Shunsuke Yamamoto

Transcriptional regulation of the human osteopontin promoter: functional analysis and DNA-protein interactions

Synthesis of cell attachment proteins and cytokines, such as osteopontin (OPN), can promote tumor cell remodeling of the extracellular matrix into an environment that promotes tumor cell attachment and migration. We investigated the transcriptional regulation of OPN in the U-251MG and U-87MG human malignant astrocytoma cell lines. Deletion and mutagenesis analyses of the OPN promoter region identified a proximal promoter element (−24 to −94 relative to the transcription initiation site) that is essential for maintaining high levels of OPN expression in the tumor cells. This element, designated RE-1, consists of two cis-acting elements, RE-1a (−55 to −86) and RE-1b (−22 to −45), which act synergistically to regulate the activity of the OPN promoter. Gel shift assays using nuclear extracts of U-251MG cells demonstrated that RE-1a contains binding sites for transcription factors Sp1, the glucocorticoid receptor, and the E-box-binding factors, whereas RE-1b contains a binding site for the octamer motif-binding protein (OCT-1/OCT-2). Inclusion of antibodies directed toward Myc and OCT-1 in the gel shift assays indicated that Myc and OCT-1 participate in forming DNA-protein complexes on the RE-1a and RE-1b elements, respectively. Our results identify two previously unrecognized elements in the OPN promoter that act synergistically to promote upregulation of OPN synthesis by tumor cells but are regulated by different transcription factors.

Possible Involvement of Toll-Like Receptor 4 in Endothelial Cell Activation of Larger Vessels in Response to Lipopolysaccharide

Toll-like receptors (TLRs) serve as recognition and signaling elements for bacterial substances. To examine the role of TLRs in endothelial cells of larger vessels in lipopolysaccharide (LPS)-induced signaling, the expression and function of TLRs in human umbilical vein endothelial cells (HUVEC) were analyzed. A high level of TLR4 mRNA expression was found in HUVEC, human peripheral blood mononuclear cells (PBMC) and human monocyte cell line THP-1 cells. Little or no TLR2 mRNA expression was observed in HUVEC. In contrast, strong TLR2 mRNA expression was observed in PBMC and THP-1 cells. Moderate and high levels of TLR1 mRNA expression were found in HUVEC, PBMC and THP-1 cells, respectively. TLR3 mRNA expression was moderate in PBMC but weak in HUVEC and THP-1 cells. Little or no TLR5 and RP105 mRNA expression was observed in HUVEC, whereas a moderate level was detected in PBMC and THP-1 cells. The LPS-induced E-selectin expression in HUVEC was significantly inhibited by pretreatment with an anti-TLR4 mAb. Preincubation of HUVEC with an anti- TLR4 mAb significantly reduced the LPS-induced IL-6 production. LPS induced E-selectin and IL-6 production by HUVEC only in the presence of human serum, suggesting the involvement of soluble CD14. Anti-CD14 mAb strongly inhibited the LPS-induced E-selectin and IL-6 production by HUVEC. The inhibition with the concomitant treatment with anti-TLR4 and anti-CD14 mAbs was stronger than that with anti-CD14 mAb only, although it was slight. These results show that TLR4 in the presence of soluble CD14 plays a major role in the signaling of LPS in endothelial cells of larger vessels.

CD156 Transgenic Mice

CD156 (ADAM8) is part of the ADAM family of proteins with the catalytic site consensus sequence of metalloprotease and disintegrins. To examine the role of CD156 in vivo, we generated mutant CD156 (eCD156) transgenic mice expressing the ectodomain of CD156 under the control of the α1-antitrypsin (AT) promoter. One of the transgenic mice designated ATMS2-TG18 expressed a 1.84 kb mRNA which was predicted to be a truncated CD156. The expression of the transgenic CD156 mRNA in ATMS2-TG18 mice was abundant in the liver and slight in kidney. Turpentine oil (TO) and lipopolysaccharide (LPS) markedly upregulated the expression. Soluble CD156 (sCD156) was produced constitutively, and increased after the treatment with TO. Casein-induced peritoneal leukocyte infiltration was significantly less extensive in ATMS2-TG18 than non-transgenic mice. The expression of L-selectin in neutrophils (PMN) from peripheral blood leukocytes (PBL) was more strongly downregulated in ATMS2-TG18 than non-transgenic mice, suggesting that L-selectin in PMN from ATMS2-TG18 mice was shed by sCD156. In contrast, oxazolone (Ox)-induced contact hypersensitivity reactions (CHR) were more marked in ATMS2-TG18 than non-transgenic mice. The expression of E-selectin mRNA was detected in inflammatory skin sites from ATMS2-TG18, but not non-transgenic mice, suggesting that sCD156 may activate the endothelial cells and lead to the upregulation of E-selectin. These results suggest that CD156 regulates leukocyte infiltration directly or indirectly.

