Bacterial Lipopolysaccharide Induces PD-L1 Expression and an Invasive Phenotype of Oral Squamous Cell Carcinoma Cells.

BACKGROUND: Expression of programmed death ligand-1 (PD-L1) is related to the prognosis of many solid malignancies, including oral squamous cell carcinoma (OSCC), but the mechanism of PD-L1 induction remains obscure. In this study, we examined the expression of PD-L1 and partial epithelial-mesenchymal transition (pEMT) induced by bacterial lipopolysaccharide (LPS) in OSCC. METHODS: The expression of Toll-like receptor 4 (TLR4) recognizing LPS in OSCC cell lines was analyzed. Moreover, the induction of PD-L1 expression by Porphyromonas gingivalis (P.g) or Escherichia coli (E. coli) LPS and EMT was analyzed by western blotting and RT-PCR. Morphology, proliferation, migration, and invasion capacities were examined upon addition of LPS. PD-L1 within EXOs was examined. RESULTS: PD-L1 expression and pEMT induced by LPS of P.g or E. coli in TLR4-expressing OSCC cell lines were observed. Addition of LPS did not change migration, proliferation, or cell morphology, but increased invasive ability. Moreover, higher expression of PD-L1 was observed in OSCC EXOs with LPS. CONCLUSION: Oral bacterial LPS is involved in enhanced invasive potential in OSCC cells, causing PD-L1 expression and induction of pEMT. The enhancement of PD-L1 expression after addition of LPS may be mediated by EXOs.

Incidence of antiresorptive agent-related osteonecrosis of the jaw: A multicenter retrospective epidemiological study in Hyogo Prefecture, Japan.

Background/purpose: The incidence of medication-related osteonecrosis of the jaw is increasing worldwide, mostly due to the use of antiresorptive agents (ARAs) such as bisphosphonate (BP) and denosumab (Dmab). However, the proportion of BP-related osteonecrosis of the jaw (BRONJ) and Dmab-related osteonecrosis of the jaw (DRONJ) among all ARA-related osteonecrosis of the jaw (ARONJ) cases is not clear; this hinders appropriate treatment, recurrence-prevention planning, and avoidance of unnecessary Dmab withdrawal. Moreover, the causative drug administered at each disease stage remains unknown. Therefore, we conducted a retrospective study of patients with ARONJ who visited oral and maxillofacial surgery departments at hospitals in Hyogo Prefecture, Japan, over 3 years to classify and compare patient characteristics with those having BRONJ and DRONJ. We sought to identify the proportion of DRONJ in ARONJ. Materials and methods: After excluding stage 0 patients, 1021 patients were included (471 high-dose; 560 low-dose). ARA treatment for bone metastases of malignant tumors and multiple myeloma was considered high dose, while that for cancer treatment-induced bone loss and osteoporosis was low dose. Results: Low doses of BP and Dmab accounted for >50% patients; the results differed from those in other countries. DRONJ accounted for 58% and 35% of high-dose and low-dose cases, respectively. Stage 3 ARONJ cases comprised 92 (19.5%) low-dose BRONJ, 39 (20.1%) high-dose BRONJ, 24 (30%) low-dose DRONJ, and 68 (24.5%) high-dose DRONJ. Eighty-nine patients who received switch therapy were divided into BRONJ or DRONJ, but there was no difference in the ratio of each stage compared to the non-switch therapy. Conclusion: To the best of our knowledge, this is the first study to clarify the proportion of BRONJ and DRONJ cases, causative drug, and its doses by disease stages. DRONJ accounted for approximately 30% of the ARONJ, approximately 60% of which was due to high doses.

G­CSF delays tooth extraction socket bone healing via the inhibition of bone turnover in mice.

