protein [CRP] level, body temperature [BT]). RESULTS: WBC, NWR, CRP level, and BT were increased in both groups the day following surgery. Thereafter, CRP level, WBC, NWR, and BT on various days after surgery in the oral care group showed greater decreases as compared to the non-oral care group. CONCLUSIONS: Perioperative oral health care can decrease postoperative inflammation in patients undergoing heart valve surgery and may be important to ensure a better outcome in those patients.

Age-related changes in oral tactile and thermal sensation throughout adulthood.

Although the life expectancy of women is over 80 years in many countries, oral sensation has scarcely been compared between adults ≥ 80 years and younger age groups. The purpose of this study was to clarify age-related changes in oral sensation throughout adulthood. After exclusion of individuals with factors that might have confounded somatosensory performance, 123 female participants were divided into four age groups: 20-39 years, 40-59 years, 60-79 years, and 80-96 years. Perceptions of tactile and thermal sensations were examined at points on the anterior and posterior palate, anterior and posterior tongue, lower labial-attached gingiva, lower lip, and buccal mucosa; two-point discrimination was examined only on the tongue. The tactile and two-point discrimination thresholds for the anterior and posterior tongue were significantly higher in the 80-96-year-old group than in any other age group (p < 0.05). The tactile threshold for the buccal mucosa was significantly higher in the 80-96-year-old group than in the 60-79-year-old group (p < 0.05). The percentage of participants able to perceive a warm stimulus (50 °C) in the buccal mucosa was significantly lower in the 80-96-year-old group than in the 20-39-year-old group (p < 0.05). Only the topography of the warm sensation perception changed with age. This cross-sectional study suggests that oral tactile and thermal sensation for warm stimuli deteriorates with age in a site-specific manner, especially after the age of 80 years, but the same does not occur with cool stimuli.

Measurement of bristle splaying of toothbrushes using digital imaging and evaluation of plaque removal efficacy over 3 months: A randomized controlled trial (RCT).

OBJECTIVE: The aim of this study was to develop an objective method to assess the degree of bristle splaying of used manual toothbrushes and to investigate their plaque removal efficacy. METHODS: A randomized controlled trial targeting Hiroshima University students was performed to assess the plaque removal efficacy of polybutylene terephthalate (PBT) manual toothbrushes. Participants were randomly assigned to the soft toothbrush group (n = 40) or the medium toothbrush group (n = 40). A small number of participants discontinued the intervention for personal reasons in both the medium (n = 6) and soft (n = 2) toothbrush groups. Toothbrushes were collected immediately after first use (T0: baseline), after 1 month of use (T1: month 1), after 2 months of use (T2: month 3) and after 3 months of use (T3: month 6), following the allocation of a new toothbrush. The bristle surface area was measured using digital software. RESULTS: The surface area of the bristles was significantly greater at T1, T2 and T3 than at T0 in the medium toothbrush group (n = 34) and soft toothbrush group (n = 38) (P < .001). Importantly, plaque removal efficacy, calculated from a modified plaque control record score and modified patient hygiene performance score, was significantly lower at T2 than at T0 in both groups. CONCLUSIONS: Our method for evaluation of bristle splaying is considered to be reliable and reproducible. PBT toothbrushes may become less effective after two months of use.

Candida albicans ß-Glucan-Containing Particles Increase HO-1 Expression in Oral Keratinocytes via a Reactive Oxygen Species/p38 Mitogen-Activated Protein Kinase/Nrf2 Pathway.

