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AIM: To investigate the hepatoprotective effect of MK615, a Japanese apricot extract, in an animal model, and its clinical therapeutic effect. METHODS: Wistar rats were administered physiological saline (4 mL/kg) or MK615 solution (4 mL/kg) for 7 d. On the sixth d, acute hepatic injury was induced by administering a single intraperitoneal injection (ip) of D-galactosamine hydrochloride (D-GalN) (600 mg/kg). Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined, and liver tissues were used for histopathological analysis. Fifty-eight patients with liver disorders [hepatitis C (n = 40), non-alcoholic fatty liver disease (n = 15), and autoimmune liver disease (n = 3)] were orally administered commercially available Misatol ME-containing MK615 (13 g/d) daily for 12 wk. Blood and urine were sampled immediately before and 6 wk, 12 wk, and 16 wk after the start of intake to measure various biochemical parameters. The percentage change in ALT and AST levels after 12 wk from the pre-intake baseline served as a primary endpoint. RESULTS: D-GalN effectively induced acute hepatic injury in the rats. At 48 h after the ip injection of D-GalN, the plasma levels of ALT (475.6 ± 191.5 IU/L vs 225.3 ± 194.2 IU/L, P < 0.05) and AST (1253.9 ± 223.4 IU/L vs 621.9 ± 478.2 IU/L, P < 0.05) in the MK615 group were significantly lower than the control group. Scattered single cell necrosis, loss of hepatocytes, and extensive inflammatory cell infiltration were observed in hepatic tissue samples collected from the control group. However, these findings were less pronounced in the group receiving MK615. At the end of the clinical study, serum ALT and AST levels were significantly decreased compared with pre-intake baseline levels from 103.5 ± 58.8 IU/L to 71.8 ± 39.3 IU/L (P < 0.05) and from 93.5 ± 55.6 IU/L to 65.5 ± 34.8 IU/L (P < 0.05), respectively. A reduction of ≥ 30% from the pre-study baseline ALT level was observed in 26 (45%) of the 58 patients, while 25 (43%) patients exhibited similar AST level reductions. The chronic hepatitis C group exhibited significant ALT and AST level reductions from 93.4 ± 51.1 IU/L to 64.6 ± 35.1 IU/L (P < 0.05) and from 94.2 ± 55.5 IU/L to 67.2 ± 35.6 IU/L (P < 0.05), respectively. A reduction of ≥ 30% from the pre-study baseline ALT level was observed in 20 (50%) of the 40 patients. ALT levels in both the combined ursodeoxycholic acid (UDCA) treatment and the UDCA uncombined groups were significantly lower after Misatol ME administration. MK615 protected hepatocytes from D-GalN-induced cytotoxicity in rats. Misatol ME decreased elevated ALT and AST levels in patients with liver disorders. CONCLUSION: These results suggest that MK615 and Misatol ME are promising hepatoprotective agents for patients with liver disorders.

Free radicals play an important role in the pathogenesis of a variety of diseases; thus, they are an attractive target for therapeutic intervention in these diseases. Compounds capable of scavenging free radicals have been developed for this purpose and some, developed for the treatment of cerebral ischemic stroke, have progressed to clinical trials. One such scavenger, edaravone, is used to treat patients within 24 h of stroke. Edaravone, which can diffuse into many disease-affected organs, also shows protective effects in the heart, lung, intestine, liver, pancreas, kidney, bladder and testis. As well as scavenging free radicals, edaravone has anti-apoptotic, anti-necrotic and anti-cytokine effects in various diseases. Here, we critically review the literature on its clinical efficacy and examine whether edaravone should be considered a candidate for worldwide development, focusing on its effects on diseases other than cerebral infarction. Edaravone has been safely used as a free radical scavenger for more than 10 years; we propose that edaravone may offer a novel treatment option for several diseases.

High-mobility group box 1 (HMGB1) is a nuclear factor and a secreted protein. During inflammation, HMGB1 is secreted into the extracellular space where it can interact with the receptor for advanced glycation end products and trigger proinflammatory signals. Extracellular HMGB1 plays a critical role in several inflammatory diseases such as sepsis and rheumatoid arthritis. Valproic acid (VPA) is one of the most frequently prescribed antiepileptic drugs. The present study was undertaken to investigate the effect of VPA on secretion of HMGB1 in systemic inflammatory responses induced by lipopolysaccharide. Pretreatment with VPA increased the susceptibility of mice to lipopolysaccharide in endotoxemia. Valproic acid induced HMGB1 release and nuclear factor κB activation in RAW-blue cells. Valproic acid promoted the phosphorylation of ERK1/2 but not that of p38 or JNK. The MEK1/2 inhibitor PD98059 also suppressed HMGB1 release and activation of nuclear factor κB induced by VPA. Valproic acid induced expression of γ-aminobutyric acid receptors in macrophages, and picrotoxin, a γ-aminobutyric acid A receptor antagonist, inhibited the VPA-activated phosphorylation of ERK and VPA-induced HMGB1 release. These results suggest that VPA may exacerbate innate immune responses to endotoxin through enhanced release of HMGB1.

Summary. Background and aims: In the present study, we describe a newly developed microchip-based analytical system to evaluate white thrombus formation (WTF). Efficacies of various antithrombotic agents were compared under different flow conditions. Methods: Whole blood containing corn trypsin inhibitor was perfused over a microchip coated with collagen and tissue thromboplastin at the lower and higher shear rates of 240 and 600 s−1, and WTF process inside the microchip was quantified by monitoring a flow pressure. Parameters of T10 (time to 10 kPa), T10–80 (time from 10 to 80 kPa) and OT (occlusion time; time to 80 kPa) were used to evaluate the onset and the growth rate of WTF, and the capillary occlusion, respectively. Results: After perfusion was started, white thrombus composed of activated platelets and fibrin was formed on the coated surface. Thrombus gradually increased in size and eventually occluded the capillary. Among anticoagulants, heparin (0.5–1.0 U mL−1) potently prolonged T10 at both shear rates, whereas low molecular weight heparin (1.0–2.0 IU mL−1) inhibited the growth of WTF at the lower shear rate. Among antiplatelet agents, abciximab (1–2 μg mL−1) significantly reduced the size and number of thrombi, which was additively enhanced in the presence of heparin (0.5 U mL−1). OS-1 (specific GPIbα-antagonist) prevented the complete capillary occlusion. Conclusion: The novel monitoring system of WTF may be useful in preclinical and clinical evaluations of different types of antithrombotic strategies, and their effects in combination.

