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.

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.

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.

LL-37-dsRNA Complexes Modulate Immune Response via RIG-I in Oral Keratinocytes.

Recognition of nucleic acids as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) promotes an inflammatory response. On the other hand, LL-37, an antimicrobial peptide, is a multifunctional modulator of immune response, though whether it modulates inflammatory responses induced by nucleic acids in oral keratinocytes is unknown. In this study, we firstly investigated the effect of LL-37 on CXCL10 induced by DAMPs and PAMPs in immortalized oral keratinocytes, RT7. Furthermore, the effects of LL-37 on translocation of exogenous nucleic acids into cytoplasm as well as cytosolic receptor, RIG-I on immune responses mediated by LL-37-nucleic acid complexes were examined. From these results, LL-37 enhanced necrotic cell supernatant (NCS)-induced CXCL10 expression in RT7, while the response was decreased by RNase. Complexes of LL-37 and double-stranded (ds) RNA, Poly(I:C) enhanced CXCL10 expression in comparison with each alone, which were associated with NF-κB activation. Furthermore, LL-37 was shown to bind with ds nucleotides and translocate into cytoplasm. Knockdown of RIG-I decreased expression of CXCL10 induced by LL-37-Poly(I:C) complexes, and RIG-I were co-localized with Poly(I:C) entered by LL-37 in cytoplasm. LL-37 modulates dsRNA-mediated inflammatory response via RIG-I in oral keratinocytes, which may play an important role in the pathogenesis of oral inflammatory diseases.

Effects of CEACAM1 in oral keratinocytes on HO-1 expression induced by Candida ß-glucan particles.

OBJECTIVE: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the carcinoembryonic antigen family. Although its expression has been found in chronic oral inflammatory epithelium, this study aimed to know whether CEACAM1 in oral keratinocytes participates in host immune response against Candida albicans . METHODOLOGY: We investigated CEACAM1 expression in oral keratinocytes induced by C. albicans as well as by Candida cell wall component ß-glucan particles (ß-GPs). Furthermore, the effects of CEACAM1 on ß-GPs-induced heme oxygenase-1 (HO-1) expression and its related signals were examined. RESULTS: Fluorescence staining showed CEACAM1 expression in oral keratinocytes (RT7) cells, whereas quantitative reverse transcription (RT)-PCR indicated that both live and heat-killed C. albicans increased CEACAM1 mRNA expression in RT7 cells. Examinations using quantitative RT-PCR and western blotting indicated that CEACAM1 expression was also increased by ß-GPs derived from C. albicans . Specific siRNA for CEACAM1 decreased HO-1 expression induced by ß-GPs from C. albicans as well as the budding yeast microorganism Saccharomyces cerevisiae . Moreover, knockdown of CEACAM1 decreased ß-GPs-induced ROS activity in the early phase and translocation of Nrf2 into the nucleus. CONCLUSION: CEACAM1 in oral keratinocytes may have a critical role in regulation of HO-1 for host immune defense during Candida infection.

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.

Inhibition of angiogenesis and tumor progression of MK-0429, an integrin αvß3 antagonist, on oral squamous cell carcinoma.

PURPOSE: Integrin αvß3 is an essential molecule for tumor angiogenesis. This study aimed to investigate the anti-tumor effect of MK-0429, an integrin αvß3 antagonist, on oral squamous cell carcinoma (OSCC) through its inhibitory effect on angiogenesis. METHODS: In this study, we investigated the effect of MK-0429 on cellular function and angiogenesis in vitro with the use of an immortalized human umbilical vein endothelial cell, HUEhT-1, which is immortalized by the electroporatic transfection of hTERT. The effect of MK-0429 on the integrin αvß3 signaling pathway was examined by FAK, MEK1/2 and ERK 1/2 phosphorylation. The anti-angiogenic effect of MK-0429 was evaluated by in vitro tube formation assay. The anti-tumor effect on OSCC was assessed by administrating MK-0429 to mouse oral cancer xenografts. RESULTS: MK-0429 inhibited cell proliferation, migration, and adhesion of HUEhT-1 in a dose-dependent manner. FAK, MEK and ERK phosphorylation were significantly blocked by MK-0429 treatment. Tube formation was suppressed by MK-0429 in dose-dependent manner. Tumor progression was significantly suppressed by MK-0429 administration in mouse oral cancer xenografts. Histological study revealed that MK-0429 decreased tumor vascularization. CONCLUSION: These results indicated integrin αvß3 as a therapeutic target for OSCC and suggested that MK-0429 might be clinically applicable as an anti-tumor agent with potent anti-angiogenic activity.

