Osteoinductive potential of recombinant BMP-9 in bone defects of mice treated with antiresorptive agents.

The aim of the present study was to investigate the effects of recombinant human (rh)BMP-9 on bone regenerative potential in a mouse model of antibody-mediated antiresorptive therapy (AMART). A monoclonal anti-murine receptor activator of nuclear factor-kappa B ligand (RANKL) antibody (mAb) was used to create an AMART model in mice. rhBMP-9 combined with collagen membrane was implanted in calvarial defects in mAb-treated mice. After 4 weeks, the bone formative potential in the defects was evaluated by micro-computed tomography and histological approaches. The groups implanted with rhBMP-9-containing collagen membranes demonstrated substantial osteopromotive potential, with significantly greater new bone volume (Sham + BMP-9 group; 0.86 ± 0.29 mm3 and mAb + BMP-9 group; 0.64 ± 0.16 mm3) than control PBS-membranes (Sham + PBS group; 0.44 ± 0.29 mm3 and mAb + PBS group; 0.24 ± 0.12 mm3) in both sham and mAb-treated mice. In line with in vivo study, bone marrow cells isolated from both sham and mAb-treated mice confirmed greater osteogenic potential upon stimulation with rhBMP-9 in vitro. These findings suggest for the first time that local rhBMP-9 administration might be a strategy to accelerate bone regeneration in the context of AMART.

Double sentinel lymph node mapping with indocyanine green and 99m-technetium-tin colloid in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) frequently metastasizes to cervical lymph nodes, which is the most known prognostic factor. Screening methods to identify sentinel lymph nodes (SLNs) are therefore of great interest for the management of potential neck metastasis. The purpose of this study was to evaluate the clinical benefit of double SLN mapping with indocyanine green (ICG) and 99m-technetium-tin colloid ((99m)Tc-tin colloid) for sentinel node navigation surgery (SNNS). Between 2007 and 2010, 16 patients diagnosed with OSCC were investigated by SLN biopsy using the double mapping method. (99m)Tc-tin colloid was injected into the peri-tumoural region on the preoperative day, and ICG was administered intraoperatively in the same position to assist in detecting nodes during surgery. Based on the gamma-ray signal and near-infrared (NIR) fluorescence of ICG, SLNs were identified and thereafter assessed pathologically and genetically for cancer involvement. Radio-guided detection was successful for all patients. ICG mapping identified a relatively larger number of nodes, suggesting that several non-SLNs were potentially involved. The double mapping method assisted surgeons to explore SLNs. Since the ICG fluorescence was shielded by the subcutaneous fatty tissue and the muscle layer including platysma and sternocleidomastoid, it was necessary to retract the tissue away from nodes.

Detection of p16 promoter methylation in the serum of oral cancer patients.

P16 promoter methylation occurs frequently in oral squamous cell carcinoma (OSCC). For the early detection of tumour-related aberrant DNA, we examined p16 methylation using the methylation-specific polymerase chain reaction (MSP) in tumour and serum samples of 17 OSCC patients. Aberrant p16 methylation was detected in 11 (64.7%) cases of primary OSCC. Of these 11 patients, 6 (54.5%) showed the same alteration in their serum. No methylation was found in control groups. Interestingly, DNA was detected in the serum of 3 out of 4 patients with recurrence. These results suggest that the MSP may be a sensitive and useful method for detecting recurrent OSCC.

Response of diffuse sclerosing osteomyelitis of the mandible to alendronate: follow-up study by 99mTc scintigraphy.

We report a case of diffuse sclerosing osteomyelitis of the mandible responded to alendronate, after a poor response to intravenous antibiotics, antibiotic irrigation-perfusion, and decortication. The patient was given an intravenous infusion of 10mg of alendronate. Pain resolved within 24 h. There were no severe adverse events. Increased uptake of 99mTc in the mandible almost completely disappeared 3 months after treatment.

In vivo proliferation of differentiated pancreatic islet beta cells in transgenic mice expressing mutated cyclin-dependent kinase 4.