Objectives To investigate whether high mobility group box 1 (HMGB1) and S100B in cerebrospinal fluid (CSF) and the serum predict the neurological outcome in patients resuscitated from out-of-hospital cardiac arrest (OHCA). Materials and methods This study was designed as a prospective observational study. Twenty-five patients, who received standard cardiopulmonary resuscitation and post-resuscitation intensive care, were enrolled in this study. The patients were divided into two groups according to Glasgow-Pittsburgh Cerebral Performance categories (CPCs) at 6 months after return of spontaneous circulation (ROSC), Group G ( n =7, CPC 1 or 2) and Group P ( n =18, CPC≥3). Their blood samples were taken at 6, 24, and 48h after ROSC. The patients, whose CSF was sampled at 48h, were also divided into either sub-Group G ( n =6) or sub-Group P ( n =8) at 6 months after ROSC. Results HMGB1 and S100B in CSF in sub-Group P were significantly higher than those in sub-Group G (HMGB1, P =0.009; S100B, 2.68 vs. 84.2ng/ml, P =0.007, respectively). HMGB1 in CSF was strongly correlated with S100B ( σ =0.81, P =0.001). HMGB1 was elevated in serum at 6h and normalized within 48h after ROSC without any significant differences between the two groups. Serum S100B in Group P was significantly higher than that in Group G at each time point. Conclusions The significant elevations of HMGB1 and S100B in CSF, and S100B in serum are associated with the neurologically poor outcome in OHCA patients.

The systemic and pulmonary inflammatory response associated with pneumonectomy performed via minithoracotomy versus that after open posterolateral thoracotomy is uncertain.Groups consisting of 7 randomly assigned mice underwent a) minithoracotomy (with 5-mm long incisions and sparing of the muscles) alone, b) posterolateral thoracotomy (with 20-mm long incisions) alone, c) pneumonectomy via minithoracotomy, or d) pneumonectomy via posterolateral thoracotomy. The animals' daily food intake, body weight changes and spontaneous activity were monitored for 10 days, and lung water accumulation and vascular hyperpermeability in the remaining right lung were measured at 24 h after surgery. Concentrations of high mobility group box 1 protein (HMGB1), a mediator of inflammation and shock, were measured in the bronchoalveolar lavage fluid.Compared with posterolateral thoracotomy, pneumonectomy via minithoracotomy was associated with significantly less weight loss (p0.05), despite a similar daily food intake among the groups. Spontaneous activity after pneumonectomy via minithoracotomy returned earlier than after posterolateral thoracotomy. Pulmonary vascular hyperpermeability and water retention in the residual lung were significantly less prominent after pneumonectomy performed via minithoracotomy than after pneumonectomy via posterolateral thoracotomy (both comparisons p0.05). HMGB1 concentrations in the bronchoalveolar lavage fluid collected from the residual lung were significantly lower (p0.05) after minithoracotomy than after posterolateral thoracotomy.Based on postoperative weight loss, spontaneous activity, and the degree of pulmonary capillary injury in the residual lung, pneumonectomy via minithoracotomy was less invasive than posterolateral thoracotomy. The lower increase in HMGB1 associated with minithoracotomy might result in lower pulmonary vascular hyperpermeability and reflect less surgical invasiveness.

ART-123 is a recombinant soluble human thrombomodulin (hTM) with potent anticoagulant activity, and is available for developing antithrombogenic surfaces by immobilization. We focused on improving blood compatibility on the dialyzer surface by the physical adsorption of ART-123 as a safe yet simple method without using chemical reagents. The physical adsorption mechanism and anticoagulant activities of adsorbed hTM on the surface of a polysulfone (PSF) membrane containing polyvinylpyrrolidone (PVP) as a model dialyzer were investigated in detail. The PVP content of the PSF-PVP films was saturated at 20 wt% after immersion in Tris-HCl buffer, even with the addition of over 20 wt% PVP. The surface morphology of the PSF-PVP films was strongly influenced by the PVP content, because PVP covered the outermost surface of the PSF-PVP films. The adsorption speed of hTM slowed dramatically with increasing PVP content up to 10 wt%, but the maximum adsorption amount of hTM onto the PSF-PVP film surface was almost the same, regardless of the PVP content. The PSF-PVP film with the physically adsorbed hTM showed higher protein C activity as compared to the PSF film, it showed excellent blood compatibility due to the protein C activity and the inhibition properties of platelet adhesion. The physical adsorption of hTM can be useful as a safe yet simple method to improve the blood compatibility of a dialyzer surface.

Edaravone was originally developed as a potent free radical scavenger and has been widely used to treat cerebral infarction in Japan since 2001. Several free radical scavengers have been developed and some of them have progressed to clinical trials for the treatment of cerebral infarction. One such scavenger, edaravone, has been approved by the regulatory authority in Japan for the treatment of patients with cerebral infarction. Of particular interest is the ability of edaravone to diffuse into the central nervous system in various neurologic diseases. Aside from its hydroxyl radical scavenging effect, edaravone has been found to have beneficial effects on inflammation, matrix metalloproteinases, nitric oxide production and apoptotic cell death. Concordantly, edaravone has been found to have neuroprotective effects in a number of animal models of disease, including stroke, spinal cord injury, traumatic brain injury, neurodegenerative diseases and brain tumors. The proven safety of edaravone following 9 years of use as a free radical scavenger suggests that it may have potential for development into an effective treatment of multiple neurologic conditions in humans.