Granulocyte colony-stimulating factor (G-CSF) regulates the survival, proliferation and differentiation of all cells in the neutrophil lineage, and is consequently used for neutropenic conditions. Upon G-CSF administration, osteoblasts and osteocytes are suppressed, and the support system allowing hematopoietic stem cells to remain in the microenvironment is diminished. The present study focused on and investigated G-CSF as a regulatory factor of bone remodeling. The aim of the present study was to investigate the effect of G-CSF administration on the bone healing of tooth extraction sockets. Significant differences in the bone volume fraction, and trabecular separation of the proximal femurs and alveolar septa were observed between the G-CSF and control (saline-treated) groups. The trabecular bone of the femur and alveolar septa was reduced in the G-CSF group compared with that in the control group. In addition, serum procollagen type 1 N-terminal propeptide levels, a marker of bone formation, were lower in the G-CSF group compared with in the control group. Fibrous connective tissues and immature bone were observed in the extraction socket, and bone healing was delayed in the G-CSF group compared with that in the control group. The bone area in the extraction socket 6 days after tooth extraction was significantly smaller in the G-CSF group (23.6%) than that in the control group (45.1%). Furthermore, G-CSF administration reduced the number of canaliculi per osteocyte and inhibited the connection of osteocyte networks. Consequently, osteoblast activation was inhibited and bone remodeling changed to a state of low bone turnover in the G-CSG group. Analysis of bone formation parameters revealed that the G-CSF group exhibited a lower mineral apposition rate compared with in the control group. In conclusion, these findings indicated that G-CSF may delay bone healing of the socket after tooth extraction.

Usefulness of quantitative bone SPECT/CT for evaluating medication-related osteonecrosis of the jaw treatment response.

OBJECTIVE: For assessment of therapeutic response in medication-related osteonecrosis of the jaw (MRONJ) cases, the clinical usefulness of quantitative bone single-photon computed tomography-computed tomography (SPECT/CT) results was investigated. MATERIALS AND METHODS: Sixteen patients (18 lesions) with a clinical diagnosis of MRONJ underwent bone SPECT/CT scanning before and during/after anti-inflammatory therapy given for 3 or more months. The GI-BONE software package was used to determine standard uptake values (SUVs), including maximum (SUVmax), peak (SUVpeak), and mean (SUVmean), and metabolic bone volume (MBV) and also total bone uptake (TBU). In both responders (downstage) and non-responders (upstage or no change), differences in quantitative values between the first and second SPECT/CT examinations were analyzed using a Wilcoxon test. RESULTS: Following therapy, significant reductions in SUVmax, SUVpeak, SUVmean, MBV and TBU values for 11 lesions were noted in the responders after therapy (p = 0.003, p = 0.006, p = 0.004, p = 0.003, and p = 0.002, respectively). On the other hand, those for the seven lesions in the non-responder group were not significantly different (p = 0.17, p = 0.16, p = 0.26, p = 0.96, and p = 0.12, respectively). Results for SUVmax change showed sensitivity and specificity values of 45.5% and 85.7%, respectively, for differentiating responders from non-responders, with - 37.3% the optimal cutoff value. Those for MBV change were 72.7 and 85.7%, respectively, with - 29.4% the optimal cutoff value. Those for TBU change were 81.8% and 85.7%, respectively, with - 36.3% the optimal cutoff value. CONCLUSION: The present findings showed that therapeutic response in MRONJ cases could be determined by use of quantitative SUV, MBV, and TBU values based on bone SPECT/CT findings.

Characterization of the Second Apoptosis Inhibitor Encoded by Guinea Pig Cytomegalovirus.