AIMS/HYPOTHESIS: It has previously been hypothesised that highly differentiated endocrine cells do not proliferate or regenerate. However, recent studies have revealed that cyclin-dependent kinase 4 (CDK4) is necessary for the proliferation of pancreatic islet beta cells. The aim of this study was to determine whether activation of CDK4 can potentially be used as a radical treatment for diabetes without malignant transformation. METHODS: We generated transgenic mice expressing mutant CDK4 under the control of the insulin promoter to examine the effect of activated CDK4 overexpression in the postnatal development of pancreatic islets. RESULTS: In the transgenic mice, total CDK4 protein expression was increased by up to 5-fold, with a concomitant increase in CDK4 activity indicated by the detection of phosphorylated Rb protein in pancreatic islets. Histopathologically, many cells tested positive for proliferating cell nuclear antigen, and pancreatic islets displayed hyperplasia due to the extreme proliferation of beta cells containing a large number of insulin granules. Pancreatic islet alpha, delta and PP cells did not increase. Over an 18-month observation period, the transgenic mice did not develop insulinoma. Levels of expression of GLUT1 and c-myc were comparable to those in the littermates of the transgenic mice. GLUT2 expression was identified in the pancreatic islets of transgenic mice. No significant differences in telomerase activities were detected between transgenic mice and their littermates. Transgenic mice were superior to their littermates in terms of glucose tolerance and insulin secretion in response to the intraperitoneal injection of glucose, and hypoglycaemia was not observed. CONCLUSIONS/INTERPRETATION: Activated CDK4 stimulates postnatal pancreatic beta cell proliferation, during which the highly differentiated phenotypes of pancreatic islet beta cells are preserved without malignant transformation.

Intraoperative real-time genetic diagnosis for sentinel node navigation surgery.

Sentinel node navigation surgery (SNNS) has received considerable attention for its role in deciding whether to perform neck dissection in patients with early oral cancer. However, diagnostic accuracy and its intraoperative availability of results remain important concerns. First, we shortened the examination time required for genetic diagnosis. Second, we assessed the quality of the extracted mRNA. Third, 10 patients with early N0 oral cancer underwent SNNS, using our new technique for genetic diagnosis to determine whether neck dissection was required. The examination time of our one-step reverse-transcriptase polymerase chain reaction method using a minicolumn and LightCycler was successfully shortened to 2 h, permitting intraoperative genetic diagnosis. The extracted mRNA was of high quality. Six sentinel nodes in four patients were diagnosed to be metastatic on genetic diagnosis; these patients underwent neck dissection. The other six patients avoided unnecessary surgery. We conclude that intraoperative genetic diagnosis of micrometastasis holds promise of being a sensitive method that can be used to support SNNS.

Use of the curved linear-array echo endoscope to identify gastrorenal shunts in patients with gastric fundal varices.

BACKGROUND AND STUDY AIMS: : Balloon-occluded retrograde transvenous obliteration (B-RTO) has emerged as an effective, minimally invasive treatment for fundal varices. B-RTO requires a spontaneously developed gastrorenal shunt as a pathway for the balloon catheter to reach the fundal varices. We used a curved linear-array (CLA) echo endoscope in patients with fundal varices to identify gastrorenal shunts, and compared the detection rate with the gold standard, contrast-enhanced computed tomography (CECT). PATIENTS AND METHODS: A total of 40 patients with fundal varices were examined with both CLA echo endoscopy and CECT. The CECT images were retrospectively and independently evaluated by two gastroenterologists who were unaware of the clinical details, including the results of the CLA echo endoscopy. RESULTS: CLA echo endoscopy identified gastrorenal shunts in 26/40 patients with fundal varices. It visualized the shunt in a longitudinal direction and provided images of the connections of the shunt at both ends, the fundal varices and the left renal vein/branch of the inferior adrenal vein. The kappa index for CLA echo endoscopy and CECT for the identification of gastrorenal shunt was 0.9 (95 % CI, 0.6 to 1.0). When the cutoff point for the diameter of the gastrorenal shunt detected by the CLA echo endoscope was set at equal to or greater than 5 mm, the kappa index was 1.0 (95 % CI, 0.7 to 1.0). CONCLUSIONS: These results suggest that CLA echo endoscopy can successfully identify gastrorenal shunt and provide detailed morphological information. It also efficiently identifies patients suitable for B-RTO, particularly in cases of acute bleeding. It also has considerable potential for providing detailed information with regard to the treatment of gastric varices.

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure.

Suppressed expression of transgenes in vivo is the major obstacle in the gene therapy. For the long-term expression, we utilized a chromatin insulator from sea urchin arylsulfatase (Ars) gene locus (Ars insulator, ArsI), which has been shown to epigenetically regulate gene expression across species. ArsI was able to prevent silencing of the transgene in a myeloid cell line, HL-60, and a murine embryonic stem cell line, CCE, in an orientation-dependent manner, but not in Huh-7, K562 and MCF-7 cells, indicating that the effect of ArsI on gene silencing was cell type dependent. Although anti-silencing effect of ArsI was almost equivalent to that of chicken beta-globin insulator, incorporation of ArsI into lentiviral vector had little effect on the virus titer compared with chicken beta-globin insulator. Clonal analysis of transduced HL-60 cells revealed that ArsI protects the lentiviral vector from position effects regardless of its orientation. Furthermore, chromatin immunoprecipitation assays revealed that a high acetylation level was observed in the promoter of the insulated vector, whereas that of ArsI was independent of its anti-silencing capacity. In addition to it having little deteriorative effect on the virus titer, the identified anti-silencing effect of ArsI suggested its possibility for application in gene therapy.

