Long-term survivor of relapsed MFH on the thigh treated with autologous formalin-fixed tumor vaccine (AFTV) combined with limb-sparing surgery and radiotherapy

Malignant fibrous histiocytoma (MFH) is an aggressive spindle cell cancer of soft-tissue sarcoma type in the elderly, mostly affecting the extremities. Lesions > 5 cm, positive margins, and local recurrence are significant poor prognostic indicators. The strongest predictor for distant metastasis was tumor size (> 5 cm), and for overall survival, presence of local recurrence. Limb-sparing extensive tumor resection is preferred to achieve negative surgical margins. However, in some circumstances, amputation is inevitable. Recent studies demonstrated that adjuvant radiotherapy for microscopically positive surgical margins significantly improved local control and disease-free survival rates. Therefore, effective therapeutic strategies against locally relapsed high grade MFH are required to prevent distant metastasis and to achieve long-term disease-free survival. Here, we report local relapse of high grade MFH treated by successive application of autologous formalin-fixed tumor vaccination (AFTV) with limb-sparing surgery and postoperative radiotherapy. The patient is alive and well, disease-free and with no functional impairment, more than five years after treatment

Το προσυνέδριο της IFLA στην Αθήνα, 19-21 Αυγούστου 2009

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Cell viabilities and biodegradation rates of DNA/protamine complexes with two different molecular weights of DNA.

Two types of DNA/protamine complexes were prepared from protamine sulfate and 7000 base pair (bp) DNA or original DNA to investigate the effect of the molecular weight of DNA on zeta potential, cell viability, flowability, soft tissue response, and biodegradation rate. The 7000 bp DNA/protamine complex had a negative charge while the original DNA/protamine complex had a positive charge. The cell viabilities (90.4-106.8%) of these complexes were close to each other. The 7000 bp DNA/protamine complex became a softer dough than that of the original DNA/complex when both were kneaded with water. In vivo, the original DNA/protamine complex showed a milder tissue response. The original DNA/protamine complex almost disappeared 30 days after implantation. The 7000 bp DNA/complex disappeared approximately 2 weeks after implantation and areas where samples were implanted became empty. Thereafter, the empty space was gradually replaced by new soft tissues. The original DNA/protamine complex showed low intercalation and groove binding ratios of daunorubicin hydrochloride. Results indicate that high DNA condensation by cationic protamine protected the penetration of degradation enzymes into these complexes. It was found that a high molecular weight of DNA reduced the biodegradation rate and flowability. This study suggests that DNA/protamine complexes could be candidates for biomaterials that control biodegradation rates and flowability.福岡歯科大学2013年

Osteogenic potential for replacing cells in rat cranial defects implanted with a DNA/protamine complex paste.

Osteoinductive scaffolds are required for bone tissue engineering. The aim of the present study was to assess the osteoinductive capacity of deoxyribonucleic acid (DNA)/protamine complexes in a rat model of critical-size calvarial defects. In addition, we investigated whether cultured mesenchymal-like cells (DP-cells) outgrown from DNA/protamine complex engrafted defects could differentiate to become osteogenic cells in vitro. DNA/protamine complexes were prepared by reactions between DNA and protamine sulfate solutions with stirring. Critical-sized (8mm) calvarial defects were created in the central parietal bones of adult rats. Defects were either left empty or treated with DNA/protamine complex scaffolds. Subsequently, micro-computed tomography (micro-CT), histological, and immunohistochemical analyses were performed. Micro-CT and histological assays showed that DNA/protamine complex engrafted defects had enhanced bone regeneration. DP-cells were expanded from explants of DNA/protamine complex engrafted defects using an explant outgrowth culture system. Osteogenesis-related factors were assessed in DP-cells after treatment with an osteoblast-inducing reagent (OIR). After 3months, nearly complete healing was observed for DNA/protamine complex engrafted calvarial defects. Increased alkaline phosphatase (ALP) activity and Alizarin red staining were found for cultured DP-cells. These cells had high expression levels of osteogenic genes, including those for RUNX-2, ALP, osteopontin, and osteocalcin. These results indicated that DNA/protamine complexes could facilitate bone regeneration in calvarial defects. Moreover, in vitro osteogenic induction experiments showed that DP-cells outgrown from DNA/protamine engrafted defects had an osteogenic potential. Based on these results, we suggest that DNA/protamine complexes may recruit osteocompetent cells in these defects, where they differentiate to osteogenic cells.福岡歯科大学2015年

Flavobacterium okayamense sp. nov. isolated from surface seawater

Strain KK2020170T, a Gram-stain negative, yellow colony-forming bacterium, was isolated from surface seawater sampled in Kojima Bay, Okayama, Japan. Phylogenetic analysis based on the 16S rRNA gene revealed that strain KK2020170T belongs to the genus Flavobacterium, with Flavobacterium haoranii LQY-7T (98.1% similarity) being its closest relative, followed by Flavobacterium sediminis MEBiC07310T (96.9%) and Flavobacterium urocaniciphilum YIT 12746T (96.0%). Whole-genome shotgun sequencing showed that strain KK2020170T, when paralleled with F. haoranii LQY-7 T, had 81.3% average nucleotide identity, and 24.6% in silico DNA–DNA hybridization values, respectively. The DNA G + C content of strain KK2020170T was 31.1 mol%. The most abundant fatty acids (> 10%) of strain KK2020170T were iso-C15: 0, iso-C17: 0 3-OH and iso-C15: 1 G. The dominant respiratory quinone of the strain was menaquinone MK-6. Based on the phylogenetic and phenotypic analysis results, we propose that strain KK2020170T represents a novel species, for which the name Flavobacterium okayamense sp. nov. has been proposed. The type strain is KK2020170T (= ATCC TSD-280 T = NBRC 115344 T)

Salmon DNA Accelerates Bone Regeneration by Inducing Osteoblast Migration.

