PDZ-binding kinase inhibitor OTS514 suppresses the proliferation of oral squamous carcinoma cells.

OBJECTIVE: PDZ-binding kinase (PBK) has been reported as a poor prognostic factor and is a promising molecular target for anticancer therapeutics. Here, we aimed to investigate the effect of specific PBK inhibitor OTS514 on the survival of OSCC cells. METHODS: Four OSCC cell lines (HSC-2, HSC-3, SAS, and OSC-19) were used to examine the effect of OTS514 on cell survival and apoptosis. DNA microarray analysis was conducted to investigate the effect of OTS514 on gene expression in OSCC cells. Gene set enrichment analysis was performed to identify molecular signatures related to the antiproliferative effect of OTS514. RESULTS: OTS514 decreased the cell survival of OSCC cells dose-dependently, and administration of OTS514 readily suppressed the HSC-2-derived tumor growth in immunodeficient mice. Treatment with OTS514 significantly increased the number of apoptotic cells and caspase-3/7 activity. Importantly, OTS514 suppressed the expression of E2F target genes with a marked decrease in protein levels of E2F1, a transcriptional factor. Moreover, TP53 knockdown attenuated OTS514-induced apoptosis. CONCLUSION: OTS514 suppressed the proliferation of OSCC cells by downregulating the expression of E2F target genes and induced apoptosis by mediating the p53 signaling pathway. These results highlight the clinical application of PBK inhibitors in the development of molecular-targeted therapeutics against OSCC.

Inhibition of VEGFR2 and EGFR signaling cooperatively suppresses the proliferation of oral squamous cell carcinoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently overexpressed in oral squamous cell carcinoma (OSCC), and EGFR-targeting therapeutics have been widely employed to treat patients with a variety of carcinomas including OSCC. Here, we aimed to investigate alternative signaling for OSCC survival under the disruption of EGFR signaling. METHODS: OSCC cell lines, namely HSC-3 and SAS, were utilized to investigate how EGFR disruption affects cell proliferation. Gene set enrichment analysis was performed to examine how EGFR disruption affects oncogenic signaling in OSCC cells. Disruption of KDR gene was performed using CRISPR/Cas9 techniques. A VEGFR inhibitor, vatalanib was used to research the impact of VEGFR inhibition on OSCC survival. RESULTS: EGFR disruption significantly decreased the proliferation and oncogenic signaling including Myc and PI3K-Akt, in OSCC cells. Chemical library screening assays revealed that VEGFR inhibitors continued to inhibit the proliferation of EGFR-deficient OSCC cells. In addition, CRISPR-mediated disruption of KDR/VEGFR2 retarded OSCC cell proliferation. Furthermore, combined erlotinib-vatalanib treatment exhibited a more potent anti-proliferative effect on OSCC cells, compared to either monotherapy. The combined therapy effectively suppressed the phosphorylation levels of Akt but not p44/42. CONCLUSION: VEGFR-mediated signaling would be an alternative signaling pathway for the survival of OSCC cells under the disruption of EGFR signaling. These results highlight the clinical application of VEGFR inhibitors in the development of multi-molecular-targeted therapeutics against OSCC.

Tumor-infiltrating FoxP3+ T cells are associated with poor prognosis in oral squamous cell carcinoma.