Free radicals play major roles in the pathogenesis of many diseases, including neurologic diseases, making them an attractive target for therapeutic intervention. Several free radical scavengers have been developed, and some have progressed to clinical trials for the treatment of ischemic stroke. One such scavenger, edaravone is currently used to treat patients who present within 24 h of an attack. Edaravone can diffuse into many affected organs. Edaravone also exerts protective effects against brain and spinal cord injuries. Beyond its direct free radical scavenging effect, edaravone has anti-apoptotic and anti-inflammatory effects in various diseases. Here, we critically review the literature on experimental animal model and clinical studies of edaravone efficacy, and examine whether it should be considered a candidate for worldwide development. Edaravone has proven safe during 10 years of use as a free radical scavenger to treat ischemic stroke. In addition to ischemic stroke treatment, animal data suggest that edaravone may be an effective treatment option for several neurologic diseases, but additional clinical trials are necessary to verify its efficacy.

We assayed plasma myoglobin and creatine kinase to elucidate the usefulness of rapid assessment of myoglobin for detecting coronary reperfusion in 31 patients with acute myocardial infarction. Reperfusion was achieved in 20 patients by thrombolytic therapy or angioplasty, and it was not in 11 patients. Blood sampling was performed before and 43 +/- 15 (+/- SD) min after the start of treatment. In the reperfused group, blood samples were obtained before and 26 +/- 10 min after reperfusion. Myoglobin was assayed by a new quantitative test based on latex agglutination turbidimetry which required an assay time of 10 min. After treatment, the rate of increase of plasma myoglobin was significantly higher than that of plasma creatine kinase in the reperfused group (9.7 +/- 9.5 and 2.8 +/- 1.6-fold), but not in the occluded group (1.8 +/- 0.6 and 1.5 +/- 0.3-fold). When a 3.0-fold or greater increase in myoglobin (1.9-fold or greater increase in creatine kinase) was taken as evidence of coronary reperfusion, the sensitivity and specificity were 95% and 100% (70% and 82% in creatine kinase), respectively. In conclusion, using the rate of increase of myoglobin, as measured by latex agglutination turbidimetry, coronary reperfusion can be diagnosed within 1 h after reperfusion.

The original version of the paper [1] reports that "This ACTIVE I study was supported by Pfizer" (Page 18905). However, the sponsors of the ACTIVE I study were actually Bristol-Myers Squibb and Sanofi-Aventis rather than Pfizer. [...]

The Japanese apricot, a commonly consumed food called 'Ume' in Japan, has been used for a traditional Japanese medicine for centuries. MK615, an extract of compounds from 'Ume', has strong antitumorigenic and antiinflammatory effects including the induction of apoptosis and autophagy, and inhibition of cytokine production mediated via the inhibition of MAPKs signaling including ERK-1/2, JNK and p38MAPK. The inhibitor of DNA binding 1 (Id-1), a basic helix-loop-helix (bHLH) transcription factor family, is essential for DNA binding and the transcriptional regulation of various proteins that play important roles in the development, progression and invasion of tumors. In melanoma, Id-1 is constitutively expressed in the late and early stages, suggesting it as a therapeutic target in patients with melanoma. This study reports that MK615 profoundly reduced both the mRNA- and protein expression levels of Id-1 and inhibited cell growth in A375 melanoma cells. MK615 markedly inhibited the phosphorylation of ERK1/2, which is associated with Id-1 protein expression in A375 cells. Id-1-specific RNAi induced the death of A375 cells. Moreover, the expression of Bcl-2 was decreased by both MK615 and Id-1-specific RNAi in A375 cells. The results suggest that MK615 is a potential therapeutic agent for treating malignant melanoma.

The efficacy of intravenous immunoglobulin G in the treatment of patients with severe sepsis or septic shock is still being debated. We investigated the impact of high-dose immunoglobulin administration on the survival rate and serum high-mobility group box chromosomal protein 1 (HMGB1) level in a rat model of sepsis created by cecal ligation and puncture (CLP).Rats received either CLP-induced sepsis or had additional immunoglobulin treatment in 1,500 or 300 mg/kg. After induction of sepsis and respective treatment conditions, pulmonary and renal tissues were examined histologically for pathological changes at postoperative hour (POH) 4, and serum cytokine and HMGB1 levels were measured at POH 4, 8, 20, and 44. Using other rats, we also observed the survival rate after CLP for 7 days.Treatment with immunoglobulin significantly improved survival rate at postoperative day 7 (73% in the high-dose group vs. 33% in the control group; p = 0.037). The serum lactate dehydrogenase, endotoxin, creatinine, and blood urea nitrogen levels were significantly lower in the high-dose group than in the other groups. The serum HMGB1 level had increased at 4 h postoperatively in the control group (10.2 ± 3.3 ng/mL) and low-dose group (10.3 ± 4.0 ng/mL), but it was significantly reduced in the high-dose group (4.2 ± 0.8 ng/mL) compared with the control group (p = 0.03).Our results suggest that high-dose immunoglobulin therapy may improve the serum endotoxin and HMGB1 levels and overall survival rate in sepsis by inhibiting the inflammation.

The pathogenesis of acute exacerbation of idiopathic pulmonary fibrosis (IPF) remains to be elucidated. To evaluate the roles of inflammatory mediators in acute exacerbation, the concentrations of high mobility group protein B1 (HMGB1), a chief mediator of acute lung injury, and 18 inflammatory cytokines were measured in the bronchoalveolar lavage fluid, serially sampled from seven IPF patients after the onset of acute exacerbation. HMGB1 gradually increased in the alveolar fluid after the onset of acute exacerbation, in positive correlation with monocytes chemotactic protein-1 (MCP-1), a potent fibrogenic mediator. In the lung tissues of eight IPF patients autopsied after acute exacerbation, intense cytoplasmic staining for HMGB1 was observed in the alveolar epithelial cells in alveolar capillary augmented lesions, where the capillary endothelial cells remarkably reduced the expression of thrombomodulin, an intrinsic antagonist of HMGB1. These results suggest pathogenic roles for HMGB1 and MCP-1 in the late phase of acute exacerbation of IPF.

