Development of prediction models for the sensitivity of oral squamous cell carcinomas to preoperative S-1 administration.

S-1 is an anticancer agent that is comprised of tegafur, gimeracil, and oteracil potassium, and is widely used in various carcinomas including oral squamous cell carcinoma (OSCC). Although an established prediction tool is not available, we aimed to develop prediction models for the sensitivity of primary OSCC cases to the preoperative administration of S-1. We performed DNA microarray analysis of 95 cases with OSCC. Using global gene expression data and the clinical data, we developed two different prediction models, namely, model 1 that comprised the complete response (CR) + the partial response (PR) versus stable disease (SD) + progressive disease (PD), and model 2 that comprised responders versus non-responders. Twelve and 18 genes were designated as feature genes (FGs) in models 1 and 2, respectively, and, of these, six genes were common to both models. The sensitivity was 96.3%, the specificity was 91.2%, and the accuracy was 92.6% for model 1, and the sensitivity was 95.6%, the specificity was 85.2%, and the accuracy was 92.6% for model 2. These models were validated using receiver operating characteristic analysis, and the areas under the curves were 0.967 and 0.949 in models 1 and 2, respectively. The data led to the development of models that can reliably predict the sensitivity of patients with OSCC to the preoperative administration of S-1. The mechanism that regulates S-1 sensitivity remains unclear; however, the prediction models developed provide hope that further functional investigations into the FGs will lead to a greater understanding of drug resistance.

Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-κB signaling pathway.

Polymeric immunoglobulin receptor (pIgR) plays an important role in mucosal immune systems. Secretory immunoglobulin A, composed of secretory component of pIgR and a dimeric form of immunoglobulin A, is secreted on mucosal surfaces and serves as a biological defense factor. pIgR gene expression is reportedly induced by activation of the transcription factor nuclear factor (NF)-κB. On the other hand, secretory leukocyte protease inhibitor (SLPI) is a glycoprotein that functions as a serine protease inhibitor. In alveolar epithelial cells, SLPI increases the level of IκBß, which indicates that it is an inhibitor of NF-κB at the protein level. Taken together, SLPI may regulate pIgR expression; however, the specific mechanism by which this occurs is unclear. Therefore, the aim of this study was to elucidatethe influence of SLPI on pIgR expression.SLPI and pIgR localized in goblet cells and ciliated epithelial cells of the gastrointestinal tract, respectively. No cells were detected in which SLPI and pIgR were co-expressed. In addition, recombinant human SLPI stimulation of an epithelial cell line (HT-29) decreased the pIgR expression. The pIgR expression was also higher in SLPI-deficient Ca9-22 cells than in wild-type Ca9-22 cells. Furthermore, a luciferase assay using a NF-κB reporter plasmid and real-time RT-PCR analysis indicated that when SLPI was present, the transcriptional activity of NF-κB protein was suppressed, which was accompanied by anincrease in the protein, but not the mRNA,expression of IκBß. These results demonstrate that SLPI down-regulates pIgR expression through the NF-κB signaling pathway by inhibiting degradation of IκBß protein.

Angiotensin II suppresses osteoblastic differentiation and mineralized nodule formation via AT1 receptor in ROS17/2.8 cells.

INTRODUCTION: Angiotensin II (Ang II) not only regulates systemic blood pressure through a vasoconstrictive effect, but also promotes bone resorption. We recently reported that Ang II (10(-6) M) stimulated the production of matrix metalloproteinases via the AT1 receptor in osteoblastic ROS17/2.8 cells, but suppressed alkaline phosphatase activity. However, the roles of Ang II in osteoblastic differentiation and the function of osteogenesis in osteoblasts are unclear. Therefore, we examined the effect of Ang II on the expression of osteogenesis-related transcription factors and extracellular matrix (ECM) proteins, as well as mineralized nodule formation in ROS17/2.8 cells. MATERIAL AND METHODS: ROS17/2.8 cells were cultured with 0 (control) or 10(-6) M Ang II in the presence or absence of the AT1 receptor blocker losartan. Mineralized nodule formation was detected by Alizarin Red staining. Gene and protein expression levels of transcription factors and ECM proteins were determined using real-time PCR and Western blotting, respectively. RESULTS: Runx2, Msx2, and osteocalcin expression significantly decreased with Ang II compared to the control, whereas AJ18 expression significantly increased. Osterix, Dlx5, type I collagen, bone sialoprotein, and osteopontin expression was unaffected. Mineralized nodule formation and calcium content in mineralized nodules decreased with Ang II. Losartan blocked suppressive or stimulatory effects of Ang II on Runx2, Msx2, osteocalcin, and AJ18 expression. CONCLUSIONS: These results suggest that Ang II suppresses osteoblastic differentiation by altering the expression of osteogenesis-related transcription factors via the AT1 receptor and the function of osteogenesis in ROS17/2.8 cells.