OBJECTIVES: Squamous cell carcinoma is the most common malignancy in the oral cavity. Moreover, human papillomavirus (HPV) infection has been recently implicated in the onset of oral squamous cell carcinoma (OSCC). Regulatory T cells (Tregs) are Forkhead box P3 (FoxP3) positive and are normally involved in the mechanism by which organisms escape attacks from their own immune system; however, in tumors, these cells are known to suppress antitumor immunity and block the attack against tumors. The present study evaluated the associations of the number of Tregs and HPV infection with prognoses in patients with OSCC. MATERIAL AND METHODS: Samples from 106 patients diagnosed with OSCC were evaluated by immunohistochemical staining for the identification of FoxP3+ Tregs and HPV. The relationship between the observed number of Foxp3-positive cells, the presence/absence of HPV infection and associations with clinicopathological indicators were analyzed. RESULTS: Tissues were classified into high (High) and low (Low) Treg count groups, with 69 patients classified as High and 37 classified as Low. The prognoses were significantly better in the Low group compared with the High group (p = 0.04). FoxP3 expression may have had some effect on nodal metastases (p = 0.09). HPV antigens were detected in 65 patients, but there were no significant associations with prognosis (p = 0.34). HPV-infected tumors were more common in the gums and tongues than in the lips, cheeks, and floor of the mouth (p = 0.05). CONCLUSIONS: These results indicate that Tregs in tumor sites are associated with worsened prognoses of patients with OSCC and suggest potential therapies targeting Tregs in OSCC.

Evaluation of skeletal muscle mass using prediction formulas at the level of the 12th thoracic vertebra.

OBJECTIVES: People with cancer have a high risk of cachexia and sarcopenia, which are associated with worse clinical outcomes. We evaluated the prediction accuracy of the Matsuyama et al. and Ishida et al. formulas using computed tomography (CT) slices from the twelfth thoracic vertebra (Th12) level in people with cancer. METHODS: This retrospective study included patients with advanced cancer who underwent thoracic and abdominal CT scans (n = 173). The cross-sectional area (CSA) on CT images was measured at the levels of Th12 and the third lumbar vertebra (L3). The Matsuyama et al. formula used the Th12 CSA, whereas the Ishida et al. formula used only the Th12 CSA of the spinal erectors; thus, the measurements were performed separately. The correlation between predicted and actual L3 CSA was assessed using r and the intraclass correlation coefficient. A prediction-accuracy analysis of the predicted values was also performed. RESULTS: The mean participant age was 66.2 ± 12.8 y; 50.3% of participants were women and 49.7% were men. Strong correlations were observed between the predicted and measured L3 values calculated from the two prediction formulas. The prediction-accuracy analysis using previously reported cutoff values showed that the Ishida et al. method had high sensitivity and the Matsuyama et al. method had high specificity for low skeletal muscle index determined by the predicted and measured L3 skeletal muscle index. CONCLUSIONS: Both the Matsuyama et al. and Ishida et al. formulas had good reliability on CT slices at the Th12 level in people with advanced cancer, indicating that these formulas can be applied in clinical practice.

Assessing skeletal muscle mass based on the cross-sectional area of muscles at the 12th thoracic vertebra level on computed tomography in patients with oral squamous cell carcinoma.

OBJECTIVES: This study aimed to create a formula to estimate the third lumbar vertebra (L3)1 level skeletal muscle cross-sectional area (CSA), known as a standard value to evaluate skeletal muscle mass on computed tomography (CT), using the twelfth thoracic vertebra (Th12) level skeletal muscle CSA on chest CT. MATERIALS AND METHODS: This retrospective observational study included patients aged 40 + years with a diagnosis of oral squamous cell carcinoma (n = 164). Skeletal muscle CSA on CT images was measured using the Th12 and the L3 levels of pretreatment CT scans. The predictive formula was created based on the five-fold cross-validation method with a linear regression model. Correlations between the predicted L3-level CSA and the actual L3-level CSA were evaluated using r and Intraclass Correlation Coefficients (ICC). RESULTS: The predictive formula for L3-level CSA from Th12-level CSA was: CSA at L3 (cm2) = 14.143 + 0.779 * CSA at Th12 (cm2) - 0.212 * Age (y) + 0.502 * Weight (kg) + 13.763 * Sex. Correlations between the predicted and measured L3-level CSA were r = 0.915 [0.886-0.937] and ICC = 0.911 [0.881-0.934]. CONCLUSION: We developed a formula for predicting skeletal muscle mass from the Th12-level CT slice. The predicted L3-level CSA correlated with the measured L3-level CSA.

Discovery of novel molecular characteristics and cellular biological properties in ameloblastoma.

