Kei Tomihara

Clinical Assessment of the Relationship Between the Third Molar and the Inferior Alveolar Canal Using Panoramic Images and Computed Tomography

PURPOSE To predict the relationship between lower third molars and the inferior alveolar canal (IAC) from panoramic radiographs, and to establish criteria for using computed tomography (CT). MATERIALS AND METHODS A retrospective cohort study was performed involving 443 patients (695 teeth). Predictor variables were the distance between the third molar and the IAC, and findings according to the Roods criteria. Outcome variables were the absence of cortication between the third molar and the IAC on the CT image, and injury of the inferior alveolar nerve (IAN). Statistical analysis was performed to assess the relationship between predictor and outcome variables. RESULTS All patients had preoperative panoramic radiographs, and 71 patients (119 teeth) also had CT images. On CT examination, 48 teeth (40.3%) showed absence of cortication. Injury of the IAN was reported in 7 cases (1.0%), 5 of which exhibited absence of cortication; the remaining 2 did not have CT scans. Five of the 48 cases showing absence of cortication exhibited IAN injury, and none of the cases with cortication exhibited IAN injury. On the panoramic images, the following signs were strongly correlated with absence of cortication: a superimposed relationship between the third molar and the IAC; darkness of the root; and diversion and narrowing of the IAC. CONCLUSION Presence of Roods criteria was a predictor for a contact relationship between the third molar and the IAC, and an indication for CT examination. However, a superimposed relationship and the absence of Roods criteria did not necessarily signify a separate relationship between third molar and the IAC.

Bcl-xL Gene Transfer Inhibits Bax Translocation and Prolongs Cardiac Cold Preservation Time in Rats

Background—Apoptosis is an important cause of early graft loss after heart transplantation. Bcl-xL was reported to protect the heart against normothermic ischemia and reperfusion injury. In this study, we determined whether overexpression of Bcl-xL could inhibit tissue injury resulting from prolonged cold preservation followed by warm reperfusion of heart transplants. Methods and Results—Lewis rat hearts were transduced with an adenovirus vector harboring Bcl-xL cDNA (AxCAhBclxL) 4 days before collection of tissue. After preservation in University of Wisconsin solution at 4°C for 24 hours, the heart was either perfused with a Langendorff device ex vivo or used for heterotopic heart transplantation in vivo. Bcl-xL gene transfer significantly reduced the infarct size (23.0±2.6% versus 47.7±7.0% in saline control and 48.6±6.1% in vector control, P<0.01) after 2-hour reperfusion at 37°C with the Langendorff device and significantly decreased creatine kinase release (0.82±0.27 IU, versus 1.57±0.33 and 1.50±0.37 IU in saline and vector controls, respectively; P<0.05). In heart transplantation, overexpresson of Bcl-xL inhibited Bax translocation from the cytosol to the mitochondria, resulting in decreased cytochrome c release from the mitochondria; it also significantly decreased cardiac cell apoptosis and improved graft survival rate after long cold preservation, followed by warm reperfusion. Conclusions—Bcl-xL gene transfer inhibited the translocation of Bax and prolonged the cold preservation time of cardiac transplants. This may be a potential therapeutic method in clinical practice.

Carcinoembryonic Antigen–Targeted Selective Gene Therapy for Gastric Cancer through FZ33 Fiber-Modified Adenovirus Vectors

Purpose: A major problem when using the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency in cancer cells or to side effects in normal cells. There is an urgent requirement to improve the specificity of gene delivery in the context of cancer gene therapy. Experimental Design: We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33, within the HI loop (Adv-FZ33). A remarkable degree of targeted gene delivery to gastric cancer cells was obtained with Adv-FZ33 with the fully human anti–carcinoembryonic antigen (CEA) monoclonal antibody, C2-45. Results:In vitro LacZ or EGFP gene expression after Adv-FZ33 infection via C2-45 was 20 times higher than control monoclonal antibody in MKN-45 at 1,000 viral particles/cell. We generated Ax3CAUP-FZ33 (UP-FZ33), which is an Adv-FZ33 derivative vector expressing a therapeutic gene (i.e., Escherichia coli uracil phosphoribosyltransferase), which converts 5-fluorouracil (5-FU) directly to 5-fluoro-UMP. UP-FZ33 with C2-45 enhanced the cytotoxicity of 5-FU by 10.5-fold in terms of IC50 against MKN-45 compared with control IgG4. In a nude mouse peritoneal dissemination model, tumor growth in mice treated with UP-FZ33/C2-45/5-FU was significantly suppressed, and tumor volumes were less than one-fourth of those of the control IgG4 group (P < 0.05). The median survival time of the UP-FZ33/C2-45/5-FU group was significantly longer than those treated with PBS or 5-FU only (P < 0.01). Conclusions: These data suggest that CEA-targeted FZ33 mutant adenovirus-mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers.