Despite the usefulness of guinea pig cytomegalovirus (GPCMV) for studies on congenital CMV infection, its viral mechanisms for the evasion of host defense strategies have not been fully elucidated. We reported previously that GPCMV gp38.1 functions as a viral mitochondria-localized inhibitor of apoptosis-like function, and its weak activity suggested the presence of an additional inhibitory molecule(s). Here, we identified gp38.3-2, a 42-amino-acid (aa) reading frame embedded within the gp38.3 gene that encodes a positional homolog of murine CMV (MCMV) m41. Characterization of gp38.3-2 resulted in the following findings: (i) the aa sequence of gp38.3-2 shows some similarity to that of MCMV m41.1, a viral inhibitor of oligomerization of a member of Bcl-2 family protein BAK, but there is no correspondence in their predicted secondary structures; (ii) gp38.3-2, but not gp38.3, showed inhibitory activities against staurosporine-induced apoptosis; (iii) three-dimensional protein complex prediction suggests that the N-terminal α-helix of gp38.3-2 interacts with residues in the BH3 and BH1 motifs of BAK, and analysis of gp38.3-2 and BAK mutants supported this model; (iv) guinea pig fibroblast cells infected with gp38.3-2-deficient GPCMV strain Δ38.3-2 died earlier than cells infected with rescued strain r38.3-2, resulting in lower yields of Δ38.3-2; (v) Δ38.3-2 exhibited a partial but significant decrease in monocyte and macrophage infection in comparison with r38.3-2; and, however, (vi) little difference in the viral infection of guinea pigs was observed between these two strains. Therefore, we hypothesize that gp38.3-2 contributes little to the evasion of host defense mechanisms under the experimental conditions used. IMPORTANCE Although GPCMV provides a useful animal model for studies on the pathogenesis of congenital CMV infection and the development of CMV vaccine strategies, our understanding of the viral mechanisms by which it evades apoptosis of infected cells has been limited in comparison with those of murine and human CMVs. Here, we report a second GPCMV apoptosis inhibitor (42 amino acids in length) that interacts with BAK, a Bcl-2 family proapoptotic protein. Three-dimensional structural prediction indicated a unique BAK recognition by gp38.3-2 via the BH3 and BH1 motif sequences. Our findings suggest the potential development of BH3 mimetics that can regulate inhibition or induction of apoptosis based on short ~40-amino-acid peptide molecules as with GPCMV.

Establishment of an oral squamous cell carcinoma cell line expressing vascular endothelial growth factor a and its two receptors.

Background/purpose: Vascular endothelial growth factor receptor (VEGFR) expression in oral squamous cell carcinoma (OSCC) promotes tumor growth through both autocrine and paracrine signaling. VEGF-positive OSCC cases are associated with a high depth of invasion, increased metastasis, and poor prognosis. In this study we established and then molecularly and functionally analyzed an OSCC cell line that co-expresses VEGF-A, VEGFR-1, and VEGFR-2, termed HCM-SqCC010 cells. Materials and methods: VEGF-A, VEGFR-1, and VEGFR-2 expression in HCM-SqCC010 cells were examined by immunohistochemistry and immunoblotting. Expression and inhibition of VEGF-A, VEGFR-1, and VEGFR-2 in HCM-SqCC010 cells were verified by quantitative real-time PCR. Results: Our analysis of HCM-SqCC010 cells revealed that their proliferation depended on VEGF-A, and selective inhibition of VEGFR-1 or VEGFR-2 resulted in decreased cell growth. Conclusion: We established an OSCC cell line, HCM-SqCC010, that expresses VEGF-A, VEGFR-1, and VEGFR-2. This triple-positive cell line showed no effect from a molecular targeted drug toward VEGF-A, but it did show strong cell growth inhibition in response to a VEGFR inhibitor. Thus, new therapeutic strategies against OSCC should include a VEGFR inhibitor.

Whole blood transcriptome profiling identifies gene expression subnetworks and a key gene characteristic of the rare type of osteomyelitis.

Chronic non-bacterial osteomyelitis (CNO) is a rare and severe inflammatory bone disorder that can occur in the jaw. It is often associated with systemic conditions including autoimmune deficiency. Medical management of patients and establishment of a correct diagnosis are difficult as the etiology of the disease remains unknown. Therefore, little is known about the disease characteristics at the gene expression level. Here, we explored aspects of CNO based on whole blood RNA sequencing (>6 Gb per sample) of 11 patients and 9 healthy controls in Japan and on a recently developed method that is applicable to small datasets, can estimate a directed gene network, and extract a subnetwork of genes underlying patient characteristics. We identified nine subnetworks, comprising 26 differentially regulated edges and 36 genes, with the gene encoding glycophorin C (GYPC) presenting the highest discrimination ability. The expression of the gene was mostly lower in patients with CNO than in the healthy controls, suggesting an abnormal status of red cells in patients with CNO. This study enhances our understanding of CNO at the transcriptome level and further provides a framework for whole blood RNA sequencing and analysis of data obtained for a better diagnosis of the disease.