Ameloblastoma is a rare odontogenic benign tumor accounting for less than 1% of head and neck tumors. Advanced next generation sequencing (NGS) analyses identified high frequency of BRAF V600E and SMO L412F mutations in ameloblastoma. Despite the existence of whole genomic sequence information from patients with ameloblastoma, entire molecular signature of and the characteristics of ameloblastoma cells are still obscure. In this study, we sought to uncover the molecular basis of ameloblastoma and to determine the cellular phenotype of ameloblastoma cells with BRAF mutations. Our comparative cDNA microarray analysis and gene set enrichment analysis (GSEA) showed that ameloblastoma exhibited a distinct gene expression pattern from the normal tissues: KRAS-responsive gene set is significantly activated in ameloblastoma. Importantly, insulin like growth factor 2 (IGF2), a member of KRAS-responsive genes, enhances the proliferation of an ameloblastoma cell line AMU-AM1 with BRAF mutation. In addition, Toll-like receptor 2 (TLR2) knockdown readily inactivated KRAS-responsive gene sets as well as increases caspase activities, suggesting that TLR2 signaling may mediate cell survival signaling in ameloblastoma cells. Collectively, the findings may help to further clarify the pathophysiology of ameloblastoma and lead to the development of precision medicine for patients with ameloblastoma.

Treatment outcome of oral appliance in patients with REM-related obstructive sleep apnea.

PURPOSE: Oral appliances (OA) are used to treat patients with obstructive sleep apnea (OSA). The purpose of this study is to evaluate the efficacy of OA treatment in patients with rapid eye movement (REM)-related OSA. METHODS: Forty-six patients with REM-related OSA and 107 with non-stage-specific OSA were prescribed OA treatment after diagnosis by polysomnography (PSG) and a follow-up sleep test by PSG was conducted. Efficacy and treatment outcome predictors were evaluated according to the following criteria for treatment success: #1, reduction of the apnea-hypopnea index (AHI) to less than 5 and > 50% compared with baseline; #2, AHI reduction to less than 10 and > 50% compared with baseline; and #3, > 50% AHI reduction compared with baseline. RESULTS: Success rates according to criteria #1, #2, and #3 were 45.7%, 50.0%, and 50.0% in REM-related OSA and 36.4%, 52.3%, and 63.6% in non-stage-specific OSA, respectively. No significant differences in success rate were found between the two groups. In multivariate logistic regression analysis with each criterion as the response variable, only BMI was extracted as a significant predictor. The BMI cutoff values defined based on the maximum Youden index according to the three criteria were 26.2 kg/m2, 25.6 kg/m2, and 26.2 kg/m2, respectively. CONCLUSIONS: No significant differences in success rate of OA treatment were found between REM-related OSA and non-stage-specific OSA. BMI has greater impact on treatment outcome of OA in patients with REM-related OSA.

Metronidazole-induced encephalopathy caused by hyperbaric oxygen therapy in a patient with mandibular osteomyelitis.

Metronidazole (MNZ) is prescribed for the treatment of infection caused by anaerobic bacteria and protozoa. Metronidazole-induced encephalopathy (MIE) has been known to be a side-effect, although its onset ratio is unclear. However, to the best of our knowledge, MIE associated with hyperbaric oxygen therapy (HBO) has not been previously reported. Here, we present the case of a 68-year-old man with mandibular osteomyelitis who received metronidazole for 49 days and received five times HBO therapy. He visited our hospital for evaluation and treatment of peripheral neuropathy, speech disturbance, nausea, and disturbance of gait after 47 days of initiating metronidazole treatment. Brain magnetic resonance imaging revealed hyperintense lesions in the cerebellar dentate nuclei, which was consistent with MIE. The patient's ataxic symptoms improved in 15 days after the discontinuation of MNZ. This is the first report demonstrating case of MIE could be related with HBO, as far as we had searched.

Adoptive immunotherapy combined with FP treatment for head and neck cancer: An in vitro study.