Tobacco-smoke exposure is linked to carcinogenic, oxidative and inflammatory cellular reactions. Green tea has been reported to have anti-release properties against various pro-inflammatory cytokines. To determine the effects of green tea extract (GTE) on serum high mobility group box-1 (HMGB1) levels in rats exposed to cigarette smoke (CS), we divided rats into 4 treatment groups: (1) CS only, (2) dietary supplement with GTE (3 mg/d) and CS (GCS1), (3) dietary supplement with GTE (4.5 mg/d) and CS (GCS2) and (4) a control group. HMGB1 and cotinine serum levels were analyzed by ELISA. The average serum HMGB1 level in the CS group was significantly higher than the other groups (p0.01), indicating the release of HMGB1 into the blood was stimulated by CS exposure, while GTE consumption suppressed HMGB1 levels. Rats exposed to CS had an average serum cotinine level of 37 ng/ml, indicating tobacco related compounds were present in the rats' blood. However, treatment with GTE did not reduce cotinine levels in all groups. Cotinine stimulated HMGB1 secretion in a dose- and time-dependent manner, and HMGB1 levels were suppressed by GTE in murine macrophage cell lines. Our results show GTE supplementation may offer beneficial systemic effects and suppress HMGB1 by protecting against cell inflammation.

Acute stroke, including acute ischemic stroke (AIS) and acute hemorrhagic stroke, (AHS) is a common medical problem with particular relevance to the demographic changes in industrialized societies. In recent years, treatments for AIS have emerged, including thrombolysis with tissue plasminogen activator (t-PA). Although t-PA is the most effective currently available therapy, it is limited by a narrow therapeutic time window and side effects, and only 3% of all AIS patients receive thrombolysis. Edaravone was originally developed as a potent free radical scavenger and, since 2001, has been widely used to treat AIS in Japan. It was shown that edaravone extended the narrow therapeutic time window of t-PA in rats. The therapeutic time window is very important for the treatment of AIS, and early edaravone treatment is more effective. Thus, more AIS patients might be rescued by administering edaravone with t-PA. Meanwhile, edaravone attenuates AHS-induced brain edema, neurologic deficits and oxidative injury in rats. Although edaravone treatment is currently only indicated for AIS, it does offer neuroprotective effects against AHS in rats. Therefore, we hypothesize that early administration of edaravone can rescue AHS patients as well as AIS patients. Taken together, our findings suggest that edaravone should be immediately administered on suspicion of acute stroke, including AIS and AHS.

1,5-anhydroglucitol (1,5-AG) decreases in diabetic patients and is used as a marker of glycemic control. Type 2 diabetic patients are susceptibile to lipopolysaccharides (LPS), which stimulate macrophages to release large quantities of tumor necrosis factor (TNF)-α and interleukin (IL)-6. This study examines the effects of 1,5-AG on lung inflammation induced by LPS and consequent systemic inflammation to determine whether the decrease of 1,5-AG concentration induces susceptibility to LPS. Before the challenge with LPS (1 mg/kg in vivo and 500 ng/ml in vitro), we pretreated db/db mice and RAW264.7 cells with 1,5-AG at 38.5 mg/kg and 500 μg/ml, respectively. The levels of IL-6, TNF-α, macrophage chemoattractant protein (MCP)-1 and IL-1β in the serum and in the cell supernatants were measured. We also measured macrophage recruitment and the expression of inducible nitric oxide synthase (iNOS) in pulmonary tissues. We found that 1,5-AG attenuated serum cytokine release and protected db/db mice from LPS-induced pulmonary inflammation. In addition, 1,5-AG suppressed cytokine release and iNOS expression by suppressing Akt/NF-κB activity in RAW264.7 cells. These results suggest that 1,5-AG may be a mediator in, as well as marker for diabetes, and 1,5-AG intake may confer tolerance to LPS in patients with type 2 diabetes.

High-mobility-group box chromosomal protein 1 (HMGB1) has recently been identified as a late mediator of various kinds of acute and chronic inflammation. A method for efficiently removing HMGB1 from systemic circulation could be a promising therapy for HMGB1-mediated inflammatory diseases. It is well known that the cationic portion of HMGB1 binds to heparin, which has abundant sulfates in its structure. In this study, we determined whether spherical sulfated cellulose (SC) efficiently adsorbed HMGB1, as well as other inflammatory mediators, in vitro. Then, we investigated the efficacy of hemoperfusion with the SC (SC group) or cellulose beads (control group) at adsorbing endogenous mediators, including HMGB1, in vivo. We have demonstrated that the SC adsorbed significantly larger amounts of HMGB1, interleukin (IL)-4, and IL-8 when compared with cellulose beads, in vitro. Hemoperfusion with the SC for 30 minute, starting 2 hour after an abdominal opening and closure operation, significantly reduced serum HMGB1 levels (p = 0.004) and consistently increased serum IL-10 levels, in vivo. These data suggest the potential benefits of hemoperfusion using the SC in treating HMGB1-mediated inflammatory diseases.

Granulomatous nephritis can be triggered by diverse factors and results in kidney failure. However, despite accumulating data about granulomatous inflammation, pathogenetic mechanisms in nephritis remain unclear. The DNA-binding high-mobility group box-1 protein (HMGB1) initiates and propagates inflammation when released by activated macrophages, and functions as an 'alarm cytokine' signaling tissue damage. In this study, we showed elevated HMGB1 expression in renal granulomas in rats with crystal-induced granulomatous nephritis caused by feeding an adenine-rich diet. HMGB1 levels were also raised in urine and serum, as well as in monocyte chemoattractant protein-1 (MCP-1), a mediator of granulomatous inflammation. Injection of HMGB1 worsened renal function and upregulated MCP-1 in rats with crystal-induced granulomatous nephritis. HMGB1 also induced MCP-1 secretion through mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) pathways in rat renal tubular epithelial cells in vitro. Hmgb1(+/-) mice with crystal-induced nephritis displayed reduced MCP-1 expression in the kidneys and in urine and the number of macrophages in the kidneys was significantly decreased. We conclude that HMGB1 is a new mediator involved in crystal-induced nephritis that amplifies granulomatous inflammation in a cycle where MCP-1 attracts activated macrophages, resulting in excessive and sustained HMGB1 release. HMGB1 could be a novel target for inhibiting chronic granulomatous diseases.