Expression of anti-fungal peptide, ß-defensin 118 in oral fibroblasts induced by C. albicans ß-glucan-containing particles.

OBJECTIVE: Although oral fibroblasts are thought to have the potential to enhance host defenses against Candida albicans , it is unknown whether they are able to recognize Candida cell components to increase the expression of antifungal peptides, such as defensin factors, against Candida infection. METHODOLOGY: We performed expression profiles of defensin genes induced by heat-killed C. albicans in oral immortalized fibroblasts (GT1) using cDNA microarray analysis. From those results, quantitative RT-PCR was used to examine the effects of Candida ß-glucan-containing particles (ß-GPs) on ß-Defensin 118 (DEFB 118) expression in oral mucosal cells. Furthermore, the antifungal activities of recombinant DEFB 118 against C. albicans and C. glabrata were investigated using fungicidal assays. RESULTS: Microarray analysis showed that DEFB118, ß-Defensin 129 (DEFB129), and α-Defensin 1 (DEFA1) genes were induced by heat-killed C. albicans and that their mRNA expressions were also significantly increased by live as well as heat-killed C. albicans . Next, we focused on DEFB118, and found that GT1, primary fibroblasts, and RT7 (oral immortalized keratinocytes) constitutively expressed DEFB118 mRNA expression in RT-PCR. Furthermore, C. albicans ß-GPs significantly increased the expression of DEFB118 mRNA in GT1 and primary fibroblasts. Although DEFB118 mRNA expression in RT7 was significantly induced by both live and heat-killed C. albicans, C. albicans ß-GPs failed to have an effect on that expression. Finally, recombinant DEFB118 significantly decreased the survival of both strains of C. albicans in a dose-dependent manner, whereas no effects were seen for both C. glabrata strains. CONCLUSION: DEFB118, induced by C. albicans ß-GPs from oral fibroblasts, may play an important role in oral immune responses against C. albicans infection.

Two PARP13 isoforms are associated with induction of antiviral factors in oral mucosal cells.

Innate immune systems in the oral cavity have important roles in the host defense against viral invasion of oral mucosa. Poly(ADP­ribose) polymerase 13 (PARP13), which has a strong antiviral ability, has been reported to possess two isoforms; a full­length protein, zinc­finger antiviral protein long (ZAPL), and a shorter protein (ZAPS). However, the expression and function of these two isoforms in oral mucosa remain unknown. In the present study, the expression levels of ZAPL and ZAPS induced by transfected double­stranded (ds) RNA, Poly(I:C), and dsDNA, Poly(dA:dT), in immortalized oral keratinocytes and fibroblasts (RT7 and GT1 cell lines, respectively) were investigated. Subsequently, the effects of the knockdown of ZAPL and ZAPS on transfected nucleotide­induced antiviral factors were examined. The results demonstrated constitutive expression of ZAPL and ZAPS in RT7 and GT1 cells, and their expression in both cell types was notably increased by transfection of Poly(I:C) and Poly(dA:dT) when compared with no transfection. Specific knockdown of ZAPL and ZAPS in RT7 cells decreased IFN­ß and C­X­C motif chemokine ligand 10 (CXCL10) expression induced by transfected Poly(I:C) and Poly(dA:dT). On the other hand, knockdown of ZAPL and ZAPS in GT1 cells decreased the expression of CXCL10 induced by the transfected nucleotides, whereas that had no effect on IFN­ß expression induced by Poly(dA:dT). Their knockdown was also associated with transfected nucleotides­induced IFN regulatory factor 3 phosphorylation in both cell types. Taken together, these results indicate that ZAPL and ZAPS, isoforms of PARP13, in oral mucosal cells participate in host defense against viral infection of oral mucosa.