The initial step of bone regeneration requires the migration of osteogenic cells to defective sites. Our previous studies suggest that a salmon DNA-based scaffold can promote the bone regeneration of calvarial defects in rats. We speculate that the salmon DNA may possess osteoinductive properties, including the homing of migrating osteogenic cells. In the present study, we investigated the influence of the salmon DNA on osteoblastic differentiation and induction of osteoblast migration using MG63 cells (human preosteoblasts) in vitro. Moreover, we analyzed the bone regeneration of a critical-sized in vivo calvarial bone defect (CSD) model in rats. The salmon DNA enhanced both mRNA and protein expression of the osteogenesis-related factors, runt-related transcription factor 2 (Runx2), alkaline phosphatase, and osterix (OSX) in the MG63 cells, compared with the cultivation using osteogenic induction medium alone. From the histochemical and immunohistochemical assays using frozen sections of the bone defects from animals that were implanted with DNA disks, many cells were found to express aldehyde dehydrogenase 1, one of the markers for mesenchymal stem cells. In addition, OSX was observed in the replaced connective tissue of the bone defects. These findings indicate that the DNA induced the migration and accumulation of osteogenic cells to the regenerative tissue. Furthermore, an in vitro transwell migration assay showed that the addition of DNA enhanced an induction of osteoblast migration, compared with the medium alone. The implantation of the DNA disks promoted bone regeneration in the CSD of rats, compared with that of collagen disks. These results indicate that the salmon DNA enhanced osteoblastic differentiation and induction of migration, resulting in the facilitation of bone regeneration.福岡歯科大学2016年

Influence of local anesthetics on tissue blood flow−The change of skin blood flow by subcutaneous injection into the back skin of rabbits−

We examined the influence of lidocaine, mepivacaine, ropivacaine , bupivacaine , and levobupivacaine on skin blood flow in rabbits using a laser Doppler blood flowmeter. New Zealand White rabbits were anesthetized with sodium thiopental. 0.2 ml of 0.125～ 2.0% lidocaine, 0.125～3.0% mepivacaine, 0.125～0.75% ropivacaine, 0.125～0.5% bupivacaine, 0.125～0.75% levobupivacaine, 2.0% lidocaine with adrenaline (A) (1/80,000), and 3.0% propitocaine with felypressin (0.03 IU) were injected subcutaneously into the back skin of the rabbit’s. Physiological saline (0.2 ml) was injected as a control. We measured the skin blood flow using a laser Doppler blood flowmeter before and 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, and 10 minutes after the injection. The blood flow after the injection of each local anesthetic was converted into a percentage of the control value (before injection). The mean values of the skin blood flow before the injection in each group showed no significant differences. The skin blood flow was increased 30 seconds after the injection of physiological saline and showed no significant changes from 1 to 10 minutes after the injection. The skin blood flow was significantly increased by the subcutaneous injection of 0.125, 0.25, 0.5, 0.75, 1.0, and 2.0% lidocaine, 1.0, 2.0, and 3.0% mepivacaine, 0.125, 0.25, and 0.5% bupivacaine, and 0.75% levobupivacaine. The skin blood flow was significantly decreased by the injection of 0.125, 0.25, 0.5, and 0.75% mepivacaine, 0.125, 0.25, 0.5, and 0.75% ropivacaine, 0.125, 0.25, and 0.5% levobupivacaine. Lidocaine and bupivacaine increased the skin blood flow dependent on their concentration. On the other hand, ropivacaine decreased the skin blood flow dependent on its concentration. Mepivacaine decreased the skin blood flow at concentrations lower than 0.75% and increased it at a concentration higher than 1.0%. Levobupivacaine decreased the skin blood flow at concentrations lower than 0.5%, and increased it at those higher than 0.75%. The 0.5% bupivacaine and 2.0% lidocaine led to the most marked increase in skin blood flow, and 2.0% lidocaine?A and 0.75% ropivacaine led to the most marked decrease.2013博士（歯学）松本歯科大

Comparison of Various Methods of Assaying the Cytotoxic Effects of Ethanol on Human Hepatoblastomaells (HUH-6 Line)

The sensitivity of five kinds of cytotoxicity assays using ethanol on human hepatoblastoma cells (HUH-6 line), which were cultured as monolayers or spheroids, was compared. Ethanol was chosen as a test because it acts on cell membranes directly without being metabolized and exerts its cytotoxicity. The assay methods used were as follows: 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), colony formation, cell growth and DNA assays. The sensitivity of the assays was: LDH &#60; DNA &#60; cell growth &#60; MTT &#60; colony formation. LDH assay had the advantage that the same culture could be used for multiple assays, but when a small number of cells were assayed, no significant increase in the release of LDH was detected in the assay cultures compared with the control cultures. Although the DNA and cell growth assays were more sensitive than the LDH assay, the extent of cell damage may be underestimated because the damaged cells and DNA present in the cultures are included in the assay samples. On the other hand, both MTT and colony formation assays showed a high sensitivity. The MTT assay was done within 24 h after ethanol was added to the cultures and was applicable to both monolayer and spheroid cultures, while the colony formation assay required 1-2 weeks and it was applicable only to monolayer cultures. Taken together, the MTT assay was the most suitable method to evaluate the cytotoxic effects of ethanol on HUH-6 cells cultured as either monolayers or spheroids.</p