Protocol for a multicentre, prospective observational study of elective neck dissection for clinically node-negative oral tongue squamous cell carcinoma (END-TC study).

INTRODUCTION: In early-stage oral tongue squamous cell carcinoma (OTSCC), elective neck dissection (END) is recommended when occult lymph node metastasis is suspected; however, there is no unanimous consensus on the risks and benefits of END in such cases. The management of clinically node-negative (cN0) OTSCC remains controversial. This study, therefore, aimed to evaluate the efficacy of END and its impact on the quality of life (QoL) of patients with cN0 OTSCC. METHODS AND ANALYSIS: This is a prospective, multicentre, nonrandomised observational study. The choice of whether to perform END at the same time as resection of the primary tumour is based on institutional policy and patient preference. The primary endpoint of this study is 3-year overall survival. The secondary endpoints are 3-year disease-specific survival, 3-year relapse-free survival and the impact on patient QoL. Propensity score-matching analysis will be performed to reduce selection bias. ETHICS AND DISSEMINATION: This study was approved by the Clinical Research Review Board of the Nagasaki University. The protocol of this study was registered at the University Hospital Medical Information Network Clinical Trials Registry. The datasets generated during the current study will be available from the corresponding author on reasonable request. The results will be disseminated internationally, through scientific and professional conferences and in peer-reviewed medical journals. TRIAL REGISTRATION NUMBER: UMIN000027875.

Human Papillomavirus 16 E6 Suppresses Transporter Associated with Antigen-Processing Complex in Human Tongue Keratinocyte Cells by Activating Lymphotoxin Pathway.

Infection by high-risk human papillomaviruses (hrHPVs), including HPV type 16 (HPV16), is a major risk factor for oral squamous cell carcinomas (OSCCs). However, the pathogenic mechanism by which hrHPVs promote oral carcinogenesis remains to be elucidated. Here, we demonstrated that the suppression of a transporter associated with the antigen-processing complex (TAPs; TAP1 and TAP2), which is a key molecule in the transportation of viral antigenic peptides into MHC class-I cells, is affected by the E6 protein of HPV16. Mechanistically, HPV-mediated immune evasion is principally mediated via the signal-transduction network of a lymphotoxin (LT) pathway, in particular LTα1ß2 and LTßR. Our analysis of transcriptomic data from an HNSCC cohort from the Cancer Genome Atlas (TCGA) indicated that expression of TAP genes, particularly TAP2, was downregulated in HPV-infected cases. We further demonstrated that LTα1ß2 and LTßR were upregulated, which was negatively correlated with TAP1 and TAP2 expression in HPV-positive clinical OSCC samples. Taken together, our findings imply that HPV16 E6 regulates the machinery of the antigenic peptide-loading system and helps to clarify the role of oncogenic viruses in the context of oral carcinoma.

Establishment of a patient-derived mucoepidermoid carcinoma cell line with the CRTC1-MAML2 fusion gene.

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor of the major and minor salivary glands. Surgical resection is the only curative treatment and there is no effective post-operative therapy for MEC. The present study reports an Institutional Review Board-approved case of a 45-year-old Japanese female diagnosed with low-grade MEC in the hard palate. Radical resection, supraomohyoid neck dissection and antero-lateral thigh flap reconstruction was performed. A MEC cell line was then established from the resected tumor tissue. Short tandem repeat profiling confirmed the origin and authenticity of the cell line, that harbors a CRTC1-MAML2 translocation, which is frequently observed in MEC. Amphiregulin (AREG), identified as one of the targets of the CRTC1-MAML2 fusion gene, was expressed in the cell line. The AREG receptor, epidermal growth factor receptor (EGFR) was also highly phosphorylated. The results predicted that AREG-EGFR signaling, which is required for tumor growth and survival, might be activated in the cell line in a cell-autonomous manner. As AREG expression is associated with EGFR-targeted drug resistance, this cell line might assist with the identification of novel strategies for MEC treatment.