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.

Effects of CEACAM1 in oral keratinocytes on HO-1 expression induced by Candida β-glucan particles

Abstract Objective Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the carcinoembryonic antigen family. Although its expression has been found in chronic oral inflammatory epithelium, this study aimed to know whether CEACAM1 in oral keratinocytes participates in host immune response against Candida albicans . Methodology We investigated CEACAM1 expression in oral keratinocytes induced by C. albicans as well as by Candida cell wall component β-glucan particles (β-GPs). Furthermore, the effects of CEACAM1 on β-GPs-induced heme oxygenase-1 (HO-1) expression and its related signals were examined. Results Fluorescence staining showed CEACAM1 expression in oral keratinocytes (RT7) cells, whereas quantitative reverse transcription (RT)-PCR indicated that both live and heat-killed C. albicans increased CEACAM1 mRNA expression in RT7 cells. Examinations using quantitative RT-PCR and western blotting indicated that CEACAM1 expression was also increased by β-GPs derived from C. albicans . Specific siRNA for CEACAM1 decreased HO-1 expression induced by β-GPs from C. albicans as well as the budding yeast microorganism Saccharomyces cerevisiae . Moreover, knockdown of CEACAM1 decreased β-GPs-induced ROS activity in the early phase and translocation of Nrf2 into the nucleus. Conclusion CEACAM1 in oral keratinocytes may have a critical role in regulation of HO-1 for host immune defense during Candida infection.

Expression of anti-fungal peptide, β-defensin 118 in oral fibroblasts induced by C. albicans β-glucan-containing particles

Abstract Objective: Although oral fibroblasts are thought to have the potential to enhance host defenses against Candida albicans , it is unknown whether they are able to recognize Candida cell components to increase the expression of antifungal peptides, such as defensin factors, against Candida infection. Methodology: We performed expression profiles of defensin genes induced by heat-killed C. albicans in oral immortalized fibroblasts (GT1) using cDNA microarray analysis. From those results, quantitative RT-PCR was used to examine the effects of Candida β-glucan-containing particles (β-GPs) on β-Defensin 118 (DEFB 118) expression in oral mucosal cells. Furthermore, the antifungal activities of recombinant DEFB 118 against C. albicans and C. glabrata were investigated using fungicidal assays. Results: Microarray analysis showed that DEFB118, β-Defensin 129 (DEFB129), and α-Defensin 1 (DEFA1) genes were induced by heat-killed C. albicans and that their mRNA expressions were also significantly increased by live as well as heat-killed C. albicans . Next, we focused on DEFB118, and found that GT1, primary fibroblasts, and RT7 (oral immortalized keratinocytes) constitutively expressed DEFB118 mRNA expression in RT-PCR. Furthermore, C. albicans β-GPs significantly increased the expression of DEFB118 mRNA in GT1 and primary fibroblasts. Although DEFB118 mRNA expression in RT7 was significantly induced by both live and heat-killed C. albicans, C. albicans β-GPs failed to have an effect on that expression. Finally, recombinant DEFB118 significantly decreased the survival of both strains of C. albicans in a dose-dependent manner, whereas no effects were seen for both C. glabrata strains. Conclusion: DEFB118, induced by C. albicans β-GPs from oral fibroblasts, may play an important role in oral immune responses against C. albicans infection.

Zoledronate Inhibits Osteoclast Differentiation via Suppressing Vascular Endothelial Growth Factor Receptor 2 Expression.