Transcriptional activation of cyclin-dependent kinase inhibitor, p21waf1 gene by treatment with a differentiation inducing agent, vesnarinone in a human salivary gland cancer cell line.

Recently, a new concept for cancer therapy termed "tumor dormancy therapy" has been proposed. The concept of this therapy is to prolong the survival time of cancer patients while maintaining their quality of life. We have been developing a differentiation-inducing therapy, which is included in the tumor dormancy therapy, for salivary gland cancer. In this study, we examined the effect of a differentiation-inducing drug, Vesnarinone on the growth of several cancer cells, and examined the molecular mechanism by which Vesnarinone induces the cyclin dependent kinase inhibitor, p21waf1 in the cancer cells. Vesnarinone significantly suppressed the growth of TYS (salivary gland cancer cells), PC3 (prostate cancer cells), and A431 (squamous cell cancer cells). Furthermore, Vesnarinone dose-dependently enhanced the expression of p21waf1 mRNA in TYS cells. Using the luciferase reporter assay it was found that the enhancement of p21waf1 mRNA expression by Vesnarinone was through direct transcriptional activation of the p21waf1 promoter. Thus, analyzing the molecular mechanisms of differentiation inducing drugs may lead to the development of a new therapeutic strategy for several human malignancies, including salivary gland cancer.

Vesnarinone, a differentiation inducing drug, directly activates p21(waf1) gene promoter via Sp1 sites in a human salivary gland cancer cell line.

We previously demonstrated that a differentiation inducing drug, vesnarinone induced the growth arrest and p21(waf1) gene expression in a human salivary gland cancer cell line, TYS. In the present study, we investigated the mechanism of the induction of p21(waf1) gene by vesnarinone in TYS cells. We constructed several reporter plasmids containing the p21(waf1) promoter, and attempted to identify vesnarinone-responsive elements in the p21(waf1) promoter. By the luciferase reporter assay, we identified the minimal vesnarinone-responsive element in the p21(waf1) promoter at -124 to -61 relative to the transcription start site. Moreover, we demonstrated by electrophoretic mobility shift assay that Sp1 and Sp3 transcription factors bound to the vesnarinone-responsive element. Furthermore, we found that vesnarinone induced the histone hyperacetylation in TYS cells. These results suggest that vesnarinone directly activates p21(waf1) promoter via the activation of Sp1 and Sp3 transcription factors and the histone hyperacetylation in TYS cells.

Dihydropyrimidine dehydrogenase mRNA level correlates with the response to 5-fluorouracil-based chemo-immuno-radiation therapy in human oral squamous cell cancer.

The measurement of the intra-tumoral level of thymidylate synthetase (TS), and dihydropyrimidine dehydrogenase (DPD), may be useful in predicting tumor sensitivity to 5-fluorouracil (5-FU). In this study, we examined the mRNA levels of DPD and TS in 28 oral squamous cell carcinomas (SCC), and 22 salivary gland tumors by semi-quantitative reverse transcription polymerase chain reaction. Then we examined the correlation of the responsiveness of the patients with oral SCC to 5-FU with the intra-tumoral levels of DPD and TS mRNA. All specimens were obtained at the biopsy before treatment, and then the patients were treated by oral administration of a 5-FU compound (UFT), the irradiation of cobalt-60 (upto 60 Gy) and injection of an immuno-potentiator (OK-432). Intra-tumoral levels of DPD mRNA in the patients who showed CR (complete response) and PR (partial response) were significantly lower than those in the patients who showed NC (no change). However, intra-tumoral levels of DPD mRNA did not correlate with the local recurrence of the tumor during the observation period after initial treatment with or without surgical resection of the residual tumors. On the other hand, TS mRNA levels in the tumors did not correlate with any clinico-pathological parameters. These observations suggest that intra-tumoral levels of DPD mRNA may predict the tumor response to 5-FU-based chemo-immuno-radiation therapy in the patients with oral SCC.

Hemodynamic analysis of esophageal varices using color Doppler endoscopic ultrasonography to predict recurrence after endoscopic treatment.