Usefulness of quantitative bone SPECT/CT for medication-related osteonecrosis of the jaw in clinical settings.

OBJECTIVE: This study was conducted to investigate the clinical utility of quantitative bone single-photon computed tomography-computed tomography (SPECT/CT) for detection and classification for medication-related osteonecrosis of the jaw (MRONJ). MATERIALS AND METHODS: Fifty-nine patients (69 lesions) clinically diagnosed as MRONJ by four specialists of Japanese Society of Oral Surgery according to the AAOMS diagnostic criteria and who underwent bone SPECT/CT were enrolled. One reader determined standard uptake values (SUVs), including maximum (SUVmax), peak (SUVpeak), and mean (SUVmean), as well as metabolic bone volume (MBV), representing total volume above threshold, and total bone uptake (TBU), calculated as MBV × SUVmean, using the GI-BONE software package. One-way repeated-measures analysis of variance and subsequent post hoc analysis were employed to compare quantitative values between clinical stages. To check reproducibility of values, another reader calculated these quantitative values. RESULTS: Mean SUVmax values for stage 0 (n = 21), 1 (n = 13), 2 (n = 25), and 3 (n = 10) were 5.82 ± 3.20, 5.46 ± 3.79, 8.16 ± 3.93, and 10.57 ± 8.43, respectively, while values for MBV were 9.52 ± 6.33, 11.36 ± 7.32, 12.4 ± 8.21, and 17.84 ± 16.94, respectively, and for TBU were 40.60 ± 46.97, 53.70 ± 77.26, 62.37 ± 42.91, and 102.01 ± 74.52, respectively. There were significant differences for SUVmax, SUVpeak, and SUVmean between clinical stages (p = 0.024, p = 0.027, p = 0.039, respectively). Subsequent post hoc analysis showed that SUVmax and SUVpeak of stage 3 were significantly higher than those of stage 0 (p = 0.046, 0.045, respectively). MBV and TBU showed a tendency to increase with increased stage, though differences between stages were not significant (p = 0.15, p = 0.053, respectively). Little differences of mean SUVmax, SUVpeak, SUVmean, MBV, and TBU between two readers were observed (- 3.10%, - 0.26%, - 4.24%%, 0.69%, and - 3.42%, respectively). The intraclass correlation coefficients (ICCs) of SUVmax, SUVpeak, SUVmean, MBV, and TBU were 0.985, 0.990, 0.980, 0.994, and 0.994, respectively (almost perfect for all values). CONCLUSION: As objective and reliable indicators, SUVmax and SUVpeak derived from quantitative bone SPECT/CT results are useful for detection of early status disease, as well as staging in MRONJ patients.

Usefulness of Quantitative Bone SPECT/CT for Evaluating Treatment Response in a Patient with Mandibular Osteomyelitis.

We report here a case of mandibular osteomyelitis in a 63-year-old female in which quantitative values determined using bone SPECT/CT were useful to evaluate response to antibiotic therapy, hyperbaric oxygen therapy, and sequestomy. After finishing therapy, the chief complaints were well relieved, and posttreatment Tc-99m HMDP bone SPECT/CT examination showed decreased uptake. The maximum standardized uptake value (SUV), peak SUV, mean SUV, metabolic bone volume, and total bone uptake of the untreated lesion were 6.26, 5.16, 3.97, and 11.86 mL and 42.21, respectively, which were decreased to 4.65, 3.90, 2.77, and 9.67 mL and 26.80, respectively, following hyperbaric oxygen therapy and antibiotic administration, and were moreover decreased to 4.28, 3.67, 2.75, and 6.24 mL and 17.19, respectively, after sequestomy. In comparison with pretreatment situation, those parameters were decreased by -25.7, -24.4, -30.2, -18.5, and -36.5%, respectively, following hyperbaric oxygen therapy and antibiotic administration, and moreover by -31.6, -28.9, -30.7, -47.4, and -59.3, respectively, after sequestomy, likely reflecting treatment response. Quantitative bone SPECT/CT may be useful to evaluate bone inflammatory activity and treatment response in a patient with mandibular osteomyelitis.