FP treatment, which combines 5-fluorouracil (5-FU) and cisplatin (CDDP) chemotherapy, is widely used for treatment of advanced head and neck cancer (HNC). It has been suggested that these drugs cause immunomodulation in the cancer microenvironment, for example, downregulation of immunosuppressive cells such as regulatory T-cells (Tregs) and myeloid-derived suppressive cells (MDSCs), activating dendritic cells (DCs), and upregulation of tumor antigens and major histocompatibility complex (MHC) molecules in cancer cells leads to enhancement of cancer immunity, which is important in cancer treatment, as well as providing a direct killing effect. Therefore, development of chemoimmunotherapy by combining FP treatment with immunotherapy for HNC has become a recent challenging issue. However, the direct effects of these drugs on immune effector cells, especially cytotoxic T-lymphocytes (CTLs), are not well known. We have investigated the direct actions of these drugs on CTL functions in in vitro experiments using cytomegalovirus (CMV) pp65 antigen-specific CTLs (CMVpp65-CTLs) and oral squamous cell cancer (OSCC) cell lines overexpressing CMVpp65 antigen as target cells. Although CDDP partially inhibited proliferation of memory CMVpp65-CTL in peripheral blood, the proliferation was not inhibited by 5-FU. Cytotoxicity and the IFN-γ release response of the CMVpp65-CTLs were not inhibited by these drugs, and it is important to note that these drugs, especially 5-FU, sensitized OSCC cell lines to CMVpp65-CTL. Furthermore, CMVpp65-CTL cytotoxicity to CDDP-resistant OSCC cells, HSC-3/CDDP-R1, was the same as the cytotoxicity to the parental cells. Thus, we suggest that combined immunotherapy with FP treatment is an effective novel HNC treatment.

Lidocaine Prevents Oxidative Stress-Induced Endothelial Dysfunction of the Systemic Artery in Rats With Intermittent Periodontal Inflammation.

BACKGROUND: Periodontal inflammation causes endothelial dysfunction of the systemic artery. However, it is unknown whether the use of local anesthetics during painful dental procedures alleviates periodontal inflammation and systemic endothelial function. This study was designed to examine whether the gingival or systemic injection of lidocaine prevents oxidative stress-induced endothelial dysfunction of the systemic artery in rats with intermittent periodontal inflammation caused by lipopolysaccharides (LPS). METHODS: Some rats received 1500 µg LPS injections to the gingiva during a week interval from the age of 8 to 11 weeks (LPS group). Lidocaine (3 mg/kg), LPS + lidocaine (3 mg/kg), LPS + lidocaine (1.5 mg/kg), and LPS + lidocaine (3 mg/kg, IP) groups simultaneously received gingival 1.5 or 3 mg/kg or IP 3 mg/kg injection of lidocaine on the same schedule as the gingival LPS. Isolated aortas or mandibles were subjected to the evaluation of histopathologic change, isometric force recording, reactive oxygen species, and Western immunoblotting. RESULTS: Mean blood pressure and heart rate did not differ among the control, LPS, LPS + lidocaine (3 mg/kg), and lidocaine (3 mg/kg) groups. LPS application reduced acetylcholine (ACh, 10 to 10 mol/L)-induced relaxation (29% difference at ACh 3 × 10 mol/L, P = .01), which was restored by catalase. Gingival lidocaine (1.5 and 3 mg/kg) dose dependently prevented the endothelial dysfunction caused by LPS application (24.5%-31.1% difference at ACh 3 × 10 mol/L, P = .006 or .001, respectively). Similar to the gingival application, the IP injection of lidocaine (3 mg/kg) restored the ACh-induced dilation of isolated aortas from rats with the LPS application (27.5% difference at ACh 3 × 10 mol/L, P < .001). Levels of reactive oxygen species were double in aortas from the LPS group (P < .001), whereas the increment was abolished by polyethylene glycol-catalase, gingival lidocaine (3 mg/kg), or the combination. The LPS induced a 4-fold increase in the protein expression of tumor necrosis factor-α in the periodontal tissue (P < .001), whereas the lidocaine (3 mg/kg) coadministration partly reduced the levels. Lidocaine application also decreased the protein expression of the nicotinamide adenine dinucleotide phosphate oxidase subunit p47phox, which was enhanced by the gingival LPS (5.6-fold increase; P < .001). CONCLUSIONS: Lidocaine preserved the aortic endothelial function through a decrease in arterial reactive oxygen species produced by nicotinamide adenine dinucleotide phosphate oxidase and periodontal tumor necrosis factor-α levels in rats with periodontal inflammation. These results suggest the beneficial effect of the gingival application of local anesthetics on the treatment of periodontal diseases on endothelial function of systemic arteries.