Bisphosphonate-related osteonecrosis of the jaw (ONJ) is a major oral complication; however, its pathogenesis remains unclear. Impairment of osteoclast differentiation by bisphosphonates may be associated with the pathogenesis of ONJ. In our previous study, we reported that the expression of the gene encoding nuclear factor of activated T cells c1 (NFATc1), a known osteoclast differentiation marker, was significantly silenced by zoledronate, a bisphosphonate, in mouse osteoclast precursor cells (mOCPCs) using cDNA microarray. In the present study, the expression value of the NFATc1 gene was regarded as a cut-off and genes whose expression value was significantly decreased compared with that of the NFATc1 gene were extracted in mOCPCs. For validation, CD11b-positive (CD11b+) cells were used, which were purified from human peripheral blood mononuclear cells as human OCPCs. A total of 19 genes were identified; sequential expression analysis revealed that the gene encoding vascular endothelial growth factor receptor 2 (VEGFR2) was frequently silenced by zoledronate in CD11b+ cells. Furthermore, the number of tartrate-resistant acid phosphatase-positive multinucleated cells was decreased by VEGFR2 suppression using a VEGFR2 neutralizing antibody. Zoledronate inhibits human osteoclast differentiation via suppressing VEGFR2 expression. These results suggest that low expression of VEGFR2 in OCPCs may be involved in the pathogenesis of zoledronate-induced ONJ. The understanding of the role of VEGFR2 on bone remodeling is important to elucidate the pathogenesis of bisphosphonate-related ONJ.

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.

Differential regulation by IFN­Î³ on TNF­α­induced chemokine expression in synovial fibroblasts from temporomandibular joint.

Tumor necrosis factor (TNF)­α and interferon (IFN)­Î³, are inflammatory cytokines in the synovial fluid of patients with temporomandibular joint disorder (TMD). However, it remains unknown whether they participate in the regulation of various chemokine expression levels associated with TMD. The effects of TNF­α and IFN­Î³ on the expression of several different inflammatory chemokines, including interleukin (IL)­8, C­X­C motif chemokine ligand (CXCL)1, C­C motif chemokine ligand (CCL)20, CXCL9, CXCL10, and CXCL11 in synovial fibroblasts obtained from the temporomandibular joint (TMJ) were examined. The results demonstrated that TNF­α increased the mRNA levels of all examined chemokines in synovial fibroblasts obtained from the TMJ. IFN­Î³ treatment alone increased the mRNA expression levels of CXCR3 chemokines, including CXCL10, while they were significantly enhanced when administered in combination with TNF­α compared with either treatment alone. However, the combination of IFN­Î³ and TNF­α resulted in lower mRNA expression levels of IL­8 and CXCL1 as compared with those induced by TNF­α alone. The nuclear factor­κB inhibitor, Bay 11­7082, decreased the TNF­α­mediated expression of IL­8 and CXCL10 in the absence, and presence of IFN­Î³. In addition, the JAK2 inhibitor, AG490, decreased CXCL10 expression when administered with TNF­α and IFN­Î³. Finally, the decrease in TNF­α­induced IL­8 caused by IFN­Î³ was recovered by AG490. The results of the present study suggest that TNF­α and IFN­Î³ function in a cooperative manner to regulate inflammatory chemokine expression in synovial fibroblasts, which may contribute to the pathological condition of the TMJ.

Preoperative oral health care reduces postoperative inflammation and complications in oral cancer patients.

The records of 70 patients with oral cancer who were treated at a single institution between 2008 and 2014 were reviewed. The body temperature, white blood cell count, and C-reactive protein (CRP) levels were compared between those who had received preoperative oral care (oral care group) and those who had not received any (non-oral care group). When the patients were divided into those who underwent minimally invasive surgery and those who underwent severely invasive surgery, the mean CRP level in the early postoperative period was lower in the oral care group as compared with the non-oral care group in those who underwent minimally invasive surgery as well as those who underwent severely invasive surgery. However, the mean CRP level was most evidently reduced in the severely invasive group on days 1 and 3-5. However, no significant differences were observed with regard to the percentage of postoperative infectious complications (for example, surgical site infection, anastomotic leak and pneumonia) between the oral care (13.6%) and non-oral care (20.8%) groups, though a reduced prevalence of postoperative complications following preoperative oral care was noted. The results of the present study suggest that preoperative oral care can decrease inflammation during the early postoperative stage in patients with oral cancer who undergo severely invasive surgery.