Senescent RAW264.7 cells exhibit increased production of nitric oxide and release inducible nitric oxide synthase in exosomes.

Aging cells not only cease growing, but also secrete various proteins such as inflammatory cytokines. This secretory phenomenon is known as the senescence­associated secretory phenotype (SASP). The aim of the present study was to elucidate the effects of senescence on the differentiation of osteoclast precursors (OCPs) and corresponding SASP. RAW264.7 cells were used as OCPs and were cultured to passage (P)5, P10 and P20. Cell proliferation assays, senescence­associated ß­galactosidase staining and telomere length quantification were subsequently performed, and it was revealed that replicative senescence was induced at P20. In addition, the level of tartrate­resistant acid phosphatase activity in P20 cells treated with receptor activator of nuclear factor­κB ligand was significantly lower than that in P5 and P10 cells. The SASP factors interleukin­6, tumour necrosis factor­α and nitric oxide were significantly increased in P20 culture supernatants compared with those in P5 and P10 supernatants. Furthermore, the number of exosomes at P20 was increased compared with that at P5 and P10, and inducible nitric oxide synthase (iNOS) was expressed in exosomes at P20, but not in exosomes at P5. In conclusion, the present study revealed that senescent RAW264.7 cells exhibit increased expression of SASP factors and release iNOS in exosomes.

Identification and functional analyses of a cell-death inhibitor encoded by guinea pig cytomegalovirus gp38.1 in cell culture and in animals.

Cytomegaloviruses (CMVs) employ an array of strategies designed to interfere with host defence responses against pathogens. Studies on such evasion mechanisms are important for understanding the pathogenesis of CMV diseases. Although guinea pig CMV (GPCMV) provides a useful animal model for congenital CMV infection, its evasion strategies are not fully elucidated. Here, we analysed a genome locus that may encode gene products for the GPCMV evasion mechanisms and found the following. (1) RACE analyses identified five transcripts in the GP38-gp38.4 locus, one of which was a spliced product encoding gp38.1. Similarities in the splicing pattern and gene position of gp38.1 to human CMV UL37 and its exon 1 encoding vMIA (viral mitochondria-localized inhibitor of apoptosis) suggest that the gp38.1 gene encodes an apoptosis inhibitor. (2) In a transient transfection assay, gp38.1 localized in the mitochondria and relocated BAX from the cytoplasm to the mitochondria, although its co-localization with BAK was not evident. Further, the expression of gp38.1 partially reduced staurosporine-induced apoptosis. (3) GPCMV defective in the gp38.1 ORF (Δ38.1) and the virus that rescues the defect (r38.1) were generated. Guinea pig fibroblast cells infected with Δ38.1 died earlier than r38.1-infected cells, which resulted in the lower yields of Δ38.1. (4) In animals, viral loads in the spleens of r38.1-infected guinea pigs were higher than those in the spleens of Δ38.1-infected animals. In conclusion, although GPCMV gp38.1 exerts a vMIA-like function, its inhibitory effect was not robust, suggesting the presence of additional inhibitory molecule(s), such as a BAK-specific inhibitor.

Enhancement of guinea pig cytomegalovirus infection by two endogenously expressed components of the pentameric glycoprotein complex in epithelial cells.

A better understanding of the mechanisms underlying cell tropisms and the efficiency of viral infection is critical for the development of vaccines and antiviral drugs for viral diseases. In this study, we worked on the entry mechanisms of guinea pig cytomegalovirus and found that endogenous expression of a combination of two components (GP131 and GP133) of the pentameric glycoprotein complex, which is required for non-fibroblast cell tropisms, enhanced viral infection more than 10-fold. In addition, D138A alteration in GP131 increased this enhancement by an additional 10-fold. Although differences in the efficiency of viral infection among various cell types are usually explained by differences in viral entry or traffic processes, our experimental evidences dismissed such possibilities. Instead, our findings that i) endogenous expression of GP131 and GP133 after nuclear delivery of viral DNA still enhanced infection and ii) an HDAC inhibitor overcame the need of the endogenous expression led us to hypothesize a novel mechanism that controls the efficiency of viral infection through the activation of gene expression from viral DNA delivered to the nuclei. Further studies of this unexpected phenomena warrant to understand novel but also general mechanisms for cell tropisms of viral infection and determinants that control infection efficiency.