BACKGROUND AND STUDY AIMS: The time to recurrence of esophageal varices may vary greatly between patients even after the same endoscopic therapy. To clarify the factors which contribute to recurrence after endoscopic treatment, the hemodynamics and morphology of the left gastric vein (LGV) were investigated using color Doppler endoscopic ultrasonography (EUS). PATIENTS AND METHODS: A total of 31 patients with high-risk esophageal varices underwent color Doppler-EUS before receiving endoscopic variceal ligation and endoscopic injection sclerotherapy combined therapy. Endoscopic examination was performed every 3 months after the treatment to evaluate recurrence of varices. RESULTS: A total of 18 patients responded to the therapy, while 13 patients did not respond, and had recurrence within 12 months. The hepatofugal flow velocity in the LGV trunk was significantly lower in the responders (9.9 vs. 13.9 cm/sec; P = 0.02). The branch pattern of the LGV was categorized into three groups: anterior branch dominant, posterior branch dominant, and no-dominant type. The incidence of the anterior branch dominant type was significantly less in responders (17 vs. 70 %; P = 0.01). There was no significant difference in the LGV trunk diameter and the size of the paraesophageal vein between the two groups. CONCLUSION: Risk factors for recurrence can be analyzed in detail using color Doppler-EUS. Further investigation using color Doppler-EUS may enable us to select the optimal way to treat esophageal varices to prevent recurrence.

Low power diode laser treatment using indocyanine green for eradication of esophageal varices.

BACKGROUND AND STUDY AIMS: Endoscopic variceal ligation (EVL) is an alternative to sclerotherapy for the treatment of esophageal varices, but is associated with higher rates of recurrence and subsequent bleeding than sclerotherapy. To prevent recurrence of varices after EVL, we have developed a low-dose diode laser therapy combined with the injection of indocyanine green, which allows enhanced tissue absorption of the laser beam selectively around varices. In this study we investigated the efficacy and safety of this technique. PATIENTS AND METHODS: Eight patients with F2 or F3 esophageal varices were enrolled. At 1 week after EVL, indocyanine green solution (1 mg/ml) was injected submucosally around the remaining varices. A diode laser (power 10 watts) was applied to the surface from the esophagogastric junction to 5 cm above it. The spot size was kept to 5 mm in diameter. RESULTS: Laser irradiation was performed safely, without bleeding from the varices, or perforation. There were no major complications. Endoscopy 1 month later showed F0 forms in seven patients, F1 in one patient, and no red color sign in any patient. No recurrence of varices has been observed in any of the patients during the follow-up period of at least 12 months. CONCLUSION: This technique may provide a simple, safe and effective procedure, as an additional treatment to EVL, for the prevention of recurrence of esophageal varices.

Low-dose retinoic acid enhances in vitro invasiveness of human oral squamous-cell-carcinoma cell lines.

Retinoids inhibit the proliferation of several types of tumour cells, and are used for patients with several malignant tumours. In this study, we examined the effect of retinoic acids (RAs) on the invasive potentials of the oral squamous cell carcinoma (SCC) cells, BHY and HNt. BHY cells expressed all of retinoid nuclear receptors (RARalpha, beta, gamma, and RXRalpha) and cytoplasmic retinoic acid binding proteins (CRABP1 and CRABP2). HNt cells lacked the expression of RARbeta, but expressed other nuclear receptors and CRABPs. All-trans retinoic acid (ATRA) and 13-cis retinoic acid (13-cisRA) (10(-6)and 10(-7)M) inhibited the growth of the cells, but low-dose ATRA and 13-cisRA (10(-8)M) marginally affected the growth of the cells. Surprisingly, low-dose RAs enhanced the activity of tissue-type plasminogen activator (tPA), and activated pro-matrix metalloproteinases (proMMP2 and proMMP9). Activation of proMMP2 and proMMP9 was inhibited by aprotinin, a serine-proteinase, tPA inhibitor. Furthermore, low-dose RAs enhanced the in vitro invasiveness of BHY cells. These results indicate that low-dose RAs enhances the in vitro invasiveness of oral SCC cells via an activation of proMMP2 and proMMP9 probably mediated by the induction of tPA.

Role of HGF/c-met system in invasion and metastasis of oral squamous cell carcinoma cells in vitro and its clinical significance.