Case report of Pierre Robin sequence with severe upper airway obstruction who was rescued by fiberoptic nasotracheal intubation.

BACKGROUND: Pierre Robin sequence (PRS) refers to the association of micrognathia, glossoptosis, and airway obstruction. Cases with severe dyspnea due to upper airway obstruction immediately after birth are very rare. We here report two cases with PRS who developed severe dyspnea due to morphological abnormality immediately after birth and were rescued by fiberoptic nasotracheal intubation. CASE PRESENTATION: The patient in case 1 had micrognathia and cleft palate, and his tongue protruded into the nasal cavity via a cleft palate. His invaginated tongue was considered an extreme type of glossoptosis and he was diagnosed as Pierre Robin sequence. The patient in case 2 also had micrognathia and cleft palate same as case 1 and accompanied some anomalad. Her chromosome analysis confirmed a diagnosis of 1p36 deletion syndrome and she diagnosed as 1p36 deletion syndrome complicated with Pierre Robin sequence. In both cases, tongue protruded into the nasal cavity via a cleft palate occupied pharynx and nasal cavity, resulting in severe dyspnea. Only the backside of the tongue was visible by laryngoscopy and oropharyngeal intubation was impossible. Therefore, fiberoptic nasotracheal intubation was done to secure the airway for resuscitation. CONCLUSION: We conclude that extreme type of glossoptosis in PRS concludes tongue invaginated into nasal cavity which have not reported before and that such cases require resuscitation by fiberoptic intubation immediately after birth. As such, neonatologists should obtain the skill of fiberoptic intubation.

Inhibition of Nox1 induces apoptosis by attenuating the AKT signaling pathway in oral squamous cell carcinoma cell lines.

NADPH oxidases, also known as the Nox family, are major sources of reactive oxygen species generation that regulate redox-sensitive signaling pathways. Recent studies have implicated the Nox family in cancer development and progression. However, the involvement of its members in the development of oral squamous cell carcinoma (OSCC) remains to be elucidated. To clarify this issue, we first analyzed mRNA expression of Nox/Duox family members (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2) in five OSCC cell lines. Nox1 and Nox4 mRNAs were highly expressed in four OSCC cell lines. Western blot analysis revealed that the protein expression level of Nox1 was higher than that of Nox4 in the OSCC cell lines. In addition, knockdown of Nox1, but not Nox4, significantly suppressed cell viability and induced apoptosis in the HSC-2 and HSC-3 cells. We also found that a specific AKT inhibitor, perifosine, dose-dependently suppressed OSCC cell growth. Notably, Nox1 knockdown significantly attenuated the phosphorylation level of AKT. Furthermore, both Nox1 knockdown and perifosine treatment markedly enhanced the cisplatin-induced cytotoxic effect. Taken together, our results highlight that the Nox1/AKT signaling pathway plays an important role in cell survival in OSCC cells.

Intermittent local periodontal inflammation causes endothelial dysfunction of the systemic artery via increased levels of hydrogen peroxide concomitantly with overexpression of superoxide dismutase.