Endocannabinoids including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB2 receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM251 and AM630, which are selective antagonists of CB1 and CB2, respectively. CP55940 (CB1/CB2 agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing.

Well-aligned nanopatterns of various serum, antithrombogenic and cell adhesive proteins, such as gamma-globulin, fibrinogen, thrombomodulin, fibronectin and type I collagen, were fabricated on a highly-oriented block copolymer lamellar surface, and these bioactive protein nanoarrays will be useful in biological research.

Malignant melanoma (MM) is an aggressive chemoresistant skin cancer characterized by rapid metastasis and a poor prognosis. Therefore, the development of innovative effective therapies is critical. MK615 is an extract from the Japanese apricot Prunus mume Sieb. Et Zucc (Ume). At a neutral pH, it contains natural chemical substances such as triterpenoids that exert anti-neoplastic effects in several types of cancers. We found that in patients with advanced MM, MK615 dramatically suppressed the cutaneous in-transit metastasis of the disease. Pre- and post-treatment comparison of tumors showed that the apoptotic index was significantly increased by MK615. In vitro studies, MTT assay, flow cytometric cell cycle analysis and immunofluorescence microscopy revealed that MK615 inhibited the growth of SK-MEL28 cells in a dose-dependent manner, increased the proportion of cells in sub-G1 phase and induced apoptosis. We further examined the expression of the receptor for advanced glycation end products (RAGE). RAGE is a multi-ligand receptor that binds to a novel cytokine, high mobility group box protein 1 (HMGB1), as well as advanced glycation end products. There is evidence that RAGE/HMGB1 interactions enhance cell invasion in MM. Here, we present Western blotting and immunofluorescence microscopy data indicating that MK615 inhibited the expression of RAGE in SK-MEL28 cells, and suppressed the release of HMGB1 by SK-MEL28 cells. Our findings suggest that MK615 may be a valuable tool for treating MM and other malignant tumors.

Introduction Deficiency of Von Willebrand factor (VWF)-cleaving protease (ADAMTS13) causes platelet thrombosis in the microcirculation. Intrarenal coagulation is thought to be associated with the development and progression of diabetic nephropathy. Our aim was to clarify the association between plasma ADAMTS13 antigen (ADAMTS13Ag) levels and diabetic nephropathy. Material and Methods We measured the plasma levels of VWF antigen (VWFAg) and ADAMTS13Ag, and calculated the VWF/ADAMTS13 ratio in 86 type 2 diabetic patients and 26 healthy volunteers, to investigate the relationship between these levels and renal function. With regard to diabetic macroangiopathy, the relationship between these levels and carotid intima-media thickness (IMT) was also investigated. Results and Conclusions A significant positive and negative correlation was noted between ADAMTS13Ag and the estimated glomerular filtration rate (eGFR), vWF/ADAMTS13 ratio and eGFR, respectively. The diabetic patients were divided into normoalbuminuria (n = 50), microalbuminuria (n = 8) and overt nephropathy (n = 28) groups. Compared among these three groups and the 26 healthy volunteers, ADAMTS13Ag was significantly lower only in the overt nephropathy group. The mean carotid IMT was measured in 69 patients and was significantly negatively correlated with ADAMTS13Ag and positively correlated with VWF/ADAMTS13 ratio. When these 69 patients were divided into four groups according to eGFR and ADAMTS13 levels (ADAMTS13/eGFR; low/low: n = 12; high/low: n = 7; low/high: n = 25; high/high: n = 25), the mean carotid IMT was increased in patients with a low ADAMTS13Ag in the same eGFR group. The present study suggests that reduced ADAMTS13 might be associated with diabetic nephropathy.

Isaac's syndrome is a movement disorder characterized by hyperexcitability of peripheral motor nerves. Patients with Isaac's syndrome often develop auto-antibodies to voltage-gated potassium channels (VGKCs) which block their function. However, anti-VGKC antibodies are not detected in all patients with Isaac's syndrome, suggesting the existence of another etiology. In this study, we performed immunoscreening using the serum from a patient with Isaac's syndrome and identified the novel gene named Kenae/CCDC125. Expression analysis of Kenae/CCDC125 revealed that its transcript was highly expressed in tissues associated with the immune system, such as the thymus, spleen and bone marrow. In cells stably expressing Kenae/CCDC125, delay in cell motility and deregulation of RhoGTPase (RhoA, Rac1 and cdc42) activity to extracellular stimuli were demonstrated. These results suggest that the novel gene, Kenae/CCDC125, acts as a regulator of cell motility through RhoA, Rac1 and cdc42.

The Japanese apricot, known as Ume in Japanese, has been a traditional Japanese medicine for centuries, and is a familiar and commonly consumed food. The health benefits of Ume are now being widely recognized and have been strengthened by recent studies showing that MK615, an extract of compounds from Ume, has strong anticancer and anti-inflammatory effects. However, the potential role of MK615 in the periodontal field remains unknown. Here, we found that MK615 significantly reduced the production of pro-inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) induced by Porphyromonas gingivalis lipopolysaccharide (LPS), a major etiological agent in localized chronic periodontitis, in murine macrophage-like RAW264.7 cells. MK615 markedly inhibited the phosphorylation of ERK1/2, p38MAPK, and JNK, which is associated with pro-inflammatory mediator release pathways. Moreover, MK615 completely blocked LPS-triggered NF-kappaB activation. The present results suggest that MK615 has potential as a therapeutic agent for treating inflammatory diseases such as periodontitis.