The antioxidant EPC-K1 attenuates renal ischemia-reperfusion injury in a rat model.

Background: Acute kidney injury (AKI) occurs frequently in the intensive care unit. A primary cause is renal ischemia/reperfusion (I/R) injury, during which excess reactive oxygen species (ROS) are produced. ROS subsequently damage renal cells, leading to the development of AKI. Here, we investigated whether renal I/R injury could be attenuated by the antioxidant EPC-K1. Methods: We divided male Wistar rats into the following three groups: (1) a renal I/R group, (2) an EPC-K1 + renal I/R group and (3) a control group. Rats were sacrificed 24 h after treatment (I/R or sham). To measure oxidative stress in renal tissue, histological examinations were performed and serum levels of blood urea nitrogen (BUN) and creatinine were measured. The antioxidant action of EPC-K1 was also evaluated in RAW264.7 cells stimulated with antimycin A. Results: Serum BUN and creatinine levels were elevated in the I/R group; however, this increase was significantly attenuated by EPC-K1 in the EPC-K1 + I/R group. Renal tissue injury was also significantly lower in the EPC-K1 + I/R group compared with the I/R group. In vitro experiments showed that EPC-K1 significantly attenuated the generation of ROS induced by antimycin A. Conclusion: In our study, EPC-K1 was able to attenuate AKI due to renal I/R by reducing oxidative stress. These results suggest that EPC-K1 may be effective against various types of I/R injury.

The Roles of Soluble Osteopontin Using Osteopontin-Transgenic Mice in vivo: Proliferation of CD4+ T Lymphocytes and the Enhancement of Cell-Mediated Immune Responses

We generated transgenic mice expressing osteopontin (OPN) under the control of the α1-antitrypsin promoter. These mice (OPN-T mice) expressed OPN mRNA in liver and kidney, and released a large amount of plasma OPN, which increased after stimulation with turpentine oil. Before sensitization, the number of CD4+ T cells in lymph nodes was significantly higher in OPN-T than nontransgenic mice, and that in spleen was slightly higher, whereas that of CD8+ T cells was no different between OPN-T and nontransgenic mice. After sensitization, the CD4+ T cell numbers in spleen increased significantly, while there were almost no changes in the CD8+ T cells in lymph nodes and spleen. The intensity of contact hypersensitivity responses to 2,4-dinitrofluorobenzene (DNFB) was obviously enhanced in OPN-T mice. In the delayed-type hypersensitivity (DTH) model elicited by DNFB, the number of CD8+ T cells among DNFB-2,4,6-trinitrobenzenesulfonic acid (TNBS)-peritoneal exudate cells was significantly higher in OPN-T than nontransgenic mice, while there was almost no difference in that of CD4+ T cells. Adoptive transfer experiments revealed that the enhanced reactivity is carried by CD4+ and CD8+ T cells, respectively, although the ability of transferring DTH was significantly lower in CD8+ than in CD4+ T cells. The enhancement of CD8+ T cell migration was observed in OPN-T mice. These results suggest that OPN induces a proliferation of effector CD4+ and CD8+ cells in cell-mediated reactions and plays a role in the migration of CD8+ T cells.

Removal of 17 Cytokines, HMGB1, and Albumin by Continuous Hemofiltration Using a Cellulose Triacetate Membrane: An Ex Vivo Study

BACKGROUNDnHemofiltration is often used to treat critically ill patients with renal failure and septic shock. Although hemofiltration has been reported to remove humoral mediators such as cytokines, most studies have investigated the removal of only limited kinds of cytokines. Here, we assessed the removal of 17 cytokines, HMGB1, and albumin by continuous hemofiltration (CHF) with a cellulose triacetate membrane (2.1 m(2) or 1.1 m(2)).nnnMETHODSnThe subjects were six healthy volunteers. We collected 400 mL blood into containers with heparin. After adding 1 mg/mL lipopolysaccharide, the blood was incubated at 39°C for 12 h and then filtered through a closed hemofiltration circuit (1 or 2 L/h). Sixty and 240 min after beginning hemofiltration, samples were collected from the outlet (arterial) side, inlet (venous) side, and filtrate port. Blood levels of cytokines, HMGB1, and albumin were determined at each time point.nnnRESULTSnIncreasing the flow rate significantly increased cytokine clearance. Increasing the membrane area of the hemofilter significantly changed the sieving coefficient of only five cytokines (IL-1β, IL-6, MCP-1, MIP-1β, HMGB1). For many cytokines, the sieving coefficient did not decline during the 240-min CHF procedure.nnnCONCLUSIONnAlthough all 17 cytokines, HMGB1, and albumin were detected in the filtrate, the SC and clearance varied widely. For numerous cytokines, clearance increased with the higher filtration flow rate. We demonstrated that CHF removed many cytokines and HMGB1, but was inefficient at removing albumin.