Oral keratinocytes provide the first line of host defense against oral candidiasis. We speculated that interactions of fungal cell wall components with oral keratinocytes regulate the stress response against Candida infection and examined the expression of genes induced by heat-killed Candida albicans in oral immortalized keratinocytes using a cDNA microarray technique. Of 24,000 genes revealed by that analysis, we focused on HO-1, a stress-inducible gene, as its expression was increased by both heat-killed and live C. albicans In histological findings, HO-1 expression in the superficial layers of the oral epithelium following Candida infection was elevated compared to that in healthy epithelium. We then investigated fungal cell wall components involved in induction of HO-1 expression and found that ß-glucan-containing particles (ß-GPs) increased its expression. Furthermore, ß-glucan was observed on the surface of both heat-killed C. albicans and Candida cells that had invaded the oral epithelium. Fungal ß-GPs also promoted induction of intracellular reactive oxygen species (ROS), NADPH oxidase activation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation, while those specific inhibitors inhibited the HO-1 expression induced by fungal ß-GPs. Moreover, fungal ß-GPs induced Nrf2 translocation into nuclei via p38 MAPK signaling, while the HO-1 expression induced by fungal ß-GPs was inhibited by Nrf2-specific small interfering RNA (siRNA). Finally, knockdown of cells by HO-1- and Nrf2-specific siRNAs resulted in increased ß-GP-mediated ROS production compared to that in the control cells. Our results show that the HO-1 induced by fungal ß-GPs via ROS/p38 MAPK/Nrf2 from oral keratinocytes may have important roles in host defense against the stress caused by Candida infection in the oral epithelium.

CD44(high) /ALDH1(high) head and neck squamous cell carcinoma cells exhibit mesenchymal characteristics and GSK3ß-dependent cancer stem cell properties.

BACKGROUND: CD44 and aldehyde dehydrogenase 1 (ALDH1) have been shown to be useful markers for identification of cancer stem cells (CSCs). We previously reported that glycogen synthase kinase 3ß (GSK3ß) is involved in regulation of the self-renewal ability of head and neck squamous cell carcinoma (HNSCC) CSCs. The purpose of the present study was to clarify the role of GSK3ß in CD44(high) /ALDH1(high) HNSCC cells. METHODS: Cells with greater expression of CD44 and higher ALDH1 enzymatic activity were FACS sorted from the OM-1 HNSCC cell line. The self-renewal ability of CD44(high) /ALDH1(high) cells was then examined using a tumor sphere formation assay. mRNA expressions of the stem cell markers Sox2, Oct4, and Nanog, as well as GSK3ß were evaluated by real-time RT-PCR. RESULTS: CD44(high) /ALDH1(high) cells exhibited higher tumor sphere forming ability and increased expression of stem cell markers as compared with CD44(high) /ALDH1(low) cells. Interestingly, spindle-shaped cells positive for vimentin were found in the CD44(high) /ALDH1(high) but not the CD44(high) /ALDH1(low) cell population. In addition, the ALDH1 activity and sphere forming ability of CD44(high) /ALDH1(high) cells was significantly inhibited by GSK3ß knockdown. On the other hand, CD44(high) /ALDH1(low) cells exhibited high epidermal growth factor receptor (EGFR) expression and increased cell growth. CONCLUSIONS: Our results show that GSK3ß plays a major role in maintenance of stemness of CD44(high) /ALDH1(high) HNSCC cells. Additionally, they indicate a close relationship between CSC and mesenchymal characteristics in HNSCC.

Elevation in 5-FU-induced apoptosis in head and neck cancer stem cells by a combination of CDHP and GSK3ß inhibitors.