We recently reported that excessive superoxide anion radical (O(2)(-)) was generated in the jugular vein during reperfusion in rats with forebrain ischemia/reperfusion using a novel electrochemical sensor and excessive O(2)(-) generation was associated with oxidative stress, early inflammation, and endothelial injury. However, the source of O(2)(-) was still unclear. Therefore, we used allopurinol, a potent inhibitor of xanthine oxidase (XO), to clarify the source of O(2)(-) generated in rats with forebrain ischemia/reperfusion. The increased O(2)(-) current and the quantified partial value of electricity (Q), which was calculated by the integration of the current, were significantly attenuated after reperfusion by pretreatment with allopurinol. Malondialdehyde (MDA) in the brain and plasma, high-mobility group box 1 (HMGB1) in plasma, and intercellular adhesion molecule-1 (ICAM-1) in the brain and plasma were significantly attenuated in rats pretreated with allopurinol with dose-dependency in comparison to those in control rats. There were significant correlations between total Q and MDA, HMGB, or ICAM-1 in the brain and plasma. Allopurinol pretreatment suppressed O(2)(-) generation in the brain-perfused blood in the jugular vein, and oxidative stress, early inflammation, and endothelial injury in the acute phase of forebrain ischemia/reperfusion. Thus, XO is one of the major sources of O(2)(-)- in blood after reperfusion in rats with forebrain ischemia/reperfusion.

High-mobility group box 1 protein (HMGB1) is a nuclear factor that is a potent proinflammatory mediator, and may trigger increases in other inflammatory cytokines. The inflammatory cytokines in the cerebrospinal fluid (CSF) of patients with subarachnoid hemorrhage (SAH) have been reported previously, but HMGB1 has not. In this study, we measured HMGB1 and the inflammatory cytokines in the CSF of patients with SAH.CSF samples were collected on days 3, 7, and 14 from the drainage tubes of the postaneurysm clips of 39 patients with SAH. HMGB1, interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNF-alpha) were measured in the CSF, and compared between the patients with favorable (good recovery and moderate disability) and unfavorable outcomes (severe disability, vegetative state, and death) at 3 months.In the unfavorable outcome group, HMGB1 (P = 0.017), IL-6 (P = 0.003), IL-8 (P = 0.041), and TNF-alpha (P = 0.002) were significantly increased. HMGB1 correlated significantly with IL-6, IL-8, and TNF-alpha (R = 0.672, 0.421, and 0.697, respectively).HMGB1 was increased in the CSF of SAH patients with an unfavorable outcome, as were the other cytokines. These results suggest that HMGB1 and cytokines are related to the brain damage observed after SAH. HMGB1 might play a key role in the inflammatory response in the CNS of SAH patients.

Summary Human serum contains thousands of proteolytically derived low-molecular-weight peptide fragments (serum peptidome). The concept of utilizing the serum peptidome for cancer diagnosis has been developed. A pathological serum peptidome appears when the homeostatic balance between proteases and protease inhibitors is disrupted. We hypothesize if analyses of the serum peptidome are of diagnostic value as information on which molecules are disrupted, and the pathological course it will take in unknown pathological conditions and disseminated intravascular coagulation (DIC). We analyzed the serum peptidome in 3 stages (early stage, pre-DIC and DIC stages) in one patient with POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein and skin changes) syndrome, an intractable disease with unknown pathology, using a 1-dimensional gel electrophoresis/matrix-assisted laser desorption/ionization-mass spectrometry (1-DE/MS)-based rapid quantitative approach. A very large number of peptide fragments appeared in the DIC stage, compared to pre-DIC. In addition, we identified fragments of transthyretin (ALGISPFHEHAEVVFTANDSGPR, m/z 2451.18) and α1-antitrypsin (EDPQGDAAQKTDTSHHDQDHPTFN, m/z 2691.02) that significantly increased in the DIC stage, compared to those in the pre-DIC stage. Rapid analyses of the serum peptidome may lead to a diagnostic method that can predict on-going protease activated pathological conditions and help to decide on multilateral strategies including nutritional support and drug therapy.

High mobility group box-1 protein (HMGB1), primarily from the nucleus, is released into the extracellular milieu either passively from necrotic cells or actively through secretion by monocytes/macrophages. Extracellular HMGB1 acts as a potent inflammatory agent by promoting the release of cytokines such as tumor necrosis factor (TNF)-alpha, has procoagulant activity, and is involved in death due to sepsis. Accordingly, HMGB1 is an appropriate therapeutic target. In this study, we found that an extract of Prunus mume Sieb. et Zucc. (Ume) fruit (Ume extract), an abundant source of triterpenoids, strongly inhibited HMGB1 release from lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells. The inhibitory effect on HMGB1 release was enhanced by authentic oleanolic acid (OA), a naturally occurring triterpenoid. Similarly, the HMGB1 release inhibitor in Ume extract was found to be OA. Regarding the mechanisms of the inhibition of HMGB1 release, the OA or Ume extract was found to activate the transcription factor Nrf2, which binds to the antioxidative responsive element, and subsequently the heme oxygenase (HO)-1 protein was induced, indicating that the inhibition of HMGB1 release from LPS-stimulated RAW264.7 cells was mediated via the Nrf2/HO-1 system; an essentially antioxidant effect. These results suggested that natural sources of triterpenoids warrant further evaluation as 'rescue' therapeutics for sepsis and other potentially fatal systemic inflammatory disorders.

High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death in a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.