Effects of TGF­ß1 on the migration and morphology of RAW264.7 cells in vitro.

Osteoclasts (OCs) differentiate from monocyte/macrophage­lineage hematopoietic precursor cells, which are known as OC precursors (OCPs). Several studies have investigated cell chemotaxis in the bone microenvironment; however, OCP migration ability in the bone microenvironment during OC differentiation is yet to be elucidated. As an initial investigation of this characteristic, the present study aimed to determine the effects of transforming growth factor (TGF)­ß1 on OCP migration in vitro. Pre­osteoclastic RAW264.7 cells were cultured with and without TGF­ß1 (2, 5 or 20 ng/ml), receptor activator of NF­κB ligand (RANKL; 50 ng/ml), and/or SB431542 (10 µM), a potent and specific inhibitor of TGF­ß1 receptor kinase activity. Cell proliferation was significantly inhibited in the presence of TGF­ß1 for 3 days, and the effect was reversed by SB431542. Tartrate­resistant acid phosphatase (TRAP) activity in RAW264.7 cells was significantly increased by RANKL treatment, compared with TRAP activity in control cells on day 3. The highest TRAP activity in RAW264.7 cells was induced by the combined treatment with TGF­ß1 (2 ng/ml) and RANKL. When TGF­ß1 signaling was inhibited by addition of SB431542 to the medium during culture, OC differentiation was notably suppressed. These findings suggest that TGF­ß1 accelerates RANKL­induced OC differentiation, but does not act in a dose­dependent manner. The migration of RAW264.7 cells was promoted at 24 h, but was suppressed at 72 h, during RANKL­induced osteoclast differentiation in the presence of TGF­ß1. These results were accompanied with the increased expression of small G­proteins, RhoA and Rac, at 24 h, but their expression decreased at 72 h. RAW264.7 cells treated with TGF­ß1 for 24 h underwent morphological changes, from round to polygonal morphology. Furthermore, protrusions were completely lost and the cell morphology reverted from polygonal to round after TGF­ß1 treatment for 72 h. Therefore, our findings indicated that OCP migration may be modified by differentiation in vitro.

Propagermanium Induces NK Cell Maturation and Tends to Prolong Overall Survival of Patients With Refractory Cancer.

BACKGROUND/AIM: Propagermanium (PG) inhibits the CCL2/CCR2 axis, and has been shown to function as an immune modulator. This study investigated its anti-tumor mechanism in patients with refractory cancers. MATERIALS AND METHODS: Five healthy volunteers and 23 patients with refractory oral (n=8) or gastric (n=15) cancer received PG (30 mg/day). We performed flow cytometry (FCM) of peripheral blood mononuclear cells and in vitro killing assays. RESULTS: FCM revealed that CD16+/CD56Dim NK cells (i.e., mature, cytolytic subset) increased, and the apoptosis induction rate of cancer cells increased after PG administration. Among gastric cancer patients, median OS was 172.0 days. Two patients showed complete remission of lung or liver metastasis. Survival of patients with oral cancer also tended to be prolonged. CONCLUSION: PG induces NK cell maturation, and may potentiate anti-tumor activity.

Novel insight into the management of bisphosphonate-related osteonecrosis of the jaw (BRONJ).

