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author:("matsuzaki, T.")
1.  Effective recovery of highly purified CD326+ tumor cells from lavage fluid of patients treated with a novel cell-free and concentrated ascites reinfusion therapy (KM-CART) 
SpringerPlus  2015;4:780.
For the production of tumor-specific vaccines, including dendritic cell (DC) vaccines, the tumor cells themselves are an ideal source. Floating tumor cells in the ascites fluid from patients with malignant ascites are a good candidate source, but it is not easy to obtain pure tumor cells from ascites because of various types of cell contamination as well as protein aggregates. We here report an effective method to recover pure tumor cells from malignant ascites. We used lavage fluid from 13 patients with malignant ascites who were treated with modified cell-free and concentrated ascites reinfusion therapy (KM-CART). Cellular components were separated from the lavage fluid by centrifugation, enzymatic digestion and hemolysis. Tumor cells were purified by depleting CD45+ leukocytes with antibody-conjugated magnetic beads. The tumor cell lysate was extracted by freeze-and-thaw cycles. The mean obtained total cell number was 7.50 × 107 cells (range 4.40 × 106–2.48 × 108 cells). From this fraction, 6.39 × 106 (range 3.23 × 105–2.53 × 107) CD45− cells were collected, and the tumor cell purity was over 80 % defined as CD45−CD326+. A sufficient amount of tumor lysate, average  = 2416 μg (range 25–8743 μg), was extracted from CD45−CD326+ tumor cells. We here established an effective method to produce highly purified tumor cells from KM-CART lavage fluid. The clinical feasibility of this simple preparation method for generating tumor lysate should be examined in clinical studies of DC vaccines.
PMCID: PMC4683161  PMID: 26702369
CART; Tumor lysate; Ascites
2.  Identification and Characterization of CXCR4-Positive Gastric Cancer Stem Cells 
PLoS ONE  2015;10(6):e0130808.
Diffuse-type solid tumors are often composed of a high proportion of rarely proliferating (i.e., dormant) cancer cells, strongly indicating the involvement of cancer stem cells (CSCs) Although diffuse-type gastric cancer (GC) patients have a poor prognosis due to high-frequent development of peritoneal dissemination (PD), it is limited knowledge that the PD-associated CSCs and efficacy of CSC-targeting therapy in diffuse-type GC. In this study, we established highly metastatic GC cell lines by in vivo selection designed for the enrichment of PD-associated GC cells. By microarray analysis, we found C-X-C chemokine receptor type 4 (CXCR4) can be a novel marker for highly metastatic CSCs, since CXCR4-positive cells can grow anchorage-independently, initiate tumors in mice, be resistant to cytotoxic drug, and produce differentiated daughter cells. In clinical samples, these CXCR4-positive cells were found from not only late metastasis stage (accumulated ascites) but also earlier stage (peritoneal washings). Moreover, treatment with transforming growth factor-β enhanced the anti-cancer effect of docetaxel via induction of cell differentiation/asymmetric cell division of the CXCR4-positive gastric CSCs even in a dormant state. Therefore, differentiation inducers hold promise for obtaining the maximum therapeutic outcome from currently available anti-cancer drugs through re-cycling of CSCs.
PMCID: PMC4481351  PMID: 26110809
3.  Induction of hepatic Bach1 mRNA expression by carbon tetrachloride-induced acute liver injury in rats 
Biomedical Reports  2014;2(3):359-363.
Hepatic oxidative stress is a major contributor to the pathogenesis of several acute liver diseases. Diagnostic markers of hepatic oxidative stress may facilitate early detection and intervention. Bach1 is an oxidative stress-responsive transcription factor that represses heme oxygenase 1 (HO-1), the rate-limiting enzyme in the catabolism of heme, a potent pro-oxidant. We previously demonstrated that carbon tetrachloride (CCl4) causes oxidative hepatic injury in rats, exacerbated by free heme, suggesting that CCl4 may affect Bach1 gene expression. In the present study, we used northern blot analysis to measure Bach1, HO-1 and δ-aminolevulinate synthase (ALAS1; a heme biosynthesis enzyme) mRNA expression levels during acute hepatic injury induced by CCl4 (at doses of 0.1, 1.0 and 2.0 ml/kg body weight). Oxidative injury was assessed by measuring serum alanine aminotransferase (ALT), hepatic malondialdehyde (MDA) and glutathione (GSH) content. Treatment with CCl4 induced a significant dose-dependent increase in Bach1 mRNA 1–3 h after administration. Bach1 mRNA peaked at 6 h after CCl4 treatment (1 ml/kg), followed by a rapid decrease and gradual return to baseline by 12 h after treatment. The timecourse of transient Bach1 mRNA induction roughly mirrored that of HO-1 mRNA, while ALAS1 mRNA was inversely downregulated. Serum ALT levels and hepatic MDA concentration were significantly increased at 24 h after CCl4 treatment, while the hepatic GSH content was significantly reduced within 3 h of treatment. Serum ALT levels were positively correlated with Bach1 mRNA levels. These findings indicate that Bach1 mRNA is transiently induced in rat liver by CCl4, possibly as a regulatory mechanism to restore HO-1 to baseline following free heme catabolism. Our findings also suggest that Bach1 mRNA expression may be a novel indicator of the extent of oxidative hepatic injury caused by free heme.
PMCID: PMC3990211  PMID: 24748974
Bach1; heme oxygenase-1; free heme; carbon tetrachloride; oxidative stress
4.  Two cases of variceal haemorrhage during living-donor liver transplantation 
BJA: British Journal of Anaesthesia  2011;106(4):537-539.