The effect of landiolol on cerebral blood flow in patients undergoing off-pump coronary artery bypass surgery

PurposeTo examine the effect of landiolol on cerebral blood flow in patients with normal or deteriorated cardiac function.MethodsThirty adult patients who were diagnosed with angina pectoris and who underwent elective off-pump coronary artery bypass surgery were studied. Patients were divided into two groups, one with a preoperative left ventricular ejection fraction (EF) of 50% or higher (normal EF group; nxa0=xa015) and the other with an EF of less than 50% (low EF group; nxa0=xa015). The mean cerebral blood flow velocity (Vmca) and pulsatility index (PI) in the middle cerebral artery were recorded using transcranial Doppler ultrasonography (TCD). Individual hemodynamic data were obtained using a pulmonary arterial catheter.ResultsIn both groups, landiolol produced a significant decrease in heart rate (HR), which then returned to baseline 15xa0min after administration was completed. A significant decrease in mean arterial pressure occurred in the low EF group, but the decrease was within 30% of the baseline. In the normal EF group, there was no decrease in cardiac index (CI), whereas in the low EF group, CI significantly decreased along with the decrease in HR. There were no significant differences in Vmca and PI between the two groups.ConclusionContinuous administration of landiolol at a dose of 0.04xa0mg/kg/min after 1 min rapid IV administration at a dose of 0.125xa0mg/kg/min decreases HR without causing aggravation of CBF during treatment of intraoperative tachycardia in patients with normal and deteriorated cardiac function.

Anesthetic management of laparoscopic adjustable gastric banding in Japanese patients with morbid obesity

Laparoscopic adjustable gastric banding (LAGB) is a common type of bariatric surgery worldwide, though not so in Japan. Here we report the anesthetic management of LAGB in ten Japanese patients with morbid obesity. General anesthesia was induced with propofol, fentanyl, and vecuronium bromide and maintained with sevoflurane in oxygen and air (or nitrous oxide in some cases). In a limited number of patients, perioperative epidural analgesia was performed, with fentanyl injected intravenously for analgesia in the remaining patients. Although some special considerations were needed, in perioperative management, including thromboprophylaxis, there were no severe complications in any of the patients.

Mouse Soluble CD14 Truncated at Amino Acid 71 in Transgenic Mice: Preventive Effect on Endotoxin-Mediated Toxic Shock

Mouse soluble CD14 truncated at amino acid 71 (N71) contains the lipopolysaccharide (LPS)-binding sequence. Transgenic mice carrying α1-antitrypsin (AT) promoter-N71 fusion genes, designated AT363-1 and AT363-2, were produced. These mice constitutively produced elevated levels of N71. The concentration of LPS in sera after intraperitoneal LPS injection was lower in AT363-1 mice than in nontransgenic mice. The expression of N71 mRNA was enhanced by subcutaneous turpentine oil injection. The levels of serum LPS and tumor necrosis factor-alpha (TNF-α) after intraperitoneal LPS injections were lower in AT363-1 mice than in nontransgenic mice. Cell surface TNF-α and CD14 expression in exudate peritoneal macrophages prepared by intraperitoneal injection of proteose peptone and then LPS were higher in AT363-1 mice than in nontransgenic mice. Neutrophil infiltration in the liver after induction of the generalized Shwartzman reaction was lower in AT363-1 mice than in nontransgenic mice. Lethality of the Shwartzman reaction was significantly lower in AT363-1 than in nontransgenic mice. These findings suggest that the endotoxin-binding protein (N71) from CD14 prevents endotoxin-mediated toxic shock.

Conversion of atrial flutter to sinus rhythm during landiolol infusion

A 73-year-old male patient with a past history of hypertension and atrial premature contraction underwent endoscopic restoration of the left bubonocele. Sinus rhythm was confirmed by preoperative electrocardiography, but paroxysmal atrial flutter developed when abdominoscopy was started. Continuous administration of landiolol hydrochloride at a dose of 0.005xa0mgxa0kg−1xa0min−1 after a loading dose of 0.04xa0mgxa0kg−1xa0min−1 for 1xa0min resulted in control of heart rate without a decrease in blood pressure. Atrial flutter was converted to sinus rhythm 3xa0min after the start of administration. Landiolol hydrochloride was administered continuously until the morning of the following day and sinus rhythm was maintained postoperatively.