Bisphosphonate-related osteonecrosis of the jaw (BRONJ), characterized by refractory bone exposure, has recently emerged as a serious side effect of bisphosphonate (BPs) treatment. We discuss novel insights that may help to improve the efficacy of BRONJ treatment and prevention. Our report highlights the following: (1) The presence of exposed bone in patients taking BPs does not necessarily reflect BRONJ, and diagnoses of oral ulceration with bone sequestration and malignancy must be excluded. (2) Osteonecrosis type of BRONJ is difficult to avoid using preventive dental measures alone. However, as with osteomyelitis type of BRONJ, preventive dental measures are indispensable for reducing the risk of secondary infection and disease progression. (3) The importance of tooth extraction as a risk factor for BRONJ among patients taking BPs has been overstated, particularly when they are administered at low doses. Delaying tooth extraction may increase the risk for the onset and progression of osteomyelitic BRONJ. (4) In patients taking low doses of BPs, dental implant surgery is not necessarily contraindicated if there are no other risk factors, such as combined use of corticosteroids or concomitant diabetes. However, the risk of BRONJ due to peri-implantitis must be explained when obtaining patient consent.

Osteonecrosis of the jaws caused by bisphosphonate treatment and oxidative stress in mice.

Aging is a significant risk factor for the development of bisphosphonate-related osteonecrosis of the jaws (BRONJ). Accumulating evidence suggests that bone aging is associated with oxidative stress (OS), and OS is associated with osteonecrosis. To elucidate the mechanisms of the onset of BRONJ, the present study focused on OS and the effects of treatment with the pro-oxidant DL-buthionine-(S,R)-sulfoximine (BSO), an oxidative stressor, on healing of a surgically induced penetrating injury of the palate. Six-week-old C57BL/6J mice were randomly divided into four groups (n=5 each) and treated with or without zoledronic acid (ZOL) and with or without BSO (experimental groups: ZOL, BSO, and ZOL+BSO; control group: saline solution). A penetrating injury of the midline palate was surgically created using a root elevator. ZOL (250 µg/kg/day) was injected intraperitoneally every day from 7 days prior to the surgical treatment to 4 days following the surgical treatment. BSO (500 µg/kg/day) was administered 7 days prior to the surgical treatment as a single intraperitoneal injection. The maxillae were harvested at 5 days following the surgical treatment for histological and histochemical studies. The presence of empty osteocyte lacunae in the palatal bone was increased by ZOL and BSO treatment. The highest number of empty osteocyte lacunae was observed in the ZOL+BSO group. The number of tartrate-resistant acid phosphatase-positive cells was decreased by ZOL treatment and increased by BSO treatment. The number of canaliculi per osteocyte lacuna was significantly decreased by BSO treatment. The mineral apposition rate was significantly lower in the treatment groups than the control group. Bisphosphonates and OS suppressed bone turnover. The present study has demonstrated that BSO treatment affects osteocytes, and OS in osteocytes exacerbates impairment of the osteocytic canalicular networks. As a result, bisphosphonates and OS may induce osteonecrosis following invasive dentoalveolar surgery. OS has been identified as an additional risk factor for the development of BRONJ.

A Multicenter Retrospective Study of Elective Neck Dissection for T1-2N0M0 Tongue Squamous Cell Carcinoma: Analysis Using Propensity Score-Matching.

BACKGROUND: This multicenter retrospective study aimed to determine whether elective neck dissection (END) can be performed for T1-2N0M0 tongue cancer. METHODS: Patients with T1-2N0M0 tongue squamous cell carcinoma who received treatment between January 2000 and December 2012 were enrolled at 14 multicenter study sites. The 5-year overall survival (OS) and 5-year disease-specific survival (DSS) were compared between the propensity score-matched END and observation (OBS) groups. RESULTS: The results showed that the OS rates among the 1234 enrolled patients were 85.5% in the END group and 90.2% in the OBS group (P = 0.182). The DSS rates were 87.0% in the END group and 94.3% in the OBS group (P = 0.003). Among the matched patients, the OS rates were 87.1% in the END group and 76.2% in the OBS group (P = 0.0051), and the respective DSS rates were 89.2% and 82.2% (P = 0.0335). CONCLUSION: This study showed that END is beneficial for T1-2N0M0 tongue cancer. However, END should be performed for patients with a tumor depth of 4-5 mm or more, which is the depth associated with a high rate of lymph node metastasis. The use of END should be carefully considered for both elderly and young patients.