Gene transfer of the CD40-ligand to human dendritic cells induces NK-mediated antitumor effects against human carcinoma cells

The CD40‐ligand (CD40L) is a key molecule for the activation of dendritic cells (DCs), followed by the induction of DC maturation and cytokine production. Here we found that DC infected with adenovirus vector encoding human CD40L (CD40L‐DC) displayed significantly higher levels of immune accessory molecules and IL‐12 production than did uninfected cells, and that CD40L‐DC produced much higher levels of IFN‐γ. To investigate whether CD40L‐DC‐derived these soluble factors could stimulate NK cells without physical cell‐to‐cell contact, we cocultured NK cells with CD40L‐DC in transwell culture plates. NK cells showed up‐regulated cytotoxic activity toward various squamous oral cell carcinoma (OSC‐70, HSC‐2, HSC‐3), and we determined that both IL‐12 and IFN‐γ contributed to the CD40L‐DC‐mediated NK cell activation. NK cells stimulated with CD40L‐DC resulted in the induction of the cell surface expression of TRAIL, the production of IFN‐γ and intracellular accumulation of granzyme B. The cytotoxic activity of NK cells stimulated with CD40L‐DC could be mostly inhibited by neutralizing antibody for TRAIL and completely abrogated by the combination of antibody and exocytosis inhibitor, indicating that this was mainly mediated by a TRAIL‐TRAIL‐receptor interaction and granule exocytosis. Moreover, CD40L‐DC‐activated NK cells could induce up‐regulation of a death‐receptor TRAIL‐R2 (DR5) and down‐regulation of a decoy receptor TRAIL‐R3 (DcR1) on carcinoma cells. Overall, these results have revealed that CD40L‐DC could activate an innate immune reaction by stimulating NK cells followed by carcinoma cells, supporting that administration of CD40L‐DC may have potential as an anticancer therapy.

Squamous cell carcinoma of the buccal mucosa in a young adult with history of allogeneic bone marrow transplantation for childhood acute leukemia

Secondary cancers are severe complications in patients who have had allogeneic bone marrow transplantation for childhood leukemia. We describe here a case of squamous cell carcinoma (SCC) of the buccal mucosa in a young adult patient who had had allogeneic bone marrow transplantation for childhood acute leukemia.

Sendai viral vector mediated angiopoietin-1 gene transfer for experimental ischemic limb disease

Sendai virus vector is emerging as a promising vector for gene therapy, and angiopoietin-1 (Ang-1) has been reported to improve the blood flow recovery in the ischemic limb or heart. In this study, we constructed a human Ang-1-expressing Sendai viral vector (SeVhAng-1) and injected it into the ischemic limb of rats. We found that SeVhAng-1 improved the blood flow recovery and increased the capillary density of the ischemic limb, compared with the controls. We also found that SeVhAng-1 increased p-Akt during the early period of limb ischemia, and decreased apoptosis in ischemic limb. It suggests that SeVhAng-1 may serve as a potential therapeutic tool in ischemic limb disease.

Gene transfer of noncleavable cell surface mutants of human CD154 induces the immune response and diminishes systemic inflammatory reactions.

CD154 (CD40-ligand) is a critical transmembrane molecule with potent immune-stimulatory properties that is used in clinical applications of gene therapy for leukemia and lymphoma. However, CD154 is cleaved into a soluble form, and high levels of sCD154 contribute to systemic inflammatory and cardiovascular diseases, suggesting a deleterious side effect of CD154 gene therapy. In this study, we engineered noncleavable mutants of human CD154 with point mutations to develop a potentially less toxic molecule in vivo. In contrast to wild-type CD154 (CD154-WT) subsequently released as sCD154, both mutants CD154-M3 and CD154-M4 were resistant to cleavage in tumor cells. Also, CD40-expressing leukemia B cells transfected with CD154-M3 mutant were highly effective stimulators in a mixed lymphocyte-leukemia reaction, indicating that CD154-M3 mutant did not lose biologic activity. In mice transplanted with tumors expressing CD154-WT, we found increased plasma levels of human sCD154 followed by various systemic inflammatory reactions such as glomerulonephritis and an increased number of infiltrating mononuclear cells in the liver. However, CD154-M3 mutant did not induce any systemic inflammatory effects in vivo. As such, the noncleavable mutant of CD154 is fully capable of inducing the immune response with less toxic properties and is a useful tool for CD154 immune gene therapy.