CD105 expression in oral capillary hemangiomas and cavernous hemangiomas.

Capillary hemangioma (capillary lobular hemangioma) and cavernous hemangioma (venous malformation) are relatively common oral tumors/malformations and are characterized by increased numbers of normal and abnormal blood vessels. However, the causes of these lesions are not well understood. CD105 (endoglin) is predominantly expressed in proliferating blood endothelial cells (ECs). We analyzed expressions of CD105, CD34, von Willebrand factor, Ki-67, cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF)-A in 31 capillary hemangiomas and 34 cavernous hemangiomas. Staining scores were calculated as the product of the proportion score and intensity score. Morphologically normal oral mucosa specimens (n = 10) were simultaneously evaluated as normal controls. As compared with cavernous hemangiomas and normal controls, capillary hemangiomas had higher staining scores for CD105, VEGF-A, and COX-2. The Ki-67 labeling index was significantly higher in capillary hemangiomas than in cavernous hemangiomas and normal controls (P < 0.01). These findings suggest that the biological characteristics of capillary and cavernous hemangiomas are quite different. The ECs of capillary hemangiomas actively proliferated and were generally regulated by VEGF-A. In contrast, the ECs of cavernous hemangiomas lacked proliferative activity. These results suggest that angiogenesis and vasodilatation of pre-existing blood vessels are important in the development of capillary hemangioma and cavernous hemangioma, respectively.

Pigmented oral carcinoma in situ: a case report and literature review.

Oral melanotic lesions, including melanin pigmentation, melanocytic nevus, and malignant melanoma, are well-recognized pathologic entities. However, melanocytic proliferation within malignant oral mucosal lesions is not well documented. We report the unusual case of a 53-year-old Japanese man who developed oral carcinoma in situ (CIS) with melanocytic proliferation and melanin pigmentation in the epithelial layer. The patient, a nonsmoker and an opportunistic drinker, presented with a brown tongue lesion. Initial examination found a large brown pigmented area and multiple small white patchy areas on the right tongue border. The pigmentation had an ill-defined border with uneven color distribution. Physical examination found no abnormalities. Ultrasonography did not find a deeply infiltrating lesion. Oral mucosal malignant melanoma in situ was diagnosed, and partial tongue resection was performed. Histopathologic examination found oral pigmented CIS. To the best of our knowledge, this is only the third reported case of oral pigmented CIS.

Epiregulin is critical for the acinar cell regeneration of the submandibular gland in a mouse duct ligation model.

Acinar cell regeneration from tubular structures has been reported to occur in duct-deligated salivary glands. However, the detailed process of acinar cell regeneration has not been clarified. We have developed a mouse duct ligation model to clarify the mechanisms underlying acinar cell regeneration, and we analyzed the epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF) ligands using the model. We studied these ligands expressions in the course of acinar cell regeneration using immunohistochemistry and RT-PCR methods. In the duct-ligated portion of the submandibular gland (SMG) that underwent atrophy, newly formed acinar cells were observed arising from the tubular structures after the release of the duct obstruction. The constitutive expression of EGFR was observed by immunohistochemistry in both the duct-ligated and duct-deligated animals as well as in normal controls. The EGFR phosphorylation detected on the tubular structures after duct ligation paralleled the acinar cell regeneration. RT-PCR showed an increase in the epiregulin and heparin-binding EGF levels from day 0 to day 3 after the release of the duct obstruction. The EGF level was increased only after day 7. In vitro, cultured cells isolated from ligated SMGs proliferated and produced EGF ligands following the addition of epiregulin to the culture medium. These findings suggest that the tubular structures localized in an atrophic gland are the source of acinar cell regeneration of the salivary gland. The induction of EGF ligands, in particular epiregulin, may play an important role in acinar cell regeneration in this model.

Detection of fascin and CCR-7 positive mature dendritic cells in oral lichen planus.