Edaravone, a potent free radical scavenger, is clinically used for the treatment of cerebral infarction in Japan. Here, we examined the effects of edaravone on the dynamics of high-mobility group box-1 (HMGB1), which is a key mediator of ischemic-induced brain damage, during a 48-h postischemia/reperfusion period in rats and in oxygen-glucose-deprived (OGD) PC12 cells. HMGB1 immunoreactivity was observed in both the cytoplasm and the periphery of cells in the cerebral infarction area 2 h after reperfusion. Intravenous administration of 3 and 6 mg/kg edaravone significantly inhibited nuclear translocation and HMGB1 release in the penumbra area and caused a 26.5 +/- 10.4 and 43.8 +/- 0.5% reduction, respectively, of the total infarct area at 24 h after reperfusion. Moreover, edaravone also decreased plasma HMGB1 levels. In vitro, edaravone dose-dependently (1-10 microM) suppressed OGD- and H(2)O(2)-induced HMGB1 release in PC12 cells. Furthermore, edaravone (3-30 microM) blocked HMGB1-triggered apoptosis in PC12 cells. Our findings suggest a novel neuroprotective mechanism for edaravone that abrogates the release of HMGB1.

Objective.Dendritic cells (DC) contribute to autoimmune disease progression and pathogenesis. Mature DC have been reported to secrete high mobility group box protein (HMGB-1), a novel inflammatory cytokine, via p38 mitogen-activated protein kinase (MAPK) activation. We investigated whether DC are involved in progression of autoimmune diseases followed by secretion of HMGB-1 via p38 MAPK activation in a lupus-prone mouse model.Methods.FR167653, a specific inhibitor of p38 MAPK, was given orally from 3 months of age in MRL-Faslpr mice. Cultured DC, treated with or without FR167653, were stimulated with tumor necrosis factor-α.Results.Inhibition of p38 MAPK led to a reduction in the number of CD11c-positive cells, including those with the mature phenotype, in the diseased kidney and spleen, which resulted in improvement of kidney pathology in MRL-Faslpr mice. The number of CD11c-positive cells in circulation was also reduced. HMGB-1 protein and transcripts detected in the diseased kidney, and the number of cells dual-positive for HMGB-1 and CD11c, were reduced by inhibition of p38 MAPK. Maturation of cultured DC and increased cytokines, including HMGB-1, in the supernatant were inhibited by FR167653 treatment. These results suggest that DC are involved in the progression of autoimmune kidney diseases in MRL-Faslpr mice followed by HMGB-1 secretion via p38 MAPK activation.Conclusion.Our results indicated that DC secrete HMGB-1 via p38 MAPK activation to participate in autoimmunity in MRL-Faslpr mice.

To investigate the pathogenesis of human T cell lymphotropic virus type I (HTLV-I)-associated chronic inflammatory arthropathy (HAAP), we sought to detect proviral DNA in the articular lesions. For the detection of proviral DNA, we used the polymerase chain reaction (PCR). Proviral DNA was detected not only in the peripheral blood mononuclear cells (PBMCs) and synovial fluid cells (SFCs), but also in the T lymphocyte-depleted cultured synovial cells (CSCs). These findings suggest that the infection by HTLV-I might occur in vivo in non-T cells. Furthermore, we detected HTLV-I tax1/rex1 messenger RNA in fresh synovial tissues and CSCs but not in fresh PBMCs and fresh SFCs using reverse transcription and PCR. Immunohistochemically, the CSCs from HAAP patients were also shown to express the HTLV-I antigens. These data indicate that HTLV-I in the non-T synovial cells can be transcribed and expressed. Moreover, the sequences of pXII regions in the CSCs demonstrated 97.5-99.4% homology to that in MT-2 cells, HTLV-I-infected cell line. This confirmed that the PCR-amplified bands reflect HTLV-I itself. These results suggest that this organ-specific inflammation can be attributed to non-T cell virus infection in articular lesions.

Prostacyclin and beraprost sodium (beraprost), a stable analogue of prostacyclin, increased cyclic AMP (cAMP) levels of cultured human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner. The elevation of cAMP by beraprost was sustained longer than that by prostacyclin. The expression of thrombomodulin (TM) on membrane surface of HUVEC was enhanced by beraprost and prostacyclin, and the persistence of the increase in TM expression by beraprost was greater than prostacyclin. Dibutyryl cAMP (db-cAMP) mimicked the effects of beraprost and 3-isobutyl-1-methylxanthine enhanced the effects. Beraprost, prostacyclin and db-cAMP also effectively blocked the interleukin-1- and tumor necrosis factor-induced depression of TM expression substantially. These results suggest that TM expression is positively regulated by cAMP in HUVEC, and that beraprost may be potentially effective for reducing thrombotic events through the mechanism which initiates the stimulation of cAMP/TM system in vascular endothelial cells.

A clonal human skeletal muscle cell line showing acid maltase deficiency (AMD) was established through the transfection of origin-defective SV40 DNA. The low acid alpha-glucosidase activity and glycogenosomes in this clone corresponded to AMD. This clone, in spite of loading glycogenososmes, was competent not only as to proliferation without contact inhibition but also as to myogenic differentiation to some extent. Dexamethasone promoted the formation by the transformant of multinucleated myotubes, which expressed acetylcholine receptors. The existence of glycogenosomes did not seem to affect the proliferation or differentiation of myoblasts. The aberrant acid alpha-glucosidase expressed in the transformed myogenic clone was shown to be biochemically identical to that in AMD fibroblasts. This transformant should be of great value for investigating the pathogenesis of AMD because of the possibility of supplying semi-permanently a uniform myogenic cell line expressing AMD.

We previously reported that the expression of thrombomodulin on the MEG-01, a cell line from human megakaryoblastic leukemia, was increased by agents that increase intracellular cAMP. In this paper we examine the effect of these agents on cultured human umbilical vein endothelial cells (HUVEC) and mouse hemangioma cells. Incubation of the cells with 3 mM dibutyryl cAMP (dbcAMP) increased functionally active thrombomodulin by about 2-fold on HUVEC and 4-fold on hemangioma cells. This effect was observed from 1 hour after the incubation and continued up to 24 hours. Dot hybridization of mRNA demonstrated a dose dependent increase in thrombomodulin mRNA in response to dbcAMP. Treatment of HUVEC with 20 microM forskolin or 100 microM isobutylmethylxanthine (IBMX) also increased cell-surface thrombomodulin on HUVEC. These agents prevented the interleukin I (IL-I) or tumor necrosis factor (TNF)-induced decrease in thrombomodulin on HUVEC. These data suggest that the expression of thrombomodulin on HUVEC and mouse hemangioma cells may be regulated by intracellular cAMP level.