BACKGROUND: The present study was designed to examine whether the intermittent local periodontal inflammation induces endothelial dysfunction of the systemic artery caused by oxidative stress and if increased levels of hydrogen peroxide coexisted with overexpression of superoxide dismutase (SOD) as well as NADPH oxidase contribute to the oxidative stress. METHODS: The rats in lipopolysaccharides (LPS) group received 1500µg LPS injection to bilateral gingiva of the lower jaw a week interval from eight- to eleven-week-old. Isolated mandibles or aortas were subjected to the evaluation of histopathological changes, isometric force recordings, reactive oxygen species using 2',7'-dichlorofluorescin diacetate (10(-5)mol/L) and protein expression of NADPH oxidase subunits and SOD, respectively. RESULTS: Mandible sections demonstrated the periodontal inflammation only in the LPS group at three days, but not seven days, after the LSP injection. Acetylcholine (10(-9) to 10(-5)mol/L)-induced relaxation was reduced only in aortas from the LPS group. Gp91ds-tat and PEG-catalase restored the impaired dilation in arteries from the LPS group. Levels of reactive oxygen species were enhanced in aortas from the LPS group, whereas the increment was abolished by the treatment with gp91-ds-tat or PEG-catalase. Expression of a NADPH oxidase subunit p47phox and CuZn-SOD increased in the LPS group. CONCLUSIONS: The intermittent local periodontal inflammation induces systemic endothelial dysfunction caused by overproduction of reactive oxygen species in the systemic artery of rats and that overexpression of CuZn-SOD as well as a NADPH oxidase cytosolic subunit contributes to increased levels of hydrogen peroxide in blood vessels of this animal model.

Activated cytotoxic T-lymphocyte immunotherapy is effective for advanced oral and maxillofacial cancers.

Conventional cancer treatments are surgery, radiotherapy, and chemotherapy, but treatment efficiency is insufficient and cancer recurrence is common. Immunotherapy has been added as an important cancer treatment component, but no reports on its efficacy in oral and maxillofacial cancers exist. We evaluated the clinical efficacy of adoptive immunotherapy using ex vivo-activated cytotoxic T lymphocytes (CTL) in the treatment of 7 patients with advanced oral and maxillofacial cancers with stage IV disease at diagnosis. The mean follow-up period was 26.2 months. Phenotype of the lymphocyte assay revealed that the percentage of CD4(+) T cells decreased and that of CD8(+) T cells increased among infused lymphocytes compared to that in unstimulated peripheral blood mononuclear cells (PBMCs), and infused lymphocytes produced a significantly higher level of IFN-γ than PBMCs or tumor cells alone. In a representative patient who refused surgery tumor regression was confirmed after CTL infusion. Computed tomography clearly indicated a significant reduction in tumor size followed by the complete disappearance of the tumor. Histological examination showed that the cancers in patients receiving CTL therapy were heavily infiltrated with lymphocytes. The other 2 patients who received CTL therapy as adjuvant therapy showed neither recurrent disease nor new disease lesions. The 1-year survival rates showing response and those with progressive disease were 100 and 25%, respectively. Moreover, no significant adverse reactions were reported during the study period. CTL therapy remains in the early stages of treatment options, but it has potential as a valuable treatment and improvement of quality of life for patients with otherwise incurable cancers.

Combined arsenic trioxide-cisplatin treatment enhances apoptosis in oral squamous cell carcinoma cells.

BACKGROUND: Oral squamous cell carcinoma (OSCC) accounts for the majority of oral cancers. Despite recent advances in OSCC diagnostics and therapeutics, the overall survival rate still remains low. Here, we assessed the efficacy of a combinatorial arsenic trioxide (ATO) and cisplatin (CDDP) treatment in human OSCC cells. METHODS: The combinatorial effect of ATO/CDDP on the growth and apoptosis of OSCC cell lines HSC-2, HSC-3, and HSC-4 was evaluated using MTT and annexin V assays, respectively. Chou-Talalay analyses were preformed to evaluate the combinatorial effects of ATO/CDDP on the dose-reduction index (DRI). To clarify the mechanism underlying the ATO/CDDP anticancer effect, we also examined the involvement of reactive oxygen species (ROS) in ATO/CDDP-induced apoptosis. RESULTS: Combination index (CI) analyses revealed that a synergistic interaction of ATO and CDDP elicits a wide range of effects in HSC-2 cells, with CI values ranging from 0.78 to 0.90, where CI < 1 defines synergism. The CI values in HSC-3 and HSC-4 cells ranged from 0.34 to 0.45 and from 0.60 to 0.92, respectively. In addition, ATO/CDDP yielded favorable DRI values ranging from 1.6-fold to 7.71-fold dose reduction. Compared to mono-therapy, ATO/CDDP combinatorial therapy significantly augmented the loss of mitochondrial potential, caspase-3/7 activity and subsequent apoptosis. These changes were all abrogated by the antioxidant N-acetylcysteine. CONCLUSIONS: This study provides the first evidence for a synergistic ATO/CDDP anticancer (apoptotic) activity in OSCC cells with a favorable DRI, thereby highlighting its potential as a combinational therapeutic regime in OSCC.