BACKGROUND: Cancer stem cells (CSCs) are involved in both tumourigenesis and in tumour recurrence after therapy. In head and neck squamous cell carcinoma (HNSCC), there are two biologically different CSC phenotypes both of which express high levels of CD44 but differ in their expression levels of epithelial-specific antigen (ESA). One phenotype is CD44(high)/ESA(high) and has epithelial features (Epi-CSCs), while the other is CD44(high) /ESA(low), has undergone epithelial to mesenchymal transition (EMT-CSCs), has mesenchymal features and is migratory (Biddle et al., 2011). CSCs are resistant to therapeutically induced apoptosis but the molecular mechanisms by which they develop apoptotic resistance remains unclear. However, glycogen synthase kinase 3ß (GSK3ß) contributes to regulation of both the self-renewal and switching of these two CSC phenotypes (Shigeishi et al., 2013). METHODS: CD44(high) /ESA(low), CD44(high) /ESA(high) and CD44(low) cells were FACS sorted from the HNSCC cell line LUC4, and 5-FU-induced apoptosis was analysed by Annexin V staining followed by flow cytometry analysis. RESULTS: CD44(high) /ESA(low) cells exhibited marked resistance to 5-FU-induced apoptosis and had high expression of dihydropyrimidine dehydrogenase (DPD). The DPD inhibitor, 5-chloro-2, 4-dihydroxypyridine (CDHP) significantly enhanced 5-FU-induced apoptosis of CD44(high)/ESA(low) cells. Inhibition of GSK3ß induced CD44(high) /ESA(low) cells to undergo mesenchymal-to-epithelial transition (MET) to CD44(high)/ESA(high) cells and pre-existing CD44(high) /ESA(high) cells to differentiate. Apoptosis induced by 5-FU was thus facilitated. Combination of both CDHP and GSK3ß inhibitors markedly enhanced 5-FU-induced apoptosis of CD44(high) /ESA(low) cells. CONCLUSIONS: Our results suggest potentially new approaches for the elimination of the therapy resistant HNSCC CSC population.

Expression and function of RIG-I in oral keratinocytes and fibroblasts.

BACKGROUND: Innate immune response by oral mucosal cells may be the first line of host defense against viral infection. Retinoic acid-inducible gene-I (RIG-I) recognizes viral dsRNA in the cytoplasm, and RIG-I-mediated signaling regulates antiviral type I IFN, and inflammatory chemokine production. Here, we tested the hypothesis that oral mucosal cell participation in host defense against viral infection via RIG-I. METHODS: RIG-I expression was detected in immortalized oral keratinocytes (RT7), oral fibroblasts (GT1) using and RT-PCR and immunohistochemistry. RT7 and GT1 were exposed to dsRNA virus mimic Poly I:C-LMW/LyoVec (PLV). Expression of IFN-ß and CXCL10 via RIG-I was examined by Real-time RT-PCR and ELISA. Phosphorylation of IRF3 and STAT1 were detected by western blotting. RESULTS: RT7 and GT1 constitutively expressed RIG-I in the cytoplasm. Furthermore, PLV increased IFN-ß and CXCL10 productions in both RT7 and GT1 via RIG-I concurrent with phosphorylation of IRF3 and STAT1. PLV-induced CXCL10 production was attenuated by neutralization of IFN-ß and blocking of IFN-α/ß receptor (IFNAR), indicating primal IFN-ß production via the RIG-I-IRF3 axis, which eventually induces CXCL10 production via the IFNAR -STAT1 axis. CONCLUSION: We propose that RIG-I in oral keratinocytes and fibroblasts may cumulatively develop host-defense mechanisms against viral infection in oral mucosa.

Maintenance of stem cell self-renewal in head and neck cancers requires actions of GSK3ß influenced by CD44 and RHAMM.

Cells sorted from head and neck cancers on the basis of their high expression of CD44 have high potency for tumor initiation. These cells are also involved in epithelial to mesenchymal transition (EMT) and we have previously reported that cancer stem cells (CSCs) exist as two biologically distinct phenotypes. Both phenotypes are CD44(high) but one is also ESA(high) and maintains epithelial characteristics, the other is ESA(low) , has mesenchymal characteristics and is migratory. Examining CD44-regulated signal pathways in these cells we show that CD44, and also RHAMM, act to inhibit phosphorylation of glycogen synthase kinase 3ß (GSK3ß). We show that inhibitory phosphorylation reduces the formation of both "tumor spheres" and "holoclone" colonies, functional indicators of stemness. GSK3ß inhibition also reduces the expression of stem cell markers such as Oct4, Sox2, and Nanog and upregulates expression of the differentiation markers Calgranulin B and Involucrin in the CD44(high) /ESA(high) cell fraction. Transition of CSCs out of EMT and back to the epithelial CSC phenotype is induced by GSK3ß knockdown. These results indicate that GSK3ß plays a central role in determining and maintaining the phenotypes and behavior of CSCs in vitro and are likely to be involved in controlling the growth and spread of tumors in vivo.