We examined the role of the hepatocyte growth factor (HGF)/c-met system on invasion and metastasis of oral squamous cell carcinoma (SCC) cells. In monolayer culture, exogenous HGF marginally affected the growth of oral SCC cells (BHY, HN, IH) and human gingival epithelial cells (GE). In type I collagen matrix, however, HGF significantly enhanced the invasive growth of the cancer cells (p < 0.05). We detected the expression of c-met (HGF receptor) mRNA in all of the cancer cells, but not in human gingival fibroblasts (GF). Oral SCC cells did not secret HGF protein into the medium, but GF secreted a large amount of HGF protein (15 ng/ml). Furthermore, HGF markedly enhanced the migration of cancer cells in a Transwell invasion chamber. Then, we examined the serum levels of HGF in oral SCC patients, or HGF concentrations in oral cancer tissues. Serum levels of HGF in the patients were significantly higher than those in healthy volunteers (p < 0.05). After initial treatment, all of the tumor-free survivors showed a marked decline in the serum HGF levels. Furthermore, HGF concentrations in metastatic cancer tissues were significantly higher than those of non-metastatic cancer tissues and normal gingiva (p < 0.01). These results suggest that HGF plays an important role in invasion and metastasis of oral SCC cells as a paracrine factor, and an elevated HGF level in the cancer tissue can be a predictive marker for metastasis formation in patients with oral SCC.

Feedback inhibition of amidophosphoribosyltransferase regulates the rate of cell growth via purine nucleotide, DNA, and protein syntheses.

To clarify the contributions of amidophosphoribosyltransferase (ATase) and its feedback regulation to the rates of purine de novo synthesis, DNA synthesis, protein synthesis, and cell growth, mutated human ATase (mhATase) resistant to feedback inhibition by purine ribonucleotides was engineered by site-directed mutagenesis and expressed in CHO ade (-)A cells (an ATase-deficient cell line of Chinese hamster ovary fibroblasts) and in transgenic mice (mhATase-Tg mice). In Chinese hamster ovary transfectants with mhATase, the following parameters were examined: ATase activity and its subunit structure, the metabolic rates of de novo and salvage pathways, DNA and protein synthesis rates, and the rate of cell growth. In mhATase-Tg mice, ATase activity in the liver and spleen, the metabolic rate of the de novo pathway in the liver, serum uric acid concentration, urinary excretion of purine derivatives, and T lymphocyte proliferation by phytohemagglutinin were examined. We concluded the following. 1) ATase and its feedback inhibition regulate not only the rate of purine de novo synthesis but also DNA and protein synthesis rates and the rate of cell growth in cultured fibroblasts. 2) Suppression of the de novo pathway by the salvage pathway is mainly due to the feedback inhibition of ATase by purine ribonucleotides produced via the salvage pathway, whereas the suppression of the salvage pathway by the de novo pathway is due to consumption of 5-phosphoribosyl 1-pyrophosphate by the de novo pathway. 3) The feedback inhibition of ATase is more important for the regulation of the de novo pathway than that of 5-phosphoribosyl 1-pyrophosphate synthetase. 4) ATase superactivity leads to hyperuricemia and an increased bromodeoxyuridine incorporation in T lymphocytes stimulated by phytohemagglutinin.

Identification of two novel mutations in the CLCN5 gene in Japanese patients with familial idiopathic low molecular weight proteinuria (Japanese Dent's disease).

Two Japanese patients, belonging to unrelated families, with idiopathic low-molecular-weight proteinuria (LMWP; Japanese Dent's disease) showed novel mutations of the gene encoding renal-specific chloride channel 5 (CLC-5). Proteinuria was first noticed at the ages of 2 and 3 years in patients 1 and 2, respectively. During follow-up, marked increases in urinary ss(2)-microglobulin levels, hypercalciuria, and high levels of urinary excretion of growth hormone were observed in both patients. Nephrocalcinosis was detected in patient 2. Renal biopsy specimens from both patients showed minimal alterations in glomeruli and tubulointerstitium, except for mild mesangial proliferation in patient 2. DNA sequence analysis of the entire 2,238-bp coding region and exon-intron boundaries of the CLCN5 gene showed the presence of two novel mutations in exon 10, consisting of one missense mutation (I524K) in patient 1 and one nonsense mutation (R637X) in patient 2. DNA analysis and measurement of urinary ss(2)-microglobulin levels in family members indicated an X-linked mode of inheritance in patient 1 and sporadic occurrence in patient 2. These results have expanded our understanding of the association between idiopathic LMWP (Japanese Dent's disease) and mutations of the CLCN5 gene.

Inhibition of the Wnt signaling pathway by Idax, a novel Dvl-binding protein.

In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of beta-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax in Xenopus embryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not beta-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.