Some patients with cirrhosis experience rupture of venous varices before operation, and liver transplantation is a therapy of last resort for these patients. However, we have experienced two cases of intraoperative rupture in whom no abnormalities of the venous varices were seen on endoscopy before operation. One patient with ruptured gastrointestinal varices was treated by direct surgical ligation and the other with ruptured oesophageal gastric varices, spontaneously recovered with a Sengstaken–Blakemore tube. These cases suggest that acute variceal haemorrhage should always be considered as a possibility during living-donor liver transplantation in patients with a history of upper gastrointestinal bleeding. Careful observation of the nasogastic tube is important during clamping of the hepatic portal vein.
PMCID: PMC3060377  PMID: 21324927
living-donor liver transplantation; portal hypertension; variceal haemorrhage
5.  RANKL-induced DC-STAMP Is Essential for Osteoclastogenesis 
Osteoclasts are bone-resorbing, multinucleated giant cells that are essential for bone remodeling and are formed through cell fusion of mononuclear precursor cells. Although receptor activator of nuclear factor–κB ligand (RANKL) has been demonstrated to be an important osteoclastogenic cytokine, the cell surface molecules involved in osteoclastogenesis are mostly unknown. Here, we report that the seven-transmembrane receptor-like molecule, dendritic cell–specific transmembrane protein (DC-STAMP) is involved in osteoclastogenesis. Expression of DC-STAMP is rapidly induced in osteoclast precursor cells by RANKL and other osteoclastogenic stimulations. Targeted inhibition of DC-STAMP by small interfering RNAs and specific antibody markedly suppressed the formation of multinucleated osteoclast-like cells. Overexpression of DC-STAMP enhanced osteoclastogenesis in the presence of RANKL. Furthermore, DC-STAMP directly induced the expression of the osteoclast marker tartrate-resistant acid phosphatase. These data demonstrate for the first time that DC-STAMP has an essential role in osteoclastogenesis.
PMCID: PMC2213286  PMID: 15452179
osteoclast; cell fusion; adhesion; seven-transmembrane receptor; TRAP
6.  Hydroxyapatite and bFGF Coating of Detachable Coils for Endovascular Occlusion of Experimental Aneurysm 
Interventional Neuroradiology  2004;9(Suppl 1):29-33.
The purpose of this study was to evaluate the effect of hydroxyapatite (HAp) and fibroblast growth factor-basic (bFGF) coating on Guglielmi detachable coils (GDCs) in an experimental aneurysm model A total of 18 aneurysms were experimentally made in the common carotid arteries of swine. Embolization was done on these aneurysms using standard GDCs and coated GDCs with HAp (GDC-HAp) and with bFGF (GDC-HAp-bFGF). The animals were then killed 14 days after embolization. The development of tissue scarring and coverage the aneurysm’s orifice were evaluated macroscopically. No significant difference of volume ratio of the coils exited in each groups. Macroscopically, covering ratio of fibrous membrane at the neck of aneurysms were 88.3 ± 14.7% in a group with GDC-HAp-bFGF, while it were 26.7 ± 15.3% in a group with standard GDC and it was 41.7 ± 31.7% in a group with GDC-HAp. These results indicated that coating by hydroxyapatite and bFGF might facilitate a wound healing in an experimental aneurysm model.
PMCID: PMC3553474  PMID: 20591225
experimental aneurysm, embolization, hydroxyapatite, bFGF
7.  Hydroxyapatite Coating of Detachable Coils for Endovascular Occlusion of Experimental Aneurysm 
Interventional Neuroradiology  2002;7(Suppl 1):105-110.
The purpose of this study was to evaluate the effect of hydroxyapatite (HAp) coating on Guglielmi detachable coils (GDCs) in an experimental aneurysm model A total of 12 aneurysms were experimentally made in the common carotid arteries of swine using a microsurgical technique. Embolization was done on these aneurysms using standard GDCs and GDCs coated with HAp (GDC-HAp). The animals were then killed 14 days after embolization. The physical properties of coated coils and the development of tissue scarring and coverage the aneurysm's orifice were evaluated macroscopically and microscopically. Macroscopically, a scar formation and coverage at the neck of aneurysms were observed in a group with GDC- HAp> while such findings were not seen in a group with GDC. With light microscope, fibroblasts were seen in the neck of the aneurysms in a group using GDC-HAp, whereas only a fibrin-like net was seen in a group using GDC. In a group with GDC-HAp, inflammatory response was more intense in the dome of the aneurysm with faster re-endothelial coverage of the neck of the aneurysm than the ones in a group with GDC. These results indicated that GDC-HAp might create a clinically beneficial biological surface in an experimental aneurysm model.
PMCID: PMC3627227  PMID: 20663386
experimental aneurysm, embolization, hydroxyapatite coating
8.  Cloning and Molecular Analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) Biosynthesis Genes in Pseudomonas sp. Strain 61-3 
Journal of Bacteriology  1998;180(24):6459-6467.
Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer {poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]} consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In the phb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), β-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed by phbBPs-phbAPs-phbCPs. Upstream from phbBPs was found the phbRPs gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbRPs gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbRPs in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of β-galactosidase activity from a transcriptional phb promoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPs is a positive regulatory protein controlling the transcription of phbBACPs in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) were flanked by a PHA depolymerase gene (phaZPs), and two adjacent open reading frames (ORF1 and phaDPs), and the gene order was ORF1, phaC1Ps, phaZPs, phaC2Ps, and phaDPs. Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida and Ralstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.
PMCID: PMC107745  PMID: 9851987

Results 1-8 (8)