Prevalence of oral Capnocytophaga species and their association with dental plaque accumulation and periodontal inflammation in middle­aged and older people.

Capnocytophaga species are commonly found in human oral microbiome. The aim of the present study was to understand the association of the prevalence of oral Capnocytophaga species with oral hygiene and periodontal inflammation. A total of 136 patients (median age 72 years) who visited the Hiroshima University Hospital (Hiroshima, Japan) between April 2021 and June 2023 were enrolled. Swab samples were obtained from the tongue surface. DNA from Capnocytophaga species (C. ochracea and C. sputigena) was detected by real-time PCR analysis. Dental plaque accumulation was observed to assess the oral hygiene condition of participants. Additionally, clinical periodontal inflammation was assessed with periodontal inflamed surface area (PISA) scores. Clinical confounding factors such as age, sex, lifestyle-related disease, remaining teeth and denture wearing between Capnocytophaga species-positive and -negative groups were adjusted with a propensity score matching method. Mann-Whitney U and χ2 or Fisher's exact test were employed for statistical analysis. The prevalence rate was 67.6% for oral C. ochracea and 83.1% for C. sputigena. C. ochracea-positive participants showed significantly higher plaque control record scores (an indicator of dental plaque accumulation) than C. ochracea-negative participants (P=0.03). Additionally, C. ochracea/C. sputigena dual-positive participants exhibited significantly higher plaque control record and PISA scores than non-dual-positive participants (P=0.01 and P=0.04, respectively). Propensity score matching was conducted in the C. ochracea/C. sputigena dual-positive group and the non-dual-positive group for adjustment of clinical factors, resulting in 51 matched patient pairs. C. ochracea/C. sputigena dual-positive participants had significantly higher plaque control record scores than non-dual-positive participants (P=0.02). The present results suggest that the prevalence of both oral C. ochracea and C. sputigena is associated with poor oral hygiene in middle-aged and older people.

Relationship of oral bacterial number with medical hospitalization costs in analysis of Diagnosis Procedure Combination database from single institution in Japan.

Oral bacteria are known to be associated with perioperative complications during hospitalization. However, no presented reports have clarified the relationship of oral bacterial number with medical costs for inpatients. The Diagnosis Procedure Combination (DPC) database system used in Japan provides clinical information regarding acute hospital patients. The present study was conducted to determine the association of oral bacterial numbers in individual patients treated at a single institution with length of hospital stay and medical costs using DPC data. A total of 2369 patients referred by the medical department to the dental department at Hiroshima University Hospital were divided into the low (n = 2060) and high (n = 309) oral bacterial number groups. Length of hospital stay and medical costs were compared between the groups, as well as the associations of number of oral bacteria with Charlson comorbidity index (CCI)-related diseases in regard to mortality and disease severity. There was no significant difference in hospital stay length between the low (24.3 ± 24.2 days) and high (22.8 ± 20.1 days) oral bacterial number groups. On the other hand, the daily hospital medical cost in the high group was significantly greater (US$1456.2 ± 1505.7 vs. US$1185.7 ± 1128.6, P < 0.001). Additionally, there was no significant difference in CCI score between the groups, whereas the daily hospital medical costs for patients in the high group treated for cardiovascular disease or malignant tumors were greater than in the low number group (P < 0.05). Multivariate regression analysis was also performed, which showed that oral bacterial number, age, gender, BMI, cardiovascular disease, diabetes, malignant tumor, and hospital stay length were independently associated with daily hospitalization costs. Monitoring and oral care treatment to lower the number of oral bacteria in patients affected by cardiovascular disease or cancer may contribute to reduce hospitalization costs.