Minimally invasive endoscopic osteosynthesis for frontozygomatic fracture: a new approach.

Miniplate fixation on the lateral face of the orbital rim using existing endoscopic methods for frontozygomatic fracture still has some disadvantages, such as cosmetic disturbance from the lateral brow incision for the trocar, and abnormal palpability of the miniplate. We applied a new method of endoscopic osteosynthesis by access through temporal incisions alone and miniplate fixation on the lateral temporal face of the frontozygoma. Postoperative courses were uneventful in all four cases treated, and we achieved cosmetic improvement (minimizing incision and scars) as well as decreased palpability of the miniplate.

Clinicopathological significance of carbonic anhydrase 9, glucose transporter-1, Ki-67 and p53 expression in oral squamous cell carcinoma.

Carbonic anhydrase 9 (CA9) is a glycoprotein present on the surface of cell membranes. It is expressed in 90% of renal cancer cells, but not in normal kidney tissue. Immunotherapy targeting CA9 is underway, and our group has also conducted a clinical trial using CA9 as a cancer vaccine and confirmed the induction of cytotoxic T lymphocytes, with efficacy in some cases. Expression of CA9 antigen in oral cancer has not been reported in Japan, but our results indicate that immunotherapy targeting CA9 might be possible. We immunohistochemically observed the expression of antigens such as CA9, Ki-67, glucose transporter-1 (GLUT-1) and p53 in 107 subjects with oral squamous cell carcinoma, and examined their correlation with clinicopathological parameters. Immunostaining analysis showed expression of CA9 in 98% of oral cancer subjects, and the survival rate was significantly lower in subjects with CA9 antigen expression in 50% or more cells (P<0.05). Subjects with poorly differentiated, T4 and lymph node metastasis, or Stage IV cancer with high CA9 expression (≥50%) had a worse outcome than those with low CA9 expression. Although GLUT-1 expression was observed in 98% of subjects, similarly to CA9 expression, no significant correlation between its expression and the survival rate was seen. However, subjects with lymph node metastasis had significantly higher GLUT-1 expression, demonstrating that GLUT-1 could be an indicator of lymph node metastasis. Ki-67 was expressed in 92% of subjects, but no correlation with outcome was observed. Expression of p53 was noted in 78% of subjects, and it was found that many oral cancers have p53 genetic abnormalities, but no correlation between p53 and outcome was observed. It was confirmed that CA9 antigen is expressed in most oral cancer subjects, suggesting the possibility of immunotherapy targeting CA9 antigen in oral cancer.

Combining anti-tumor drugs with mild hyperthermia increases the cytotoxic effects of drugs on human leukemia cells in vitro.

