Yoshiaki Kazaoka

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 Sjögrens 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.

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

BackgroundOral 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.MethodsThe 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.ResultsCombination 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.ConclusionsThis 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.

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.

Effective three-dimensional evaluation analysis of upper airway form during oral appliance therapy in patients with obstructive sleep apnoea

The oral appliance (OA) is considered to be an effective treatment modality for obstructive sleep apnoea (OSA). Upper airway enlargement during OA therapy is critical, and lateral cephalometry has been used for the two-dimensional evaluation of upper airway form during this therapy. However, this method cannot provide an accurate three-dimensional (3D) view of upper airway form. To confirm the effects of OA on the upper airway in patients with OSAS, we performed CT in the presence and absence of OA in 15 Japanese patients (12 males, 3 females) who responded to OA therapy. CT in the presence and absence of OA was consecutively performed for each patient, and upper airway cross-sectional area in six arbitrary planes parallel to the palatal plane was measured. Next, 3D image reconstruction was performed; morphological changes in upper airway form were evaluated, and upper airway volume at three levels from the palatal plane to the deepest point of the epiglottis was measured. The cross-sectional area of two planes in the posterior soft palate region significantly increased in the presence of OA compared with that in the absence of OA. In the presence of OA, upper airway cross-sectional area and volume significantly increased in the posterior soft palate region compared with those in the posterior tongue region. 3D CT image reconstruction accurately confirmed morphological changes in the upper airway during OA therapy. Continued use of this 3D evaluation is expected to improve the results of OA therapy in the future.

Plumbagin suppresses tumor cell growth in oral squamous cell carcinoma cell lines

OBJECTIVES Plumbagin (PL), a naturally occurring quinoid, exerts antitumoral effects in diverse types of cancer cells. However, the effect of PL on tumor cell proliferation in oral squamous cell carcinoma (OSCC) remains poorly understood. In this study, we assessed the efficacy of PL, in human OSCC cells. METHODS The effect of PL on the cell growth and apoptosis of OSCC cell lines was evaluated using MTT and Annexin V assays, respectively. The effect of PL on mitochondrial membrane potential loss and reactive oxygen species (ROS) generation was evaluated using flow cytometry analysis. RESULTS MTT assay showed that PL dose-dependently suppressed OSCC cell growth, with IC50 values ranging from 3.87 to 14.6 μM. Flow cytometry analysis revealed that PL treatment resulted in a significant decrease in mitochondrial membrane potential and an increase in the number of apoptotic cells. Notably, ROS generation was significantly elevated after PL treatment. Furthermore, a ROS scavenger, N-acetylcysteine (NAC), clearly suppressed the decrease in mitochondrial membrane potential, increase of caspase-3/7 activity, and apoptosis after PL treatment. CONCLUSION This study provides the considerable evidence of the tumor-suppressive effect of PL, thereby highlighting its therapeutic potential for OSCC treatment.

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.

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

BackgroundPierre 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 presentationThe 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.ConclusionWe 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.

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, 10–9 to 10–5 mol/L)-induced relaxation (29% difference at ACh 3 × 10–8 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 × 10–8 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 × 10–8 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-&agr; 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-&agr; 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.

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 chemoimmuno-therapy 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.