The gene coding for plasminogen has been compared with several abnormal genes from Japanese patients by the polymerase chain reaction and DNA sequence analysis. Two types of abnormal genes coding for plasminogen were identified in these patients. In the type I mutation, a guanosine in GCT coding for Ala-601 near the active-site histidine was replaced by an adenosine resulting in ACT coding for threonine. This mutation was also shown by the loss of a cleavage site for Fnu4HI endonuclease, a restriction enzyme that recognizes GCTGC but not ACTGC. In the type II mutation, a guanosine in GTC coding for Val-355 was replaced by a thymidine resulting in TTC coding for phenylalanine. This change was readily shown by digestion with Ava II endonuclease, a restriction enzyme that recognizes GGTCC and not GTTCC. The type I mutation has been found to be identical to a plasminogen variant identified in Japanese patients by amino acid sequence analysis and also detected by isoelectric focusing, whereas the type II mutation is a unique amino acid substitution in the connecting region between the third and fourth kringles in plasminogen. DNA sequence analysis also revealed that the abnormal genes carry several silent nucleotide substitutions located primarily within introns and 5' and 3' flanking regions.

In order to elucidate the relationship between antiphospholipid antibody (APLA) and thrombotic tendency, we studied the changes in the blood coagulation system in 31 positive APLA patients using thrombin-antithrombin III complex (TAT) and β-thromboglobulin (β-TG), two sensitive molecular markers for in vivo hypercoagulability.The mean plasma levels of TAT and β-TG in the patients were not significantly elevated when compared with those in healthy subjects. And also they were not significantly correlated with anti-cardiolipin antibody titers. In two cases of the patients, plasma levels of TAT and β-TG were serially measured as the underlying disease activities went on. In spite of the disease progression, no significant changes in both plasma TAT and β-TG levels were observed. In one case of the two, however, deep vein thrombosis occurred at a markedly advanced stage of the underlying disease with a rapid increase in both plasma TAT and β-TG levels.These findings suggest that the presence of APLA per se would not directly correlate with in vivo generation of thrombin or platelet activation, but would be an important precipitating factor of thrombus formation under a physical condition closely associated with thrombin generation and/or platelet activation.

In 3 patients with hemophagocytic histiocytosis (HH) accompanied by disseminated intravascular coagulation syndrome (DIC) and multiple organ failure (MOF), a markedly significant decrease of plasma free protein S (PS) levels was found with markedly raised plasma C4b-binding protein (C4bp) levels as compared to non-HH DIC patients with MOF. Moreover, a significant elevation of serum cytokines, interleukin-lβ and tumor necrosis factorα, was observed when compared to non-HH DIC patients with MOF. Plasma free PS was inversely correlated with these two cytokines, while plasma C4bp was positively correlated with them.These results imply that abnormal plasma PS distribution, that is, markedly reduced free PS with raised C4bp-PS complex which was closely associated with hypercytokinemia would contribute to the pathogenesis of DIC and/or MOF in HH.

The levels of protein C (PC) and protein S (PS) were assessed in plasmas derived from 39 patients with various chronic liver diseases and found to be significantly decreased. These decreased levels were positively correlated with the parameters for the protein synthesis in the liver, including cholinesterase, serum albmin, and hepaplastin test, suggesting that the synthesis of PC and PS were also decreased in these diseases. Interestingly, the patients with increased levels of T·AT and manifested decreased PC and PS levels in plasma. These inverse correlations seem to indicate that the decreased levels of PC and PS in plasma could precipitate the disseminated intravascular coagulation syndrome by virtue of insufficient regulation of accelerated blood coagulation.

We established long-term cell lines of cytotoxic T lymphocytes (CTL) specific for human T cell leukemia virus type I (HTLV-I) from peripheral blood lymphocytes (PBL) of a patient with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), an HTLV-I-carrier with Sjögren syndrome, and an asymptomatic HTLV-I-carrier, by repeated stimulation with autologous HTLV-I-infected T cells in vitro. CTL derived from the patient with HAM/TSP expressed CD8 antigen, and their function was restricted by HLA-A2. They showed cytotoxic effects predominantly against the target cells expressing HTLV-I p40tax among the autologous B cell lines infected with vaccinia recombinants containing various HTLV-I genes which served as targets. These data are consistent with the previously reported findings that fresh PBL of HAM/TSP patients contain p40tax-specific CTL activity. Furthermore, CTL derived from the patient with Sjögren syndrome without neurological involvement also demonstrated cytotoxicity predominantly to p40tax. The cytotoxicity to the target cells experimentally expressing p40tax was blocked by unlabeled HTLV-I-infected cells possessing HLA-A2. HTLV-I-specific cytotoxicity was also inhibited by unlabeled B cells bearing p40tax. Thus, HTLV-I p40tax-specific cytotoxicity is mediated by the major CTL population activated by native HTLV-I antigens in patients with HAM/TSP or Sjögren syndrome. In contrast to the CTL of these patients, CTL similarly induced from the asymptomatic HTLV-I-carrier, which were highly cytotoxic to autologous HTLV-I-infected T cells, did not show significant levels of cytotoxicity to autologous B cells expressing p40tax.(ABSTRACT TRUNCATED AT 250 WORDS)

血管内皮細胞は, (1) PGI2, EDRF(内皮細胞由来平滑筋弛緩因子)を産生し,血管拡張/血小板機能を抑制する, (2) thrombomodulin (TM),ヘパリン様分子を産生し,凝固を制御する. (3) tissue plasminogen activator (t-PA)を産生放出する一方,内皮細胞表面にplasminogen, urokinaseを結合せしめて線溶を賦活する, (4)外因系のインヒビターであるLACI (lipoprotein associated coagulation inhibitor)を産生する,等の機能を有し,抗血栓的である.