Although the benefits of using a combination of hyperthermia and chemotherapy or radiotherapy in the treatment of cancer have been theoretically established, the use of such combination therapy is not widespread at the clinical level, as the application of hyperthermia is complex and maintaining a tumor temperature of 43°C or higher is exceedingly difficult. Consequently, in the present study, the effects of chemotherapy combined with mild hyperthermia at 41°C (which is easier to apply than standard hyperthermia) were examined in the NALM-6 leukemia cell line. The results were as follows: i) NALM-6 leukemia cells, like most cells, survived mild hyperthermia at 41°C, but were killed at temperatures over 43°C. ii) Low concentrations of adriamycin (0.1 µg/ml) or mild hyperthermia applied separately did not have a visible effect on the survival rate of NALM-6 cells, whereas combined treatment with these therapies decreased the survival rate of NALM-6 cells in a time-dependent manner. The anti-tumor effect after 5 h of the combination of 0.1 µg/ml adriamycin and mild hyperthermia was the same as that observed with a 10-fold higher concentration (1 µg/ml) of adriamycin alone. iii) Another anti-tumor drug, vincristine, exhibited the same behavior as adriamycin. The anti-tumor effect after 1 h of the combination of 5x10-11 M vincristine and mild hyperthermia was the same as that observed with a 10-fold higher concentration (5x10-10 M) of vincristine alone. The results indicate that it may be possible to reduce the required concentrations of anti-tumor drugs by using them in combination with mild hyperthermia. In this way, the side effects of chemotherapy may be reduced in clinical settings. Mild hyperthermia is a useful and practical heating method, and could result in the increasing clinical application of hyperthermia in the treatment of cancer.

Combinations of tumor-specific CD8+ CTLs and anti-CD25 mAb provide improved immunotherapy.

One new approach to cancer therapy is based on the adoptive transfer of tumor-specific cytotoxic T cells and anti-CD25 antibodies. In the present study, CD8+ and IFN-gamma secreting T lymphocytes (CTLs) were enriched as tumor-specific cytotoxic T cells from spleen lymphocytes of mice bearing the Renca tumor (a murine renal carcinoma line originating from a BALB/c mouse) after stimulation with tumor cells. An anti-CD25 IL-2Ralpha(anti-CD25) mAb from hybridoma PC61 was used for depletion for CD4(+)CD25(+) regulatory T (Treg) cells. Treatment-efficacy for tumor-bearing mice was compared using 4 systems: 1, whole spleen lymphocytes stimulated with tumor cells in vitro from tumor-bearing mice; 2, CTLs; 3, anti-CD25 mAbs; 4, CTLs and anti-CD25 mAbs. At the 50th day after tumor inoculation, in the group which received anti-CD25 mAb for depletion of T cells and inoculation of CTLs, tumors had disappeared and no re-growth was observed. In contrast, all mice of the non-treated and other three groups, treated with whole spleen cells alone, CTLs alone and anti-CD25 mAb alone, had died. These results showed that a combination of Treg cell-depletion using anti-CD25 mAbs and CTL administration is a feasible approach for treatment of cancers which warrants further exploration in the clinical setting.

Identification of specific autoantigens in Sjögren's syndrome by SEREX.

We carried out SEREX (serological analysis of antigens by recombinant cDNA expression cloning) using sera from patients with Sjögren's syndrome (SjS) and investigated the frequencies of autoantibodies against autoantigens identified by SEREX in the sera of healthy individuals (HI) and patients with SjS, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). IFI16 and two kelch-like proteins, KLHL12 and KLHL7, were found to be novel autoantigens in SjS by SEREX. A markedly high frequency of anti-IFI16 autoantibodies was observed in the sera of SjS (SjS, 70%; RA, 13%; SLE, 33%; HI, 0%). Interestingly, all serum samples from SjS demonstrated immunoreactivity against one or both of IFI16 and SS-B/La. The presence of autoantibodies against KLHL12 and KLHL7 in the sera was significantly specific to SjS (23% and 17%, respectively), as they were not detected in RA, SLE or HI. Furthermore, we confirmed that transcripts of these autoantigens were expressed preferentially in the salivary glands and immuno-privileged testes. Our results suggest these autoantigens may be useful as serological markers for the clinical diagnosis of SjS and may play a crucial role as organ-specific autoantigens in the aetiopathogenesis of SjS. This study warranted clinical evaluations of autoantibodies against IFI16, KLHL12 and KLHL7 in combination with anti-SS-B/La autoantibodies.