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.

Zoledronate inhibits receptor activator of nuclear factor kappa-B ligand-induced osteoclast differentiation via suppression of expression of nuclear factor of activated T-cell c1 and carbonic anhydrase 2.

Bisphosphonates (BPs) are widely used in the prevention of skeletal-related events (SRE), including osteoporosis, skeletal metastases of malignant tumours, and multiple myeloma. Osteonecrosis of the jaw (ONJ) is frequently reported as a major adverse effect induced by BP treatment. The receptor activator of the nuclear factor kappa-B ligand (RANKL) inhibitor, denosumab, has recently been used to prevent SRE, but the frequency of ONJ induced by denosumab is similar to that by BPs. This finding suggests that the inhibition of RANKL-mediated osteoclastogenesis may have a close relationship with the occurrence of ONJ. We therefore investigated the expression status of RANKL-inducible genes in zoledronate-treated mouse osteoclast precursor cells. The molecular targets of zoledronate in the RANKL signal pathway and additional factors associated with osteoclastogenesis were analysed by genome-wide screening. Microarray analysis identified that among 31 genes on 44 entities of RANKL-inducible genes, the mRNA expression level of two genes, i.e., nuclear factor of activated T-cells c1 (NFATc1) and carbonic anhydrase 2 (CAII), was decreased in zoledronate-treated cells. Subsequent analyses verified that these two genes were significantly silenced by zoledronate treatment and that their expression was restored following inhibition of zoledronate action by geranylgeraniol. Zoledronate inhibited RANKL-induced osteoclast differentiation by suppression of NFATc1 and CAII gene expression. Our results suggest that these genes might be common targets for zoledronate and denosumab in the mechanism underlying RANKL-induced osteoclast differentiation. A clear understanding of the common molecular mechanisms of bone-remodelling agents is thus essential for prevention of ONJ.

Clinicopathological analysis of salivary gland carcinomas and literature review.

Malignant salivary gland tumors are rare and exhibit a broad spectrum of phenotypic heterogeneity. The objective of this study was to investigate prognostic factors in patients with salivary gland carcinomas and review the results in light of other reports. We retrospectively reviewed 40 patients with primary salivary gland carcinomas who were diagnosed and treated at our institution between 1991 and 2014. Of the 40 tumors, 19 (47.5%) were mucoepidermoid carcinomas, 11 (27.5%) were adenoid cystic carcinomas, 7 (17.5%) were acinic cell carcinomas, 2 (5.0%) were myoepithelial carcinomas and 1 (2.5%) was a squamous cell carcinoma. Clinically positive lymph nodes were present in 4 patients (10.0%). As regards clinical stage, 15 cases (37.5%) were stage I, 13 (32.5%) were stage II, 1 (2.5%) was stage III and 11 (27.5%) were stage IVA. The majority of the patients (97.5%) were treated with surgery, of whom 25 (62.5%) received surgery alone and 14 (35.0%) underwent surgery in combination with chemotherapy or chemotherapy and radiotherapy. The median follow-up time for all the patients was 48 months. The disease-specific survival rate at 5 years was 87.1%. We identified a significant correlation between poor survival rate and histological grade (intermediate/high), tumor size (T3/T4), lymph node metastasis (node-positive) and clinical stage (III/IV) using the Kaplan-Meier method (P<0.05 for each). In addition, the Cox proportional hazards regression analysis confirmed that lymph node metastasis and tumor size were independent prognostic factors for disease-specific survival (hazard ratio = 18.7 and 15.1, respectively; P=0.023 and 0.037, respectively). Furthermore, tumor size was found to be a predictive factor regarding recurrence in the multivariate logistic regression analysis (odds ratio = 8.35; P=0.025). Our results suggest that lymph node metastasis and tumor size are significant prognostic factors for patients with salivary gland carcinomas.