BACKGROUND: Dendritic cells (DC) play a crucial role in the pathogenesis of oral lichen planus (OLP) with respect to antigens presented to T cells. We performed immunohistochemical analysis to elucidate the process of activation of DC in OLP. METHODS: Thirty biopsy specimens were obtained from the patients with OLP. The expressions of CD1a, Langerin, S-100, fascin, chemokine receptor-7 (CCR-7), D2-40, cyclooxygenase-2 (COX-2), and microsomal prostaglandin E synthase-1 (mPGES-1) in DC from OLP and disease free control were investigated using specific antibodies. The distribution and number (1 mm(2)) of DC were assessed in the intra-epithelium and the submucosa specimens. Correlation between the number of DC and epithelium thickness was also determined. RESULT: Immature DC (Langerin(+), CD1a(+), and S-100(+)) were identified in the epithelia from OLP patients and control, though the numbers of Langerin(+) and CD1a(+) positive cells were decreased in the OLP samples as compared to the control. Mature DC (fascin(+)) were identified in the submucosa specimens, not found in the epithelium from OLP or control. Double immunostaining revealed DC positive for fascin and CCR-7 in the submucosa, which had migrated into D2-40(+) lymph vessels. Furthermore, keratinocytes expressed both Prostaglandin E(2) (PGE(2)) converting enzymes, COX-2, and mPGES-1, indicating PGE(2) synthesis in the epithelial layer of the OLP specimens. CONCLUSION: Our results indicate that DC change from immature to mature in the epithelium and are then drawn out to the submucosa. We demonstrate that mature DC localized in the submucosa, it consequently migrates into lymph vessels. This maturation process of DC is an important immunopathological feature of OLP.

Behavioural characterisation of rats exposed neonatally to bisphenol-A: responses to a novel environment and to methylphenidate challenge in a putative model of attention-deficit hyperactivity disorder.

Neonatal exposure of rats to bisphenol-A, an endocrine disruptor, has recently been proposed as a possible animal model of attention-deficit hyperactivity disorder (ADHD), because such rats exhibit motor hyperactivity. To strengthen the face validity of this animal model, the present study replicated the original experiments and additionally analysed both changes in habituation to a novel environment and behavioural responses to methylphenidate, the two phenomena known to be altered in ADHD. Single intracisternal administration of bisphenol-A (20 and 40 microg) into 5-day-old male Wistar rats impaired habituation to a novel environment in the light, but not the dark, phase at 4 weeks of age. Thus, habituation as assessed by time-dependent decrease of locomotor activity, rearing, sniffing and grooming was significantly reduced in bisphenol-A-pretreated rats. Methylphenidate (1 and 3 mg/kg, i.p.) dose-dependently enhanced locomotor activity in both vehicle-pretreated and bisphenol-A-pretreated rats during both the dark and the light phases. Thus, the effects of methylphenidate did not differ between bisphenol-A-pretreated and vehicle-pretreated rats. Apart from a slight methylphenidate-induced increase in rearing and sniffing in bisphenol-A (20 microg)-pretreated rats, the overall effects of methylphenidate on rearing, sniffing and grooming were similar in both vehicle- and bisphenol-A-pretreated rats. It is concluded that neonatal exposure of rats to bisphenol-A is an animal model with limited face validity for ADHD, because the motor hyperactivity and reduced habituation to a novel environment are not accompanied by altered responses to methylphenidate.

Phosphorylation of Extracellular Signal-Regulated Kinase in medullary and upper cervical cord neurons following noxious tooth pulp stimulation.

The phosphorylated Extracellular Signal-regulated Kinase (pERK) and Fos expression and masticatory muscle activity were analyzed in rats with capsaicin-induced acute inflammation of the tooth pulp in order to clarify the role of the spinal trigeminal nucleus and upper cervical spinal cord in tooth pulp pain. Digastric and masseteric muscle activities were significantly increased following capsaicin injection into the molar tooth pulp but not after vehicle treatment. The pERK-like immunoreactive (LI) neurons were observed in the subnuclei interpolaris-caudalis transition (Vi/Vc) zone, the paratrigeminal nucleus (Pa5) and the superficial laminae of the caudal Vc/C2 zone. The pERK expression was detected as early as 2 min and peaked at 5 min after capsaicin or vehicle injection. The pERK expression in the Vi/Vc zone and Pa5 was bilateral, whereas it was predominantly ipsilateral in the caudal Vc/C2 zone. The capsaicin treatment of the whisker pad produced pERK expression in the rostro-caudal middle portion of the ipsilateral Vc, but small number of pERK-LI cells were observed after vehicle treatment. The pERK expression was similar in the Vi/Vc zone following capsaicin injection into the upper or lower molar tooth pulp, whereas the pERK expression was in the lateral portion of the caudal Vc/C2 zone after upper molar injection and restricted to the medial portion of the Vc/C2 zone after the lower molar capsaicin. These data were confirmed with Western blots. There were differences in the distribution of Fos protein-like immunoreactive (LI) cells and pERK-LI cells following tooth pulp stimulation. After capsaicin application into the upper molar tooth pulp, no pERK-LI cells were observed in the ventral part of the Vi/Vc zone, whereas many Fos protein-LI cells were expressed in this region. The difference in the distribution pattern of pERK- and Fos protein-LI cells in the Vi/Vc zone suggests their differential temporal expression profiles after capsaicin. The present findings suggest that tooth-pulp-driven neurons in the spinal trigeminal nucleus are involved in tooth pulp pain through activation of the intracellular signal transduction pathway that involves earlier ERK phosphorylation and subsequent Fos expression.